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LSU Historical Dissertations and Theses Graduate School

1994

The Production and Characterization of AlginateProduced by Pseudomonas Syringae.Richard David AshbyLouisiana State University and Agricultural & Mechanical College

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The production and characterization o f alginate produced byPseudomonas syringae

Ashby, Richard David, Ph.D.

The Louisiana State University and Agricultural ana Mechanical Col., 1994

U M I300 N. ZeebRd.Ann Arbor, MI 48106

THE PRODUCTION AND CHARACTERIZATION OF ALGINATE PRODUCED

BY PSEUDOMONAS SYRINGAE

A Dissertation

Subm itted to the Graduate Faculty of the Louisiana State University and

Agricultural and Mechanical College in partia l fulfillment of the

requirem ents for the degree of Doctor of Philosophy

in

The D epartm ent of Microbiology

byRichard David Ashby

B.S., Brigham Young University, 1987 August 1994

ACKNOWLEDGMENTS

I w ould like to express m y deep est ap p rec ia tio n to m y

advisor, Dr. Donal. F. Day, for his advise, encouragem ent, an d

valuable discussions throughout this work, and also for his help in

the preparation of this dissertation.

S incere ap p rec ia tio n is also ex ten d ed to m y com m ittee

m em bers, Drs. E. A chberger, A. Biel, D. Church, R. Laine, and J.

Larkin for valuable discussions, and suggestions on each aspect of

th is work, an d th e ir helpful insights and constructive criticism

pertain ing to the p repara tion of this d issertation . I would like to

thank Ms. C. Henk for h e r helpful assistance in photo developm ent,

Dr. B. G unn (C hem istry , LSU) fo r h e r he lp in com pu terized

m acrom olecular m odeling, and Ms. D. Sarkar (A udubon Sugar

Institu te, LSU) fo r h e r various help an d suggestions during th is

study. I would also like to thank m y lab collegues, D. Kim, J. Lee,

an d M. Ott for the ir valuable discussion, an d especially thank the

A udubon Sugar Institute for providing the facilities and funds to

complete this research.

Finally, I dedicate this work to my wife, Shelley, m y daughter,

Kailee, and to both sides of my extended family. Their love, patience

and understanding m ade this work possible.

TABLE OF CONTENTS

Page

ACKNOWLEDGMENTS.......................................................................... ii

LIST OF TABLES...................................................................................... vi

LIST OF FIGURES..................................................................................... vii

ABSTRACT............................................................................................... xi

INTRODUCTION........................................................... 1

REVIEW OF LITERATURE....................................................................... 3

I. Bacterial Exopolysaccharides: General C haracteristics.. 3

II. Alginate: O verview ................................................................ 51. Compositional Differences,

Seaweed vs Bacterial A lginate................................ 52. Properties...................................................................... 103. Applications.................... 144. Commercial M anufacture.......................................... 15

III. Alginate Biosynthesis........................................................... 211. Pathw ay. .................................................................. 212. Enzymology................................................................... 253. Regulation...................................................... 30

IV. Pseudomonas syringae Alginates.................................... 32

V. Polysaccharide Analysis....................................................... 321. Depolymerization........................................................ 322. Acid Sensitivity............................................................ 36

VI. Goals of this Study................................................................ 38

MATERIALS AND METHODS............................................................... 39

I. Organisms, Growth Conditions, and M aintenance 39

II. Analytical M ethods................................................................ 401. Cell Mass D eterm ination ........................................... 402. Total C arbohydrate Q uantitation............................. 403. Alginate Q uantitation....................... 414. Acetyl Q uantitation..................................................... 43

iii

III. Optimization of Bacterial Alginate Production............. 451. Media Composition...................................................... 45

A. Carbon Source.................................................... 45B. Nitrogen Source................................................ 45

2. pH and Tem perature ...................................... 463. Agar vs Broth Culture................................................. 46

IV. Batch Ferm entations............................................................ 48

V. Purification of Bacterial Alginate......................................... 48

VI. Deacetylation of Bacterial Alginate.................................. 49

VII. Alginate Size and Quantity D eterm inations................. 50

VIII. Sugar Sensitivity to Acid................................................... 501. Thin Layer Chrom atography.................................... 502. Ion Chrom atography.................................................. 51

IX. Identification of the Acid Hydrolysis Product of L-Gulose................................................................................... 52

1. Thin Layer Chrom atography ........................... 522. Stability of 1,6 Anhydro p-L-Gulopyranose 53

X. Alginate Reductions.................. 53

XI. Alginate Compositions......................................................... 54

XII. Properties of Bacterial Alginates and Effects of Acetylation............................................................................. 55

1. Viscosity.......................................................................... 552. W ater Holding Capacity.............................................. 563. Surface Tension.................................................. 564. Precipitation by Metal Ions....................................... 57

RESULTS................................................................................................... 58

I. Production of Bacterial Alginate...................................... 581. Media Compositions and Conditions ........ 582. Agar vs Broth Culture................................................. 62

II. Characterization of Bacterial Alginate.............................. 691. Recovery................................................................. 692. Molecular W eight......................................................... 693. Composition.......................... 71

iv

III. Functional Properties......................................................... 761. Viscosity.......................................................................... 762. Physical Effects.............................................................. 853. Cation Precipitation by Alginates.............................. 94

DISCUSSION............................................................................................ I l l

REFERENCES............................................................................................ 125

VITA........................................................................................................... 141

v

LIST OF TABLES

Page

Table 1. The structures of some bacterial exopolysaccharides. 6

Table 2. Some food applications of alginates................................ 16

Table 3. Some industrial applications of alginates...................... 17

Table 4. Estimates of U. S. consum ption and price ofpolysaccharides........................................... 20

Table 5. Effects of carbon source on alginate yield fromP. syringae ATCC 19304..................................................... 60

Table 6 . Alginate yield by P. syringae ATCC 19304 ondifferent m edia.................................................................... 67

Table 7. Molecular weights of alginates......................................... 70

Table 8 . Thin layer chrom atography (Rf values)........................ 74

Table 9. The energetics and stability of 1,6 anhydrop-L-gulopyranose................................................................ 79

Table 10. Effects of calcium concentration on the waterholding capacities of alginate gels.................................. 86

Table 11. Effects of acetylation on bead surface tension 91

Table 12. The precipitation of Macrocystis alginate andacetylated and deacetylated P. syringae alginate by m etal ions........................................................................ 1 1 0

vi

LIST OF FIGURES

Page

Figure 1. Structures of the uronic acids of alginates................... 8

Figure 2. The block structures of alginate..................................... 11

Figure 3. The "egg box" model for Ca4f induced gelation ofpoly a-L-guluronate..................... 13

Figure 4. Flow diagram for the extraction of sodium alginatefrom seaweed.................................................................... 19

Figure 5. The proposed biosynthetic pathway of bacterialalginate in Azotobacter vinlandii and Pseudomonas aeruginosa............................................... 22

Figure 6 . A com parison of the overall routes ofincorporation of fructose and glucose in alginate produced by Pseudomonas aeruginosa....................... 24

Figure 7. The relative location of the alginate (alg) geneson the chrom osom e linkage m ap of Pseudomonas aeruginosa................................................. 26

Figure 8 . The m echanism of acid hydrolysis of glycosides 34

Figure 9. The relationship between cell mass andalginate accum ulation by P. syringae ATCC19304 with time in shake flask culture...................... 59

Figure 10. The effect of the initial am m onium concentrationon cell mass, and total alginate accum ulation by P. syringae ATCC 19304 at 48 hours...................... 61

Figure 11. The effect of the initial am m onium concentrationon specific yields of alginate by P. syringae ATCC 19304 a t 48 hours............................................................ 63

Figure 12. The effect of initial pH on cell mass, andalginate accum ulation by P. syringae ATCC 19304 a t 48 hours........................................................................ 64

vii

Figure 13. The effect of tem perature on cell mass, andalginate accum ulation by P. syringae ATCC 19304 at 48 hours....................................................... 65

Figure 14. The effect of initial ferric ion (Fe^+) concentration on the specific yields of alginate by P. syringae ATCC 19304 a t 48 hours............................... 68

Figure 15. Thin layer chrom atograph of HC1 hydrolyzedD-mannose....................................................... 72

Figure 16. Thin layer chrom atograph of HC1 hydrolyzedL-gulose............................................................. 73

Figure 17. Stability of m onom eric D-mannose and L-gulose in HC1 and H2SO4 under hydrolysis conditions at 100°C, as determ ined by ion chrom atography .. . 75

Figure 18. Thin layer chrom atograph of the acid hydrolyzedproduct of L-gulose com pared to 1,6 an h y d rid e s .. . 77

Figure 19. Com puter derived image of 1,6 anhydrop-L-gulopyranose............................................ 78

Figure 20. Gulose recovered, expressed as a percent of the total sugar present in the HC1 hydrolyzed reduced alginates, from Macrocystis pyrifera, and P. syringae ATCC 19304, as determ ined by ion chrom atography.................................................. 80

Figure 21. Comparative viscosity, (N/No), of Macrocystis alginate, acetylated P. syringae alginate, and deacetylated P. syringae alginate as a function of alginate concentration................................................ 82

Figure 22. The effects of acetylation on the com parative viscosity, (N/No), of Macrocystis alginate, and P. syringae alginate........................................ 83

Figure 23. The effects of tem perature on the com parative viscosity, (N/N0), of Macrocystis alginate, acetylated P. syringae alginate, and deacetylated P. syringae alginate................................ 84

viii

Figure 24. Percent difference in w ater holding capacity of Macrocystis alginate gels m ade with ferric iron, and lead, as com pared to calcium alginate g e ls .. . . 87

Figure 25. Percent difference in w ater holding capacity of acetylated P. syringae alginate gels m ade with ferric iron, and lead, as com pared to calcium alginate gels....................... 89

Figure 26. Percent difference in w ater holding capacity of deacetylated P. syringae alginate gels m ade with ferric iron, and lead, as com pared to calcium alginate gels...................................................................... 90

Figure 27. Rate of w ater loss of calcium alginate gels m ade from Macrocystis alginate, acetylated P. syringae alginate, and deacetylated P. syringae alginate as a function of incubation time a t 50°C................... 93

Figure 28. Com parison of the w ater loss of ferric iron alginate gels of Macrocystis alginate, acetylated P. syringae alginate, and deacetylated P. syringae alginate com pared to calcium alginate gels as a function of incubation time a t 50°C.......... 95

Figure 29. Precipitation of Macrocystis alginate, acetylated P. syringae alginate, and deacetylated P. syringae alginate by uranium ( U ^ ) ions.................................. 97

Figure 30. Precipitation of Macrocystis alginate, acetylated P. syringae alginate, and deacetylated P. syringae alginate by copper (Cu2+) ions.................................... 98

Figure 31. Precipitation of Macrocystis alginate, acetylated P. syringae alginate, and deacetylated P. syringae alginate by lead (Pb2+) ions........................................ 99

Figure 32. Precipitation of Macrocystis alginate, acetylated P. syringae alginate, and deacetylated P. syringae alginate by ferric (Fe^+) ions...................................... 100

ix

Figure 33. Precipitation of Macrocystis alginate, acetylated P. syringae alginate, and deacetylated P. syringae alginate by calcium (Ca2+) ions............................. 101

Figure 34. Precipitation of Macrocystis alginate, acetylated P. syringae alginate, and deacetylated P. syringae alginate by strontium (Sr^+) ions.............................. 103

Figure 35. Precipitation of Macrocystis alginate, acetylated P. syringae alginate, and deacetylated P. syringae alginate by zinc (Zn^+) ions........................................ 104

Figure 36. Precipitation of Macrocystis alginate, acetylated P. syringae alginate, and deacetylated P. syringae alginate by m anganese (M n^+) ions......................... 105

Figure 37. Precipitation of Macrocystis alginate, acetylated P. syringae alginate, and deacetylated P. syringae alginate by cobalt (Co2+) ions...................................... 106

Figure 38. Precipitation of Macrocystis alginate, acetylated P. syringae alginate, and deacetylated P. syringae alginate by cesium (Csl+) ions.................................... 107

Figure 39. Precipitation of Macrocystis alginate, acetylated P. syringae alginate, and deacetylated P. syringae alginate by m agnesium (M g2+) ions......................... 108

x

ABSTRACT

A lginate has m any in d u stria l uses because of its u n ique

colloidal behavior, an d its ab ility to thicken, stabilize, emulsify,

suspend, form films, and produce gels. Currently, all com m ercial

alg inate com es from brow n algae, w here it exists n a tu ra lly as a

structural m aterial. Many d ifferen t types of brow n algae produce

alg inate b u t it can only be o b ta ined in sufficient q u an tity an d

quality from a lim ited num ber of species. On the basis of location,

a t least half of the w orld's resources of this polym er are potentially

a t risk due e ith e r to political in stab ility o r industria l pollution.

B acteria p ro v id e a p o ten tia lly u n lim ited a lte rn a te source fo r

alginate. Pseudomonas syringae pv phaseolicola ATCC 19304,

p roduced an acety lated alginate-like polysaccharide with a weight

average m olecu lar w eight (Mw ) o f 1.2 x 1 0 $. This b ac te ria l

polym er was com posed of 82% m annuronic acid and 18% guluronic

acid. Com positional analysis of the red u ced alg inate po lym er

showed th a t L-gulose was m ore sensitive to acid degradation than

D -m annose. The p ercen tag e of gulose reco v e red a t various

hyd ro lysis tim es was ex trap o la ted to zero hyd ro lysis tim e to

account for the loss of gulose. Acetylation affected the solution and

gelling p roperties of the polym er. A cetylated bacteria l alg inate

showed increased viscosity, and w ater holding capacity, and altered

cation precip itability over unacety lated alginates. By controlling

the degree of acetylation on the bacterial alginate, the solution and

gelling p ro p erties of the po lym er can be m an ip u la ted and the

polym er targeted to specific applications.

xi

INTRODUCTION

Alginate is a water-soluble gum used in the food and chemical

in d u s try p rim a rily as an em ulsifier, stab ilizer, o r th ickening

(gelling) agent (117, 144). The com mercial sources of alginate are

th e b ro w n m a rin e seaw eeds o f th e fam ily Phaeopliyceae,

specifically, those of the genera Ascophyllum, Ecklonia, Fusarium,

Laminaria, and Macrocystis. These alginates form viscous solutions

a t low concentrations, show polyelectrolytic behavior, and form gels

an d films (97). Typically, these alginates are block copolym ers,

linked 1-4, of p-D-m annuronic acid (M) an d its C-5 epim er a-L-

guluronic acid (G). The ratio of M/G varies from one algal species to

th e n ex t an d d ic ta tes th e gelling p ro p ertie s of the po lym er.

Alginates w ith h igh M/G ratios are m ore ex tended and p roduce

elastic, pliable gels due to the sm aller regions of poly-G blocks.

Alginates with low M/G ratios produce strong, b rittle gels, because

of the higher affinity of poly-G for C a^ , and the greater com paction

of the molecule.

Alginate is one of the few eukaryotic polysaccharides th a t

have a po ten tia l p rokaryo tic source. M any species of b ac te ria

p roduce "alginate-like" polysaccharides. This was first discovered

in studies on the "slime" produced by Pseudomonas aeruginosa (13,

75, 76) in in fec tions of cystic fibrosis p a tien ts . Azotobacter

vinlandii (51, 99) was also studied as a potential source of alginate

because of its nonpathogenic nature. At high resp iration ra tes the

m ajority of the carbon used by A. vinlandii goes to resp iration and

the form ation of poly p hydroxybutyrate (PHB, 23). This bacterium

1

2

produces alginate only during encystm ent an d does n o t p roduce

sufficient polym er for industrial production.

Other nonpathogenic pseudom onads, including the fluorescent

pseudom onads (58, 120), and the phytopathogenic pseudom onads,

specifically Pseudomonas syringae (37, 38, 91), produce alginates.

P. syringae produces an alginate conspicuously d ifferent from tha t

of the brow n seaw eeds. As w ith o th e r b ac te ria l alg inates, P.

syringae alginates are generally acetylated a t position C-2 a n d /o r C-

3 of the m annuronic acid residues. Unlike seaw eed alginate, P.

syringae polym ers do n o t contain an extensive block structu re .

The bacterial alginate produces bulky, elastic gels w ith high w ater

re te n tio n because of its lack of ex tensive poly-G blocks, O-

acetylation, and high M/G ratio (47).

The goals of th is study were to determ ine the conditions

w hich influence alg inate p ro d u c tio n from P. syringae a n d to

characterize the properties of the polym er in o rder to determ ine if

the use of this organism is feasible fo r large scale p roduction of

alginate.

REVIEW OF LITERATURE

I. Bacterial Exopolysaccharides: General Characteristics.

Most species of bacteria produce exopolysaccharides (EPS), i.e.,

polysaccharides found outside the cell wall, e ither a ttached in the

form of a capsule o r secreted into the extracellular environm ent as

a slime (135). There have been m any theories proposed to explain

the role of bacterial EPS and all re la te to the survival advantages it

offers to the organism s. Originally, these polysaccharides were

studied because of the ir link to pathogenicity. This was especially

true of the capsules of strains of Streptococcus pneumoniae, Bacillus

anthracis, an d Klebsiella pneum oniae (2). The viru lence of these

organism s is due, in part, to the protection provided by the capsular

polysaccharide surrounding the cell. Within anim al hosts, capsules

in terfere with both phagocytosis and antibody binding (87).

Some b ac te r ia secre te EPS as an aid to colonization by

assis tin g in su rface a tta c h m e n t (16, 50). T he a b ili ty o f

Streptococcus m utans and Streptococcus salivarius cells to adhere to

the surface of teeth is partia lly a function of the EPS p roduced by

these oral bac teria (87). O ther roles of EPS include p reven ting

desiccation, energy storage, concentration and up take of charged

molecules, particu larly m etal ions (11, 17, 46), p ro tec tion against

the effects of u ltrav io let rad ia tion (17), and m ediation of biofilm

and microcolony form ation (18).

B acterial EPS is species specific. Sugars a re common

com ponents in m ost bacterial EPS. D-glucose, D-mannose, and D-

galactose occur frequently , and L-rhamnose and L-fucose a re less

3

4

com m on. The presence of negative charges is a featu re of some

b ac te ria l EPS. U sually these charges a re th e re su lt o f the

incorporation of uronic acids into the polym er. The m ost com m on

negatively charged sugar p resen t in bacteria l EPS is D-glucuronic

acid which is a com ponent of hyaluronan , xan than gum, an d m ost

teichuronic acids. D -m annuronic acid, D-galacturonic acid and L-

guluronic acid are also com ponents of some bacterial polymers. D-

m annuronic acid and L-guluronic acid are associated prim arily with

b ac te ria l a lg inates. D -galactu ron ic ac id is fo u n d in som e

lipopo lysaccharides p ro d u ced by Proteus m irabilis, (77) an d

Xanthomonas campestris (142).

B iosynthesis of EPS n o rm a lly occu rs by e i th e r o f two

m echanism s. EPS m ay be produced at the cytoplasm ic m em brane

using p recursors form ed in tracellu larly , o r they m ay be form ed

fro m specific p re c u rso rs in th e e x tra c e llu la r en v iro n m en t.

G enerally, the firs t type is characteristic of heteropolysaccharide

p ro d u c tio n , w hile th e seco n d is c h a ra c te r is t ic o f c e r ta in

hom opolysaccharides including levans an d dex trans (133). Both

p a th w a y s re q u ire th e p ro d u c tio n o f a c tiv a te d n u c leo tid e

d iphosphate form s of the m onosaccharides and the p recursors for

substituent groups, specifically acetate, pyruvate, and succinate (25,

37). Acetyl CoA was recently confirm ed as the source of acetate in

xan than gum biosynthesis by Xanthomonas campestris (6 6 ). The

p rec u rso r fo r p y ru v a te is p h o sp h o en o lp y ru v a te (71). T hese

substituents introduce negative charges into the polysaccharides. A

high num ber of negative charges, coupled with a high m olecular

weight, p roduce polym ers th a t form highly h y d ra ted gels. While

5

th e ab ility to p ro d u ce EPS is w idesp read , u n d e r la b o ra to ry

conditions this ability is often unstable and lost on repea ted culture

(17).

Bacterial EPS varies enorm ously in structure an d composition,

ran g in g from sim ple hom opolysaccharides to com plex, h ighly

substitu ted and branched heteropolysaccharides (Table 1). These

polysaccharides can be divided into five distinct groups: 1) dextrans

an d levans, o r hom opolysaccharides p roduced by b ac teria using

sucrose as a specific substrate, 2 ) hom opolysaccharides o th e r than

d e x tra n a n d le v a n , i.e., cellu lose, 3) h e te ro p o ly sacch a rid e s

containing m ore than one type of m onosaccharide synthesized from

a specific carbon substrate, 4) heteropolysaccharides form ed from

re p e a t in g u n i t s tru c tu re s , i.e., x a n th a n g u m , a n d 5)

heteropolysaccharides com posed of two types of m onom er w ith no

repeating unit, i.e., alginate (134).

II. Alginate: Overview

1. Compositional Differences, Seaweed vs. Bacterial Alginate

Alginate, the m ajor s truc tu ra l polysaccharide of the brow n

algae (Phaeophyceae), was first discovered by E. C. C. S tanford in

1881 (19). It was n o t un til 30 years la ter th a t uronic acids were

identified as the m ajor com ponents (118). Initially, it was assum ed

th a t p-D -m annuronate was the only m onosaccharide p re sen t in

alginate. Subsequent research established the copolym eric n a tu re

of alginate and the presence of variable am ounts of a second uronic

acid, a-L-guluronate (41).

Table 1. The structures of some bacterial exopolysaccharides.

Species Polysaccharide Structure

Leuconostoc m esenteroides Dextran ( l i t6 glucose)nAcetobacter xylinum Cellulose (16.4 glucose)nAlcaligenes faecalis var. niyxogcnes Curdlan (163 glucose)nStreptococcus m utans Mutan (1“ 3 glucose)nStreptococcus salivarius Levan (26.6 fructose)nAzotobacter vinlandii Alginate 1-4 linked ManA and GulAPseudomonas aeruginosa Alginate 1-4 linked ManA and GulAStreptococci H yaluronan (-3GlcNAcl6.4GlcAl6.)n(Haemolytic group A)Xanthomonas campestris Xanthan (-4Glclh4Glclh4Glclli)n

I1

Aa

Abbreviations: Gal, galactose; Glc, glucose; GlcA, glucuronic acid; GulA, guluronic acid; Man, m annose; ManA, m annuronic acid; GlcNAc, N-acetylglucosamine; OAc, O-acetyl; Pyr, pyruvate.

a A= (-3«lM an(OAc)2hlGlcA4lilM an4-Pyr)

7

W ithin the last 30 years, alginate was found to be produced

by som e p ro k a ry o te s . The o b se rv a tio n th a t Pseudomonas

aeruginosa was able to synthesize an "alginate-like" polysaccharide

was f irs t rep o rted by Linker and Jones (75), a lthough m ucoid

s tra in s o f th is b ac te riu m w ere iso la ted m uch ea rlie r . They

dem onstrated th a t this polym er had com ponents identical to algal

alginate. In a subsequent study, they showed tha t an O-acetylated

a lg inate was the m ajor com ponent of P. aeruginosa slim e (76).

Carlson and Matthews (13) confirm ed the com position of bacterial

alg inates in a study of 13 d iffe ren t m ucoid isolates from cystic

fibrosis p a tien ts . M ore recen tly , the re la ted p seudom onads P.

fluorescens, P. mendocina, P. pu tida (53), and m any pathovars of P.

syringae (37) have been identified as alginate producers. The same

year th a t P. aeruginosa was reported to produce alginate, Gorin and

Spencer (51) showed tha t the polysaccharide produced by strains of

Azotobacter vinlandii was the sam e as the acety lated alginate

produced by pseudom onads. Later it was determ ined th a t alginate

b io syn thesis by A. vinlandii was closely asso c ia ted w ith the

form ation of vegetative cysts (93).

Alginates are unbranched copolymers of p-D-mannuronic acid

an d its C-5 epim er cx-L-guluronic acid, linked 1-4 (Fig. 1). The

actual composition and sequence of the polym er is dependen t upon

the source (73, 96, 130). Alginates from seaweed an d A. vinlandii

generally con tain extensive regions of hom opolym eric blocks of

m annuronic acid and guluronic acid together with some random or

a lte rn a tin g sequences. A lginates d e riv e d fro m s tra in s of

Pseudomonas contain few block hom opolym eric regions. Instead,

p-D-m annuronate

C -( i

ooc

a-L-guluronate

Figure 1 . Structures of the uronic acids of alginates.

9

they a re com posed p red o m in an tly of ran d o m sequences. In

addition, the m annuronic acid residues are highly O-acetylated (2 1 ,

125). The structure, as well as the position of acetylation in these

alginates, has been extensively studied. The O-acetyl groups are

associated exclusively with the C-2 a n d /o r C-3 positions of the D-

m a n n u ro n a te re s id u e s . n u c le a r m ag n e tic re so n a n c e

sp ec tro m etry (NMR) rev ea led th a t som e of th e m a n n u ro n a te

residues were 2,3 di-O-acetylated, although the m ono-O -acetylated

form s were m ore com m on (21, 125, 127). The reason for th e O-

acety la tion in bac teria l alg inates is n o t en tire ly clear. It was

suggested tha t acetylation m ay be p art of a control m echanism tha t

o p e ra te s du rin g b iosyn thesis th a t con tro ls the M/G ra tio by

p rev en tin g ep im eriza tion of th e ca rboxy l g ro u p on th e C-5

m an n u ro n a te carbon (128). A lternatively, it m ay play a ro le in

dictating the solution properties of the polysaccharide (129).

Seaweed and bacteria l alginates d iffer in th e ir M/G ratios.

Most alginates isolated from the brown seaweeds show M/G ratios

in the range of 0.45 (Laminaria hyperborea) to 1.85 (Ascophyllum

nodosum , 117). The m ost com mon industrially available alginate is

extracted from the brow n alga, Macrocystis pyrifera. This polym er

contains 60% 4-linked m annuronate and 40% 4-linked guluronate

residues giving an M/G ratio of 1.5. The alginates p roduced by P.

aeruginosa con tain 80% 4-linked m annuronate an d 20% 4-linked

guluronate residues, giving an M/G ratio of 4.0 (150).

Haworth projections of the (5-D-mannuronate an d the a-L-

gu luronate residues show little d ifference in the two structu res.

Epimerization of the carboxyl group at C-5 does, however produce a

10

m arked change in the th ree dim ensional conform ation of these

m onosaccharides. Since the carboxyl group is th e m ost bulky

s u b s t i tu e n t on th e r in g , th e m ost en e rg e tic a lly fav o rab le

conform ation orien ts this group to the equatorial position. As a

result, p-D-m annuronate exists preferentially in the chair form,

w hereas the a-L-guluronate residues adop t the IC4 conform ation

(Fig. 1). Chains of sugars containing com binations of these two

fo rm s p ro d u c e p o ly m ers w ith d if fe re n t th re e d im en sio n a l

structures. The p-D -m annuronate linkage positions, C -l an d C-4,

are equatorial to the p lane of the sugar ring. These linkages are

axial fo r th e a-L -guluronate residues. X-ray fib er d iffrac tio n

studies of alginates containing high p roportions of m annurona te

residues indicate a flat ribbon-like conform ation in the solid state

(5, 109, 110). This co n fo rm atio n is s im ila r to th e p 1-4

diequatorially linked polym ers such as cellulose. Alginates rich in

polyguluronate, which is 1-4 diaxially linked, ad o p t a buckled 2-

fold chain conform ation (6 , Fig. 2).

2. Properties

The physical com position of alginates dictates the solution and

gelling p ro p e rtie s of th e po lym ers. A lginate is a s tru c tu ra l

com ponent in the brow n seaweeds w here it contributes to bo th the

tensile strength and the flexibility of the algal tissue (64, 143). In

A. vinlandii it serves a s truc tu ra l function for the m etabolically

dorm ant cysts. Evidence exists tha t it is an im portan t com ponent of

bo th the cyst exine and intine, which are m icroscopically d istinct

reg ions ou tside th e cen tra l body of th e cyst (115). A lginate

11

Figure 2. The block struc tu res of alginate. A. The ribbon-like s tru c tu re of poly p -D -m annuronate. B. The b u ck led ch a in conform ation of poly a-L-guluronate. C. A lternating sequences of p- D -m annuronate and a-L-guluronate.

12

production by P. aeruginosa was related to resistance to antibiotics,

phage an d bacteriocins and pro tection against phagocytosis and

antibody attachm ent. This suggests, tha t for P. aeruginosa, alginate

functions as a general protective barrier.

Alginates with low M/G ratios produce strong bu t brittle gels,

w hereas alginates with high M/G ratios form m ore elastic gels (62,

110). This d ifference is due to the a rran g em en t of the block

structures w ithin the polym ers and is particularly evident when the

polysaccharide in teracts with cations. Alginate, being a polyanion,

is a n a tu ra l ion-exchanger w here the selectivity and streng th of

b inding depend on the cation, the conform ational characteristics,

an d the linear charge density of the polym er. Most m ultivalent

cations b ind alginates an d cross-link the polysaccharide to form a

gel m atrix . Some reg ions of the po lysaccharide form stronger

chelation com plexes th an o thers w ith divalent cations, especially

calcium ions (Ca+h). The "egg box model" was proposed to explain

this interaction (82, 83, 8 8 , 109, 110, Fig. 3,). The binding of Ca_l+ is

strong because in ad d itio n to the ionic b inding to the carboxyl

groups, various ring and hydroxyl oxygen atom s are able to chelate

th e c a tio n s . P o ly g u lu ro n a te b in d s Ca4f v e ry s tro n g ly .

Polym annuronate or mixed sequences do no t bind Ca_l+ with as high

an affinity . W hen various block sequences w ere iso lated from

in tact alginate, polyguluronate blocks showed an enhanced binding

of C a ^ in polym ers above 20 residues (61). This in d ica ted a

cooperative m echanism w here binding sites exist in an o rd ered

array, and binding of one ion facilitates binding of a second. Similar

Ca++

S^°-VCa++

Figure 3. The "egg box" model for C a^ induced gelation of poly «- L- guluronate.

14

effects were n o t seen with polym annuronate blocks o r alternating

sequences (6 8 ).

Bacterial alginates tha t contain few polyguluronate blocks (a

h igh M/G ratio ), an d th a t a re O-acetylated, p roduce relatively

bulky, flexible gels w ith high w ater re ten tio n in the p resence of

Ca++( 109). The high num ber of m annuronate residues and O-acetyl

groups reduce the cooperative binding of Ca'l+ and w eaken the gel

network. This fact, in association with the greater positive osmotic

p re s s u re cau sed by th e in c re a se d n u m b e r of d isso c ia ted

counterions, significantly enhances the w ater holding capacity of

bacterial gel m atrices (129). The resulting gel provides cells with

hydrophilic capsules th a t p ro tect them from other microorganisms,

chemicals, antibiotics, and desiccation (116). In P. syringae, alginate

is th o u g h t to p lay an im portan t role in the pathogenicity of the

bacterium , allowing adhesion to the host surface (137).

3. Applications

While alginate production is w idespread among m em bers of

the brow n seaweed (Phaeophyceae), only a few species of brow n

seaweed are used for com mercial production. The principal source

of th e w orld 's supply o f alg inate is the g iant kelp, Macrocystis

pyrifera. O ther seaweeds tha t are used in alginate m anufacture are

Ascophyllum nodosum and species of Laminaria and Ecklonia.

A lg ina te is u sed in foods a n d fo r genera l in d u s tria l

applications because of its unique colloidal behavior and its ability

to thicken, stabilize, em ulsify, suspend, form films, an d p roduce

gels. The p rim ary p ro p erties on which the w idespread use of

15

alginate is based are: 1 ) the fo rm ation of viscous so lu tions a t

relatively low concentrations, 2 ) the polyelectrolytic behavior in

solution, 3) the ability to form gels by chem ical reaction , 4) the

form ation of films on surfaces, and 5) the base exchange properties

(97).

Food p roducts in which alginates are used include frozen

foods, pastry fillings, bakery products, and syrups. Because of their

gelling ab ility , a lg inates a re also used in in s ta n t an d cooked

puddings, and pie fillings. Alginates are excellent em ulsifiers for

salad dressings an d stabilizers for beverages, w hipped toppings,

an d sauces (Table 2). Alginates are also of value in a num ber of

industrial applications, such as the m anufacture of paper, adhesives,

textiles, a ir fresheners, explosives, polishes, antifoam s, ceramics,

and cleaners (19, 97, Table 3).

Since the industria l p roduction of seaweed alginates began,

several com m ercially im portan t derivatives of alginates have been

developed. Commercial derivations include the sodium, potassium,

am monium , calcium, and mixed am monium-calcium salts of alginic

acid, propylene glycol alginate, and alginic acid. The propylene

glycol ester is the only commercial, organic derivative of alginate.

It has im proved acid stability an d resistance to p recip itation by

calcium and o ther m ultivalent m etal ions (19).

4. Commercial M anufacture

Macrocystis pyrifera grows in relatively calm m arine w aters

in large, dense beds. It is a very rapid ly growing p lan t th a t allows

for up to four cuttings p er year. At the time of harvesting, a dense

16

Table 2. Some Food Applications of Alginatesa

Property Product Perform ance

W ater holding Frozen foods

Pastry fillings Syrups

Bakery icings

Maintains texture during freeze-thaw cycles.Produces smooth, soft texture Suspends solids, controls pouring consistency Counteracts stickiness and cracking

Gelling Puddings Firms body and texture Pie and pastry fillings Acts as a cold w ater gel base;

develops soft gel body with broad tem perature tolerance; gives im proved flavor release.

Dessert gels Produces clear, firm, quick-setting gels.

Emulsifying Salad dressings Sauces

Emulsifies and stabilization Emulsifies oils and suspends solids

Stabilizing BeerJuices

syrups and toppings

W hipped toppings

Sauces an d gravies

Maintains beer foam Stabilizes pulp in concentrates and finished drinksSuspends solids; produces uniform bodyStabilizes fat dispersion, and freeze-thaw breakdow n Thickens and stabilizes for a broad range of applications

a Table was adapted from Sandford and Baird (117).

17

Table 3. Some Industrial Applications of Alginatea

Property Product Performance

W ater holding Paper coating Paper sizings

Adhesives

Textile printing

Controls rheology of coatings Improves surface properties, and ink acceptance Controls penetration to im prove adhesion and applicationProduces very fine line prints

Gelling Air freshener

Explosives

Toys

Hydromulching

Firm, stable gels are produced from cold w ater system sElastic gels produced byreaction with boratesNontoxic m aterials m ade forimpressionsHolds mulch to inclinedsurfaces; prom otes seedgermination

Emulsifying Polishes

AntifoamsLatexes

Emulsifies oils and suspends solidsEmulsifies and stabilizes Stabilizes latex emulsions; provides viscosity

Stabilizing CeramicsCleaners

Suspend solids Suspends and stabilizes insoluble solids

a Table was adapted from Sandford and Baird (117)

18

m at of fronds floats on the ocean surface. Harvesting is actually a

m assive prun ing of the kelp beds. U nderw ater blades mow the

kelp approxim ately th ree feet below the w ater surface. The kelp is

then conveyed into the hold of a barge by a moving belt.

The process fo r alg inate ex trac tion is based on an ion-

exchange process. In seaweed, the alginate is p resen t as a mixed

salt of sodium a n d /o r potassium, calcium, and m agnesium (19). The

exact com position of the polym er varies w ith the type of seaweed

bu t this does no t affect processing. The extraction process begins

by grinding the seaweed and washing it with water. A strong alkali

is th en added to the w ashed seaweed an d the m ixture is heated to

ex tract an d dissolve the alginate. The crude alginate solution is

then clarified and precip itated by the addition of calcium chloride.

The calcium alg inate is acid trea ted to p roduce an alginic acid

p recip ita te . Sodium carbonate is then ad d ed to m ake a sodium

alg inate paste w hich is d ried , an d m illed in to sodium alg inate

pow der (Fig. 4).

The extraction costs of seaweed alginate are higher than o ther

industrially available gums due to harvesting and purification costs

(117, Table 4). Because of the unique properties of alginates, and

the high num ber of potential food and industrial applications, there

are no good replacem ents for this polym er. Bacteria have been

studied as potential a lternate sources of alginate. A bacterial source

would give an unlim ited supply of alginate and may reduce costs.

C a l c i u mA lka l i + W a t e r C h l o r i d e

W a t e r * H e a t S o l u t i o n

Mi l l ing W a s h i n gW e t o r D r y

S e a w e e dC l a r i f i c a t i o n

D i s s o l u t i o n o f A lg in a te s P r e c i p i t a t i o n

C r u d e A lg in a te S o l u t i o n

W a sh in g s I n s o l u b l e R e s i d u e

C o l o r a n d O d o r S o d i u m

R e m o v a l A c i d C a r b o n a t e

C a l c i u mA l g i n a t e

A c id T r e a t m e n t

A lg i n ic A c i d P r e c i p i t a t e

W a te r a n d

D is so l v e dI m p u r i t i e s

S o d i u m A lg in a te P a s t e

D r y i n g Mil l ingD r y S o d i u m

A lg i n a t e P o w d e r

Figure 4. Flow diagram for the extraction of sodium alginate from seaweed (117).

20

Table 4. Estimates of U. S. Consumption and Price of Polysaccharides.

Polysaccharide% U. S. ConsumDtiona

Food Industrial Retail Price (per 1 0 0 g)b

Agar 0.4 0.6 $ 14.25Alginate 11.5 6.2 $ 14.55Carrageenan 11.5 0.3 $ 15.35Guar gum 19.3 53.8 $ 2 .2 0Gum arabic 29.5 10.8 $ 5.25Gum ghatti 1.3 1.6 $ 12 .2 0Gum tragacanth 1.2 0.3 $ 16.35Karaya gum 1.3 10.8 $ 2.40Locust bean gum 11.5 6.2 $ 2 .1 0Pectin 9.6 0 $ 18.10Xanthan gum 2.9 9.3 $ 11.60

a Percent U. S. consum ption is based on 1980 figures. Percentages represen t the am ount of polysaccharide used for food and industrial applications relative to each o ther (117).b Retail price is based on 1994 figures from Sigma Chemical Co., St Louis, Missouri.

21

III. Alginate Biosynthesis

1. Pathway

In 1966, the biosynthetic ro u te of alginate p roduction was

established for the m arine brow n seaweed Fucus gardneri (73, 74).

Later, a sim ilar pathw ay was proposed for alginate p roduction by

Azotobacter vinlandii (99). In 1981, Piggott e t al (98) proposed a

pathw ay fo r alginate p roduction in Pseudomonas aeruginosa (Fig.

5). This pathw ay was based on the detection of all the enzym es

necessary for b iosynthesis of bac teria l alg inate, except fo r the

epim erase and the O-acetyl transferase.

Fructose 6 -phosphate is considered to be the p recursor in the

alg inate b iosynthetic pathw ay . The p rim a ry ro u te of glucose

ca tabo lism in p seu d o m o n ad s is th ro u g h the Entner-D oudoroff

pathw ay , p roducing g lyceraldehyde 3-phosphate an d pyruvate .

Carlson and Matthews (13) showed tha t the C-6 , bu t no t the C-l of

glucose was incorporated in to alginate. This im plied th a t carbon

atom s 1, 2, an d 3 of glucose were converted to pyruvate by the 2-

keto 3-deoxyphosphogluconate aldolase reac tion an d eventually

were lost to the alginate biosynthetic pathw ay as CO2 an d acetyl

CoA. Carbon atoms 4, 5, and 6 were channeled into alginate through

g lycera ldehyde 3 -phosphate . F ru c to se 6 -p h o sp h a te can be

p roduced either th rough gluconeogenesis o r by condensation of

g lycera ldehyde 3-p h o sp h ate w ith d ihydroxyacetone phosphate.

S u bsequen t stud ies w ith a P. aeruginosa m u ta n t d e fic ien t in

fru c to se 1 ,6 d ip h o sp h a te a ld o lase show ed th a t th e re w as

p referen tia l 14c incorporation from C-6 in to a lg inate com pared

w ith C-l labeled glucose in the wildtype. Incorporation of C-6 and

22

fructose 6-phosphate

Phosphomannose isorerase

Mannose 6-phosphate

Phosohomannonutase

Mannose 1-phosphate

GDP-r.annose pyrcpncspnory 1 ase

GDP-mannose

GDP-nannose denvcrooenasc

GDP-nannuromc acid

Pol viiierase

Epinerase

Acetyl transferase-

ALGINATE

Figure 5. The p ro p o sed b io syn thetic pa thw ay of a lg in a te in A zotobacter vinlandii a n d Pseudomonas aeruginosa. The firs t 4 enzym atic steps have been iden tified in bo th organism s. The po lym erase an d epim erase have been iden tified in A. vin landii only, and the acetyl transferase has not yet been identified in either organism.

23

C-l of glucose were sim ilar in the m u tan t (7, 78). A sum m ary of

the overall ro u tes of in co rp o ra tio n of fruc tose an d glucose in

alginate can be seen in Figure 6 .

F ructose 6 -phosphate can be isom erized to m an n o se 6 -

p h o sp h a te by p hosphom annose isom erase. T ra n s fe r o f th e

phosphate group by phosphom annom utase produces m annose 1-

phosphate from m annose 6 -phosphate. M annose 1-phosphate and

GTP are th en converted in to GDP-mannose, catalyzed by GDP-

m annose pyrophosphorylase. The subsequent oxidation th rough

GDP-mannose dehydrogenase resu lts in th e fo rm atio n of GDP-

m annuronic acid. Polymerization of GDP-mannuronic acid results in

p o lym annurona te w hich is secreted from th e b ac teria l cell and

becom es th e su b s tra te fo r an ex trace llu la r ace ty lase a n d /o r

epim erase in the production of the m ature polym er (47).

The tran sp o rt m echanism of the bacterial alginate across the

cytoplasm ic m em brane is believed to be sim ilar to th a t of some

bacterial cell wall polym ers, w hich use isopreno id lip id carriers

(134). The final step in the biosynthesis of bacterial alginate is the

selective epim erization of the m annuronate residues to guluronate

by an extracellu lar epim erase. The selectivity is th o u g h t to be

d ic ta te d by th e O -ace ty la tion of the m a n n u ro n a te residues.

Acetylation is believed to inh ib it epim erization, thereby dictating

the final com position of the alginates. Acetyl CoA is the probable

source of the acetyl group in bacterial alginates (136), ju st as it is

on th e m a n n o sy l re s id u e s o f x a n th a n gum p ro d u c e d by

Xanthom onas cam pestris (6 6 ). The m echanism of b ac te ria l O

acetylation is no t well defined.

F ructose G 1ucoso >• G l u c o n a t e

2 - K e t o g l u c o n a t eF ru c t o s e 1 - p h o s p h a t e

F r u c t o s e 6 - p h o s p h a t e ■* ► G l u c o s e 6 - p h o s p h a t e

6 - P h o s p h o g l u c o n a t e

A L G I N A T E

F r u c t o s e 1 , 6 - d i p h o s p h a t e

2- Ke t o 3-deoxy p h o s p h o g l u c o n a t e

Di h y d r o x y a c e t o n e ph o s p h a t e

G l y c e r a l d e h y d e 3 - p h o sp h at e

Pyruvate

TCAc y c le

Figure 6 . A com parison of the overall routes of incorporation of fructose an d glucose in alginate produced by Ps. aeruginosa (47).

25

2. Enzymology

Only fo u r of the seven p ro p o sed enzym atic steps in th e

biosynthetic pathw ay of alginate from fructose 6 -phosphate to the

m atu re polym er have been positively iden tified in Pseudomonas

aeruginosa (98). Six of th e steps h av e b een id e n tif ie d in

Azotobacter vinlandii (8 6 ). The first four enzym es in the pathw ay

have been found in bo th organism s an d have been the subject of

m uch investigation . In P. aeruginosa the genes encoding these

enzym es a re lo ca ted a t ap p ro x im a te ly 34 m in u te s o n th e

chrom osom e linkage m ap (89, Fig. 7). W hat is know n abou t these

enzymes is sum m arized below.

P h o sp h o m a n n o se iso m era se : This enzym e catalyses the

reversib le conversion of fructose 6 -p h o sp h a te to m annose 6 -

phosphate . The enzym e is the p ro d u c t of th e algA gene in P.

aeruginosa, and has a molecular weight of approxim ately 56,000 on

th e b a s is o f so d iu m d o d ecy l su lfa te -p o ly a c ry la m id e gel

electrophoresis (SDS-PAGE, 82). O verexpression of the algA gene

p ro d u ces in c reased activ ity of the n ex t two enzym es of the

p a th w a y , i.e., p h o s p h o m a n n o m u ta s e a n d GDP m a n n o se

p y ro p h o sp h o ry la se (48 , 114). In th e o v erexp ressed state,

p h o s p h o m a n n o s e i s o m e r a s e w as s e p a r a t e d from

phosphom annom utase b u t could n o t be se p a ra te d fro m GDP

m annose pyrophosphorylase (114). The inability to separate these

two activities has led to the proposal that, in P. aeruginosa, the algA

gene encodes a single p ro te in having phosphom annose isom erase

and GDP m annose pyrophosphorylase activities.

26

Switching G e n e s " v

7570' algRI srgH

ModulatorG e n e s

a r g B

Posit iveEffectors

algAr~Z------------- \

StructuralG e n e s

Figure 7. The relative location of the alginate (alg) genes on the chromosom e linkage m ap of Pseudomonas aeruginosa. (89).

27

P h o sp h o m a n n o m u ta se : This enzym e converts m annose 6 -

phosphate to m annose 1-phosphate. The enzyme is the p roduct of

th e algC gene, and has a m olecular w eight of 38 ,000 . It was

d e tec ted in the soluble cytoplasm ic fraction of b o th m ucoid an d

nonm ucoid strains of P. aeruginosa, from clinical an d nonclinical

sources (92). Phosphom annom utase has an absolute requ irem en t

for glucose 1,6-diphosphate for its activity (114, 151). Induction of

the algA gene increases phosphom annom utase activity by abou t 10

fold.

GDP m an n o se p y ro p h o sp h o ry la se : This enzym e catalyses

the form ation of GDP m annose from m annose 1-phosphate and GTP.

This enzym e was detected in P. aeruginosa by Piggott e t al (98). It

was suggested th a t the activity of this enzym e m ay rep resen t one

of the two enzym atic activ ities o f the algA gene p roduct. This

enzym e m ay be an example of a bifunctional p ro te in w here the two

activities catalyze noncontiguous steps in a biosynthetic pathway.

GDP m an n o se d e h y d ro g e n a se : This enzym e catalyses the

oxidation of GDP m annose to GDP m annuronic acid (98, 105). The

p roduct of the algD gene, GDP m annose dehydrogenase has been

p u rified an d found to be a hexam er w ith a m olecular w eight of

290,000 (9). Its absence in alginate negative m utants suggests tha t

it is essential for alginate biosynthesis. The algD gene has been

cloned an d sequenced. It is transcrip tionally activated in m ucoid,

b u t n o t in nonm ucoid strains of P. aeruginosa (26, 27). The gene

contains regions th a t have considerable sequence hom ology with

two ou te r m em brane p ro te in genes (ompF and ompC) from E. coli

28

(9). Like ompF and ompC, expression of the algD gene is regulated

by external osm olarity (9).

The final reactions in the biosynthesis of bacterial alginate in

P. aeruginosa are n o t ye t fully understood . From the structu ra l

com position of alginate, it has been p roposed th a t the final steps

include polym erization of the m annuronate residues, O-acetylation

of some of those residues, export of the polym er, and epim erization

of th e carboxyl g roup a t C-5. The assu m p tio n is th a t the

polym erase uses GDP m annuronic acid as its substrate and tha t the

product of this reaction is polym annuronate. The evidence for this

is based on the observation th a t in Azotobacter vinlandii and the

brow n seaw eeds th e re is an ep im erase th a t converts ce rta in

m annuronate residues to gu lu ronate a t the po lym er level. The

polym erase in P. aeruginosa has p roved exceptionally difficult to

m easure and the enzyme has no t been purified. Very low levels of

activity of this enzyme have been m easured in m em brane fractions

p rep ared from cell extracts of m ucoid P. aeruginosa (47), b u t the

characterization of this polym erase is still a t a prelim inary stage.

T he m e c h a n ism b y w h ich g u lu ro n a te re s id u e s a re

incorporated into Pseudomonas alg inate rem ains a m ystery . The

assum ption has been th a t a polym annuronic acid C-5 epim erase

acts a t the polym er level to convert some m annuronate residues to

gu lu ronate residues. The epim erase from A. vinlandii has been

purified to hom ogeneity (126), an d recen tly Piggott e t al (98)

iso la ted the gene encod ing a C-5 ep im erase (algG) fro m P.

aeruginosa. The enzym e requ ired Ca44- for activity, m uch like the

enzyme isolated from A. vinlandii. AlgG m utants were found to be

29

incapable of incorporating guluronic acid residues in to bacterial

alginate.

The epim erase of P. aeruginosa appears to be d ifferen t from

th e enzym e iso la ted from A. vinlandii. The M /G ra tio of P.

aeruginosa alginate is never less than 1 .0 , and this ratio is generally

unaltered by changes in growth conditions. In A. vinlandii the M/G

ratio m ay be less than 1 .0 and the resulting block structu re m ay be

a ltered by changes in CaH+ concentrations in the grow th m edium

(70).

The alginates of P. aeruginosa are h ighly O -acetylated with

th e O -acety l g ro u p s being asso c ia ted exclusively w ith th e

m an n u ro n a te residues. It is though t th a t one of the functions of

acetylation is to p ro tec t the m annuronate residues in the polym er

from ep im erization to gu lu ronate residues (126). Recently, an

alginate m odification gene, algF, was sequenced. This gene codes

for a 28 kd p ro tein which controls the addition of O-acetyl groups to

the m annuronic acid residues. The algF gene was rep o rted to be

n o n essen tia l fo r a lg in a te b io sy n th esis , b u t is re q u ire d fo r

acetylation of the alginate polym er (44).

A m ajor difference betw een the alginates of A.vinlandii, and

P. aeruginosa is the arrangem ent of m annuronate and guluronate

residues w ithin each polym er. The polym er from A. vinlandii is

a r ra n g e d in to h o m o p o ly m eric b locks o f m a n n u ro n a te an d

guluronate, w hereas in P. aeruginosa alginate polyguluronate blocks

are absent. This difference reflects differences in O-acetylation and

ep im eriza tio n ac tiv ities betw een these two b ac te ria . T hese

30

differences m ay indicate alterations in the la tte r stages of alginate

biosynthesis in P. aeruginosa from those proposed in Figure 5.

3. Regulation

In the lungs of cystic fibrosis patien ts Alg (-) stra ins of P.

aeruginosa usually convert to the m ucoid Alg (+) form. In vitro, the

Alg (+) phenotype is unstab le an d Alg (-) revertan ts ap p ear over

time. Genetic m apping experim ents have shown th a t spontaneous

alginate conversion is based on the gene products of the algB, algR,

algS, an d algT genes (42, 45, 79, 90). The p rim ary genetic event

th a t regulates the " o n /o f f switch in alginate production is handled

prim arily by the algS an d algT genes located a t approxim ately 6 8

m inutes on the chrom osom al linkage m ap of P. aeruginosa (89, Fig.

7).

Spontaneous conversion between the m ucoid and nonm ucoid

state is the result of a genetic alteration a t the algS gene locus. The

algS gene is a genetic switch th a t controls the expression of algT

(42). Form ation of the AlgS p ro tein results in the activation of the

algT gene. The gene p roduct of algT acts as a regulatory p ro te in in

the biosynthesis of bacterial alginate in P. aeruginosa by prom oting

the activation of the structu ra l genes involved in the biosynthetic

pathw ay (42, 43).

Along with its ro le in regu la tion of the stru c tu ra l genes in

alginate production, the algT gene p roduct also is involved in the

expression of the algB gene (149). The algB gene is located at

approxim ately 12 m inutes on the chrom osom e m ap (Fig. 7). Its

gene product is no t directly involved in the biosynthetic pathw ay of

31

alginate b u t is ap p a ren tly involved in high level p roduction of

alginate in P. aeruginosa (49). The algB gene p roduct belongs to a

class of p ro te in s th a t contro l gene tran scrip tio n in response to

environm ental stimuli (149).

The la s t gene in v o lv ed in th e re g u la tio n o f alginate

b io sy n th esis is th e algR gene. This reg u la to ry gene controls

transcrip tion o f th e algD gene, which encodes fo r GDP m annose

dehydrogenase, an essential enzyme in the biosynthesis of bacterial

alg inate (98). DNA sequence analysis showed a h igh degree of

hom ology betw een th e algR gene a n d o th e r environm entally

responsive bacterial regulatory genes, including ompR, phoB, ntrC,

an d spoA (28). This ind icates th a t the p roduction of bacteria l

alg inate is affected by environm ental stimuli o r specific chem ical

com pounds presen t in the environm ent.

Recently, the m echanism of alginate p roduction contro l by

algB, algR, an d algT has been exam ined. Com pared w ith Alg (+)

strains, deletion m utations in the algB an d algT genes show ed

highly reduced transcrip tional activ ity in the b iosyn thetic gene

c luster (at 34 m inutes, 49). This ind icated th a t the pathw ay of

alg inate biosynthesis is u n d er contro l no t only by the algR gene

p roduct b u t also by the gene products of algB and algT. W hether

the algT an d algB gene products act directly on any prom oters in

the b iosynthetic gene cluster or on o ther regu la to ry genes is no t

known.

32

IV. Pseudomonas syringae Alginates

Numerous pathovars of P. syringae were rep o rted to produce

bacterial alginates (37). El Banoby and Rudolph (34) repo rted tha t

the EPS from several p lan t pathogenic pseudom onads were capable

of inducing w ater soaked lesions on com patible leaf tissue. This

suggested tha t alginate m ay be necessary for successful colonization

of p la n t h o st tissue. In com m on w ith o th e r bacteria l alg inates,

those produced by P. syringae are highly acetylated. The M/G ratio

is variable am ong strains and am ong d ifferen t p repara tions from

the sam e strain . Fett e t al (38) rep o rted th a t the percen tage of

guluronic acid in P. syringae alginates varied from < 1% to 28%

w hen grown in planta, and th a t the in planta samples h ad a higher

degree of acetylation th an the alginates p roduced in vitro. It was

also rep o rted th a t the P. syringae alginates w ere sm aller, on the

average, th an those from P. aeruginosa (37). The num ber average

m olecu lar weights range from 3.8 x 10^ fo r a lg inates from P.

syringae pv glycinea produced in vitro, to 47.1 x 1 0 ^ for P. syringae

pv papulans alginate produced in vitro, com pared to P. aeruginosa

alginates whose size was repo rted ly in the range of 1 0 ^ (37). It

appears th a t alginate biosynthesis is a com m on p ro p e rty of the

m ajority of pseudom onads in rRNA-DNA hom ology group 1 (29, 40,

94).

V. Polysaccharide Analysis

1. Depolymerization

C h a ra c te riza tio n of an y p o ly sacch a rid e s ta r ts w ith a

com positional analysis. Acid hydrolysis is com mon to m ost m ethods

33

of d e te rm in in g the physical com position of a polysaccharide.

Hydrolysis is usually conducted with dilute m ineral acids, the m ost

com mon being sulfuric acid, hydrochloric acid, or trifluoroacetic acid

a t 100°C for varying lengths of time. Many factors influence the

ra te of h y d ro ly sis o f any po lysaccharide, includ ing ring size,

configuration, conform ation, and polarity of the com ponent sugars

(8).

In studying the com position of alginic acid, m ost researchers

reduce the uronic acids to a n eu tra l po lym er to facilita te acid

h y d ro ly s is w ith o u t th e rm a l d e s tru c tio n o f th e co m p o n e n t

m onosaccharides (13, 37, 38, 91, 150). Acid hydrolysis of neu tra l

polysaccharides has been studied, and the m echanism now accepted

was first suggested by Edward (33) and is depicted in Figure 8 for

the hydrolysis of m ethyl p-D glucopyranoside. The process involves

p ro to n a tio n of the glycosidic oxygen atom to form the conjugate

acid, followed by the form ation of a cyclic carbonium -oxonium ion

which probably exists in the half chair conform ation having C-2, C-

1, O, an d C-5 in a p lane. Reaction with w ater th en gives the

p ro tonated reducing sugar and from it the reducing sugar is form ed.

Alginic acid, being a polyuronide, contains a carboxyl group at

the C-5 position of each com ponent m onosaccharide. This carboxyl

group confers acid resistance to the glycosyluronic acid linkages

(15). M any theories have been advanced as to why. T here is

evidence th a t the enhanced stability m ay be a ttrib u ted to e ith e r

steric factors or to inductive effects (145). Ranby and M archessault

(107, 108) fo rm u la ted an induction-stab ilization theory , w hich

p roposed th a t the glycosidic bond is stabilized by the inductive

Figure 8 . The m echanism of acid hydrolysis of glycosides. The carbonium ion in term ediate (3) is in the half-chair conform ation (8 ).

OJ

35

effect of the po lar carboxyl group. According to this theory, the

presence of an electronegative group, such as a carbonyl or carboxyl

group, can exert inductive influences on the glycosidic oxygen atom.

In a polyuronide, the carboxyl group would oppose the pro tonation

of the glycosidic oxygen atom s by making the electron pairs on the

glycosidic oxygen atoms less basic, resulting in a m ore stable bond.

A possib le fo rm atio n o f six m em bered rings, w here hydrogen

stabilizes the negatively charged carboxyl group and the glycosidic

oxygen atom has also been postu lated (107). A lthough the exact

explanation for the stability of glycosyluronic acid bonds has no t

been pinpointed, suffice it to say tha t these bonds are m ore difficult

to hydrolyze than the neutra l O-glycosidic bonds.

M any fa c to rs in f lu e n c e th e r a te o f h y d ro ly s is of

polysaccharides. The ease of hydrolysis a t a particu lar tem perature

an d acid concentration increases in the o rd e r g lucopyranoside <

fructopyranoside < fructofuranoside (63). This indicates th a t ring

size affects hydrolysis. T here is a d irec t rela tionsh ip betw een the

strain (or free energy) associated with a m olecule and the ra te of

hydrolysis (121). In general, aldofuranosides and aldoheptanosides

a r e h y d ro ly z e d m o re r a p id ly th a n th e co rre sp o n d in g

aldopyranosides. The five and seven m em bered rings are strained

because of the distortion of the te trahedral angle of the ring carbon

atoms. The pyranoid rings can pucker to elim inate strain (35).

The anom eric configuration in pyranosides also plays a role in

the stab ilities of these sugars u n d er acid hydrolysis conditions.

F e a th e r a n d H arris (36) u sed a n o m eric p a irs o f m e th y l

aldopyranosides to study the affects of the anom eric configuration

36

on acid stability. They found th a t an anom er w ith an equatorial

m ethoxy l g roup h y d ro ly zes m ore ra p id ly th a n an an o m e r

containing an axially oriented group. Two explanations have been

p roposed . First, eq u a to ria l bonds are considered to be m ore

accessible th an axial bonds, thus making them m ore available for

p ro to n tran sfe r from a hydron ium ion to the glycosidic oxygen

atom . A lternatively , th e g rea te r reac tiv ity of th e eq u a to ria l

su b stitu en t is due to its h igher free energy caused by a p o la r

in te rac tion betw een the equatorial m ethoxyl group an d the ring

oxygen atom (33).

2. Acid Sensitivity

M onosaccharides are degraded by acid to a g rea ter o r lesser

extent depending on the sugar an d strength of the acid (119). In

add ition to degradation , acids m ay convert sugars in to anh y d ro

derivatives. Spontaneous conversion to a 1,6 anhydroaldopyranose

occurs in acidic solutions of several aldoses an d ketoses having the

ido (100, 102, 146), a ltro (101, 112), an d gulo (131, 132)

configurations. In dilute acid solutions those sugars of the gluco,

m a n n o , a n d g a lac to co n fig u ra tio n s a re a lm o st completely

hydro lyzed to the free aldoses. Reeves (111) first suggested an

explanation for this d ifferen t behavior in term s of conformational

in teractions. He showed th a t (3-D-idose, with all hydroxyl groups

equatorial, has a h igher p ropensity to form 1,6 anhydrides than

does p-D-glucose whose hydroxyl groups are all o rien ted axially.

This system represents a good example of conform ational control of

37

an equilibrium. The position of the equilibrium is dependen t on the

steric arrangem ent of the groups no t taking part in the reaction.

The solutions of any reducing sugar contain an equilibrium

m ixture of the IC4 and 4 c i forms of the a and (3 anom er s. Only the

IC4 form of the (3 anom er can directly form the anhydride w ithout

change in configuration and conformation. 1,6 A nhydride form ation

is believed to occur in two steps: 1 ) conversion of o ther form s of the

sugar into the IC4 form of the |3-D anom er, and 2) form ation of the

anhydride . P ra tt an d R ichtm yer (104) show ed th a t an axial

hydroxyl group at C-3 is the m ost im portan t axial constituen t in

determ ination of 1,6 anhydride form ation. An axial hydroxyl group

a t C-3 can in te rac t with an anhydride bridge an d destabilize the

anhydride. The four hexoses having axial hydroxyl groups at C-3

(D-glucose, D -m annose, D -galactose, an d D -talose) fo rm less

anhydride than those hexoses whose C-3 hydroxyl group is orien ted

equatorially (D-allose, D-altrose, D-gulose, and D-idose). The overall

ability for 1,6 anhydride form ation then is:

ido > altro, gulo > talo > alio > galacto > m anno > gluco

Some debate still exists abou t the placem ent of talose com pared to

allose in the above scheme. Some researchers claim talose is more

p rone to 1,6 anhydride form ation. O thers claim allose form s 1,6

anhydrides m ore readily (103, 141).

38

VI. Goals of This Study

This s tu d y was designed to op tim ize th e cond itions fo r

p roduction of bacteria l alginate from Pseudom onas syringae pv

phaseolicola, ATCC 19304 to determ ine the feasibility of large scale

p roduction , an d to characte rize th e physical p ro p ertie s of th e

p roduct, rela ting those p ro p erties to the resu lting so lu tion an d

gelling properties of the polymer.

MATERIALS AND METHODS

I. Organisms, Growth Conditions, and M aintenance

Pseudom onas syringae subsp. phaseolicola ATCC 19304 was

obtained from the American Type Culture Collection, Rockville, MD.

It was selected fo r th is research because of its lack of hum an

pathogenicity an d its po ten tia l for producing a highly acety lated

bacterial alginate (39).

Cultures were m ain ta ined at 4°C on Dworkin Foster (DF) agar

(32) supp lem en ted w ith 2% (w/v) gluconic acid. This m edium

co n ta in ed (in gram s p e r lite r of deionized w ater): KH2PO4 , 4.0;

Na2 HTC>4 , 6.0; NaCl, 0.4; KNO3 , 9.1; (NH4 )2 S0 4 , 0.9; MgS0 4 • 7 H2 O,

0.2; gluconic acid , 20; an d agar, 15. The gluconic ac id was

aseptically added to the salts m edium after separate sterilization in

an autoclave a t 121°C, a t 15 lb s . / in ^ of p ressu re for 15 m inutes.

The pH of the agar was betw een 6.9 an d 7.0 p rio r to sterilization.

Plates w ere inocu lated w ith 0.1 ml of a standard ized 48 h o u r DF

b ro th cu ltu re of P. syringae. This culture showed an absorbance

between 1.9 and 2.0 a t 660 nanom eters. The inoculum was spread

using a flame sterilized, ben t glass rod. After growth a t 30°C for 48

hours, the p lates w ere sto red a t 4°C. Cultures w ere transferred

every fou rth week.

All b ro th sta rter cultures used in this work were p rep a red by

inoculating P. syringae ATCC 19304 from agar plates in to 100 ml of

DF b ro th in 250 ml Erlenm eyer flasks. Cultures were incubated at

30°C for 48 hours a t 180 rpm on a NBS Model G25-KC ro tary shaker

(New Brunswick Scientific Co. Inc., Edison, NJ). C ultures w ere

39

40

s ta n d a rd iz e d w ith s te rile w a te r to a n a b s o rb a n c e a t 660

n an o m ete rs betw een 1.9 a n d 2.0 on a G ilford R esponse II

spectrophotom eter (Gilford Instrum ent Lab., Oberlin, OH) p rio r to

use.

II. Analytical M ethods

1. Cell Mass D eterm ination

Dry cell weight for calculation of specific yield of alginate or

p ercen t acety lation was m easured directly . Broth cu ltu res w ere

cen trifuged at 17,000 x g for 15 m inutes in a Sorval Superspeed

M odel RC-5B centrifuge (Du Pont Co., W ilmington, DE) to rem ove

bacterial cells. Cells from agar m edia were resuspended in 100 ml

of deionized water. The suspension was cen trifuged a t 27,000 x g

for 60 m inutes to pellet the cells from the highly viscous solution.

Once the cells were separated, the pellets were trea ted equally. The

pelle ts w ere w ashed twice in 10 m l o f d e ion ized w ater. The

su p ern a tan t was d iscarded and the pellet was resu sp en d ed in an

equal volum e of deionized water. The cell suspension was p ou red

into a pre-dried, tared , alum inum weighing dish and was d ried in a

drying oven a t 100°C to a constant weight.

2. Total Carbohydrate Quantitation

The to ta l ca rb o h y d ra te p re se n t in P. syringae EPS was

determ ined by the phenol-sulfuric acid assay (31). The protocol

was as follows:

41

1) To 2 ml of carbohydrate solution containing 10 to 100 |.ig of

carbohydrate, 0.05 ml of 80% (v/v) aqueous phenol was

added.

2) The solution was shaken in a Vortex m ixer for a count of 5.

Then, 5 ml of concentrated sulfuric acid was added and the

solution mixed again for another count of 5.

3) The tubes were incubated at room tem perature for 30

m inutes to allow the color to develop. The absorbance was

m easured at 485 nanom eters on a Gilford Response II

spectrophotom eter (Gilford Instrum ent Lab., Oberlin, OH).

This value was com pared to a standard curve of sodium

alginate from Macrocystis pyrifera (Sigma Chemical Co., St

Louis, MO) for determ ination of the total carbohydrate

p resen t in the solution.

3. Alginate Q uantitation

Alginate concen tra tions were d e te rm in ed by two d iffe ren t

m ethods. These m ethods included the uronic acid assay described

by Blum enkrantz and Asboe-Hansen (10), and the carbazole assay

of K nutson an d Jeanes (69). Each m ethod has its benefits. The

uronic acid assay is rap id , bu t is only accurate up to 150 itig/ml of

uronic acid. The carbazole assay takes m uch longer to perform , bu t

it is accurate up to 1 0 0 0 ng/ml.

The uronic acid assay was used predom inately w ith alginates

produced in b ro th cultures. This assay allowed d irect testing of the

superna tan t w ithout color in terference from the rem aining salts in

solution. The protocol was as follows:

42

1) To 0.2 ml of sample containing from 0.5 to 30 f.ig uronic

acid, 1.2 ml of 12.5 mM tetraborate in concentrated sulfuric

acid was added.

2) The tubes were chilled in an ice bath for 10 minutes.

3) The m ixture was shaken in a Vortex mixer, and the tubes

heated in a w ater ba th at 100°C for 5 minutes.

4) After cooling in a water-ice bath, 20 |xl of 0.15% (w/v)

m eta-hydroxydiphenyl in 0.5% (w/v) sodium hydroxide was

added to the above mixture.

5) The tubes were shaken for a count of 5 and the absorbance

read a t 520 nm as described previously. This value was

com pared to a standard curve of sodium alginate from

Macrocystis pyrifera to obtain the concentration of uronic

acid.

The carbazole assay was used fo r h igh concentrations of

purified alginate. Experim ental e rro r dim inished w ith th is assay

because of few er d ilu tion steps. The protocol fo r the carbazole

assay was as follows:

1) 0.5 ml of a purified alginate solution containing 0 to 500 jxg

of alginate was equilibrated in a water-ice bath for 10

minutes.

2) Three ml of cold concentrated sulfuric acid was added to

this solution. The m ixture was re-equilibrated in a water-ice

bath.

3) The solution was then mixed with a Vortex mixer for a

count of 4 and heated at 55°C for 20 m inutes in a w ater bath.

43

4) The solution was then re-equilibrated in a water-ice bath

for 10 m inutes, an d 0 .2 ml of a 0 .2% (w/v) carbazole solution

(Eastman Kodak, Rochester NY) in ethanol was added.

5) The sample was mixed with a Vortex m ixer for a count of

10 and allowed to incubate at room tem perature for 3 hours

for color development.

6 ) The sample absorbance was read at 530 nm as described

previously. This value was com pared to a standard curve of

sodium alginate from Macrocystis pyrifera to determ ine

bacterial alginate concentrations.

4. Acetyl Q uantitation

The p e rc e n t ace ty la tio n was d e te rm in ed by th e m e th o d

described by McComb an d McCready (80). A s ta n d a rd curve for

percen t acetylation was p rep ared with glucose pen taaceta te (Sigma

Chemical Co., St. Louis, MO). P rior to assay, all sam ples w ere

desalted by dialysis for 48 hours against deionized w ater a t room

tem perature. The protocol was as follows:

1) One volum e of 9.4% (w/v) sodium hydroxide was added to

one volume of 3.75% (w/v) hydroxylam ine solution.

2) To 2 ml of the above mixture, 0.5 ml of the sample solution

was added with agitation.

3) After 5 m inutes, 0.5 ml of acid m ethanol was added with

agitation, then 1.3 ml of the ferric perchlorate solution was

added.

44

4) After 5 m inutes, the precipitated hydroxam ic acid and

ferric complex was rem oved by m icrocentrifugation for 3

m inutes in a Sorval Microspin 24s M icrocentrifuge (Du Pont

Co., Wilmington, DE).

5) Color intensity was determ ined by m easuring absorbance

a t 520 nm as described above. This value was com pared to

the standard curve of glucose pentaacetate to determ ine the

percent acetylation.

The reagents for acetyl quantitation were p repared as follows:

1) Acid M ethanol Solution

Chilled reagent grade absolute m ethanol was added to 35.2 ml

o f chilled 70% perchloric acid to m ake a 500 ml solution. This

solution was used as the acidic m ethanol solution.

2) Ferric Perchlorate Solution

Ferric ch lo rid e (1.93 g) was d isso lved in 5 m l o f 70%

perchloric acid and evaporated alm ost to d ryness. It was th en

d ilu ted to 100 ml with w ater for use as the stock ferric perchlorate.

Then 8.3 ml of 70% perchloric acid was ad d ed to 60 ml of stock

ferric perch lorate solution. This solution was cooled in an ice bath

an d m ade to 500 ml with chilled reagent grade absolute m ethanol.

3) Glucose Pentaacetate Standard Solution

Pure crysta lline p-D-glucose p en taace ta te (108.9 mg) was

dissolved by heating w ith gentle agitation in abou t 5 ml of ethy l

alcohol, and m ade to 50 ml with deionized w ater. 2, 4, 5, and 7 ml

of this solution were then taken and m ade to 50 ml with deionized

w ater. These solutions rep resen t 120, 240, 300, an d 420 |ng/ml of

acetyl.

45

III. Optimization of Bacterial Alginate Production

1. Media Composition:

A. Carbon Source

P. syringae ATCC 19304 was tested for its ability to p roduce

an acety la ted bacteria l alg inate w hen grown on d ifferen t carbon

sources. The carbon sources tested were fructose, glucose, sucrose,

glycerol, and gluconic acid. Each separate solution of test carbon

source was autoclaved and then aseptically added to the DF bro th .

The concen tra tion of each stock solution was 20% (w /v o r v /v ).

Each carbon source was tested a t a final concentration of 2% (w/v or

v /v ). After inoculation of DF b ro th from sta rter cultures, (3%, v /v ),

P. syringae was incubated for 48 hours w ith shaking as described

previously . At 48 hours, the cu ltu re b ro th was cen trifuged to

rem ove bacterial cells, an d the cell mass determ ined as described

previously. The cell free b ro th was analyzed by the phenol-sulfuric

assay (to ta l c a rb o h y d ra te p ro d u c tio n ), th e u ro n ic ac id assay

(alginate production), and the acetyl assay (degree of acetylation).

B. Nitrogen Source

P. syringae was tested fo r its ab ility to p roduce acetylated

bac teria l a lg inate on d iffe ren t n itrogen sources. The n itro g en

sources tested were am m onia, [(NH4 )2 9 0 4 ], n itrate, (KNO3 ), n itrite,

(KNO2 ), and urea. Each DF b ro th was m ade by incorporating only

the test com pound as a potential n itrogen source for the organism .

Each n itro g en source was p laced in to th e m ed iu m a t in itia l

concentrations of 2 mM, 5 mM, 9 mM, 12 mM, an d 15 mM (w /v).

Gluconic acid was used as the carbon source a t a final concentration

46

of 2% (w/v). After inoculation, (3%, v /v), from standardized sta rter

cu ltures, P.syringae was incubated for 48 hou rs w ith shaking as

described previously. At the ap p ro p ria te tim e the cu ltu re b ro th

was cen trifu g ed to rem ove b ac te ria l cells, an d th e cell m ass

d eterm ined as described previously. The b ro th was analyzed by

the uronic acid assay for alginate production.

2. pH and Tem perature

The effect of pH on bacterial cell yield and alginate production

was investigated in DF broth . The initial pH's were 6.0, 6.2, 6.4, 6 .6 ,

6 .8 , 7.0, and 7.2. Cell yield and alginate production were m easured,

as described previously, after incubation a t 30°C fo r 48 h o u rs a t

180 rp m in a NBS M odel G25-KC ro ta ry shaker (New Brunswick

Scientific Co. Inc., Edison, NJ). In each case, phosphate buffer (0.03

M) was used to m aintain the pH of the culture broth. Gluconic acid,

a t a final concentration o f 2% (w/v), was the carbon source.

The effect of tem peratu re on bacterial cell yield an d alginate

p roduction was investigated in DF broth . The tem pera tu res tested

w ere 25°C, 28°C, 29°C, 30°C, 31°C, and 32°C. Both cell y ield and

alginate production were m easured, as described previously. Each

culture was grown under the same conditions as the pH cultures.

3. Agar vs. Broth Culture

Cell y ie lds an d a lg ina te p ro d u c tio n w ere m easu red in 5

d ifferent media, both on agar and in b ro th culture. All m edia were

m ade up in deion ized w ater. The m ed ia inc luded DF m edia,

n u tr ien t m edia (Difco Lab. Detroit, MI), m edia com posed of 3 g/L

47

beef extract (Difco Lab. Detroit, MI), m edia containing 5 g/L peptone

(Difco Lab. Detroit, MI), and m edia containing a m ixture of 3 g/L

beef extract and 5 g/L peptone. All m edia were supplem ented with

2% gluconic acid (w/v). The gluconic acid was sterilized separately

by autoclaving as a stock solution of 2 0 % (w /v), an d added to the

cu ltu re m edia asep tically a fte r sterilization . Agar m ed ia w ere

p repared with 15 g/L agar. The pH of each test m edia was betw een

6.9 an d 7.0 p r io r to s te riliza tion . All in o cu la w ere fro m a

standard ized 48 h o u r s ta rte r cu ltu re of P. syringae grow n in DF

bro th a t 30°C with shaking as described previously. Liquid cultures

were inoculated from the standardized 48 h o u r s ta rter cu lture to a

fined concentration of 2% (v/v). Solid cultures were inoculated with

0.1 ml of the same standard ized s ta rte r cu ltu re p e r 100 x 15 m m

petri dish containing 25 ml of m edium . The inoculum was spread

using a flam e sterilized, b en t glass rod . All test cu ltu res w ere

allowed to grow for 48 hou rs a t 30°C. The liquid cultures w ere

incubated with shaking as described previously. At the appropriate

tim e, the cell yield and alginate p roduction of each cu lture were

m e asu red as d esc rib ed p rev iously . A lginate p ro d u c tio n was

m easured by the uronic acid assay. Total alginate production on

solid m edia was m easured by scraping the bacteria l growth from

half of a pe tri dish (allowing 2 m easurem ents p e r p e tri dish) and

resuspending th a t growth in 10 ml of deionized w ater. The cells

were then rem oved by centrifugation and the cell mass determ ined,

as described previously. Alginate p roduction was assayed by the

uronic acid assay. The to ta l a lg inate p ro d u c tio n fo r the solid

cultures was reported in (.ig/cm2.

48

IV. Batch Ferm entations

Batch ferm entations were conducted in 1 liter volum es in a

NBS 2.5 lite r Bioflo II b a tch /co n tin u o u s cu ltu re fe rm en te r (New

Brunswick Scientific Inc., Edison, NJ) in DF b ro th supplem ented with

2% (w/v) gluconic acid. T em perature was m aintained a t 30°C, and

the pH was m a in ta in ed a t 7.0 by titra tio n w ith 3 M sod ium

hydroxide. Air, filtered through a sterile W hatm an H epavent filter

(W hatman Inc., Clifton NJ) was supplied through a sparger a t a ra te

o f 1 s tan d ard liter p e r m inute (SLPM), an d ag itation was a t 100

rpm . Samples were periodically w ithdraw n aseptically, th roughout

the bacterial growth cycle. Growth was m easured by absorbance at

660 nanom eters as described previously. Alginate was m easured in

the cell free supernatan t by the uronic acid assay.

V. Purification of Bacterial Alginate

The bacterial alginates used for characterization studies w ere

obtained from either DF agar or N utrient agar plates supplem ented

with 2% (w/v) gluconic acid. Bacterial growth was scraped off the

agar plates using a ben t glass rod an d resuspended in 150 ml of

d e io n ized w ater. The sam ples w ere v o rtex ed u n til evenly

suspended and then the bacterial alginate was separa ted from the

cells by centrifugation as described previously. Three volum es of

isopropanol were added to one volum e of clarified su perna tan t to

p recip ita te the polysaccharide. This solution was m ixed fo r 15

m inutes an d the p rec ip ita ted alginate was rem oved by w inding

a ro u n d a glass rod . The p rec ip ita te was th en d ried in acetone

followed by a ir drying.

49

Purity was determ ined by the carbazole assay using sodium

alg in a te from M acrocystis pyrifera a s th e s ta n d a rd , an d by

w avelength scans betw een 200 nm and 300 nm in increm ents of

0.5 n m on a G ilford R esponse II sp ec tro p h o to m e te r (Gilford

Instrum ent Lab., Oberlin, OH). Absence of detectable peaks a t 260

nm an d 280 nm indicated an absence of nucleic acid an d p ro te in

respectively. A large peak a t 230 nm ind icated the presence of

large am ounts of carbohydrate. This carbohydrate was determ ined

to be alginate by the carbazole assay. The purity of the bacterial

alginate produced from P. syringae ATCC 19304 was m ore than 98%

(w/v).

VI. Deacetylation of Bacterial Alginate

The alginates from P. syringae ATCC 19304 were deacety lated

fo r com parison w ith seaw eed alg inate an d acety lated bacteria l

a lg inate . The com parisons w ere to d e te rm in e th e effects of

acetylation on the solution and gelling p roperties of the polym er.

Purified, acety lated bacteria l alginate was dissolved in deionized

w ater a t a concentration of 1 m g/m l. Three volumes of this solution

w ere m ixed w ith one volum e of 1 N sodium hydrox ide solution.

After incubation for 20 m inutes at room tem peratu re , w ith gentle

ag ita tion , one volum e o f 1 N hydroch lo ric acid was ad d e d to

n eu tra lize th e so lu tion (final pH was ab o u t 7.0) an d stop the

reac tio n . The d eace ty la ted bac teria l a lg inate was ex tensively

dialyzed against deionized water. The effectiveness of the process

was d e te rm in ed from co n cen tra tio n s of acety l g roups in the

preparation.

50

VII. Alginate Size and Q uantity Determ inations

M olecular w eights w ere d e te rm in ed by gel p e rm e a tio n

chrom atography (GPC) from alginate solutions [100 (ng/ml (w/v)] in

deion ized w ater. A lginate sizes and polydispersity indices were

d e te rm in e d by m e a su re m e n t of m u ltian g le lig h t scattering

in tensities using a DAWN-Photometer (W yatt Technology, Santa

Barbara, CA). The DAWN GPC detec to r m easures the scattering

intensities of a sample at 15 different angles and transm its the data

to a com puter for digital conversion and subsequen t processing

u n d e r con tro l of the ASTRA™ (or ASTRA 202) softw are (W yatt

Technology, Santa Barbara, CA). Acetone an d cyclohexane were

used for instrum ent calibration. Concentrations were obtained from

a W aters Model 410 D ifferential Refractom eter (M illipore Corp.,

Milford, MA). Alginate sizes were based on the GPC calculation and

the following external standards: T10, T40, and T500 (Pharm acia

Co., Uppsala, Sweden) dextrans. Sample injection volumes were 100

jil and the GPC column was an U ltrahydrogel Linear colum n (Waters,

Millipore Corp., Milford, MA). The running buffer was 0.1 M NaNC>3 ,

and the tem perature was 45 °G

VIII. Sugar Sensitivity to Acid

1. Thin Layer Chrom atography

Standards of D-mannose and L-gulose (Sigma Chemical Co., St.

Louis, MO) were p repared a t a concentration of 6 .6 6 m g/m l (w/v)

in deionized water. One m l of acid (1 N HC1 or 1 N H2SO4 ) and 1 ml

of s tan d ard sugar solution were m ixed and the solution hea ted a t

100°C for 0.5, 1, 2, 3, an d 4 hours. Each so lu tion was then

51

neutralized with 1 ml of 1 N NaOH giving a final sugar concentration

of 1.67 m g/m l (w/v) and a final pH of between 6.0 and 7.0.

T h in la y e r c h ro m a to g ra p h y was p e r fo rm e d o n th e

h y d ro ly sa tes on 0.25 m m p la tes co a ted w ith silica gel 150A

(W hatman Co., Maidstone, England), o r kieselgel 60 F254 (E. Merck

Co., D arm stadt, Germ any). The hydro lyzed sugar solutions w ere

spotted a t a concentration of 50 fig sugar/spot. The running solvent

was n -p ropanol an d w ater in a ra tio of 85:15 (v /v ). The p lates

were developed by spraying with 20% (v/v) H2SO4 in m ethanol and

charring a t 100°C for 15-20 m inutes.

2. Ion Chrom atography

Standards of D-mannose and L-gulose (Sigma Chemical Co., St.

Louis, MO) were p rep ared a t a concentration of 1 m g /m l (w/v) in

deionized w ater. One ml of sugar solution was mixed with 1 ml of

acid (1 N HC1 o r 1 N H2SO4 ). Each solution was hydrolyzed at 100°C

fo r 0.5, 1, 2, 3, and 4 hours. After hydrolysis each solution was

neutralized with 1 ml of 1 N NaOH giving a final sugar concentration

of 0.33 m g/m l (w/v) and a final pH between 6.0 and 7.0.

Q uantitative determ inations of the acid sensitivities of each

sugar w ere m ade with ion chrom atography. Ion chrom atography

was perfo rm ed on each hydrolyzed standard , using a Dionex ion

chrom atography system (Dionex Corp., Sunnyvale, CA) fitted with a

Carbopac PA1 colum n (4 x 250 mm). A 100 mM solution of NaOH

was used as e luen t an d pum ped a t 0.5 m l/m in u te by a g rad ien t

pum p. A 50 |il sample was injected and the signal was detected by

a pulse am perom etric detector. Integration was accom plished by a

52

Dionex 4400 in teg ra to r. The rela tive peak areas w ere used to

q u a n tita te th e p e rc e n t d eg rad a tio n o f each sugar u n d e r the

experim ental conditions employed.

IX. Identification of the Acid Hydrolysis Product of L-Gulose

1. Thin Layer Chrom atography

Thin layer chrom atography was used to help identify the acid

hydrolysis p ro d u c t of L-gulose. L-Gulose (Sigma Chemical Co., St.

Louis, MO) was acid hydrolyzed and separa ted on a TLC p la te as

described previously. At the end of a run , the TLC plate was air

dried . The silica gel in the region corresponding to the Rf value of

the spo t of in te re s t was scraped from th e p la te an d e lu ted in

deionized w ater for 10 m inutes. The solution was m icrocentrifuged

in a Sorval Microspin 24S microcentrifuge (Du Pont Co., Wilmington,

DE) for th ree m inutes to pellet the silica gel. The su p ern a tan t was

freeze d ried in a Flexi-Dry freeze d ry apparatus (FTS Systems, Stone

Ridge, NY).

The freeze d r ie d sam ple was resu sp en d ed in 0.2 ml of

deionized w ater and again spotted on a 0.25 m m TLC p la te coated

w ith kieselgel 60 F254 (E. Merck Co., D arm stadt, G erm any) as

described previously. The sample was ru n in n-propanol and w ater

in a ra tio of 85:15 (v /v ) w ith sam ples o f 1,6 an h y d ro (3-D-

m a n n o p y ra n o se , a n d 1,6 anhydro -p -D -g lucopyranose (Sigma

Chemical Co., St. Louis, MO) an d the m igration of the unknown

com pared to the known standards.

53

2. Stability of 1,6 Anhydro p-L-Gulopyranose

The stability of 1,6 anhydro |3-L-gulopyranose was m easured

by m olecu lar m odeling on SYBYL m olecular m odeling software

version 6.2 (Tripos Assoc. Inc., St. Louis, MO). The m olecule was

draw n using the above software and then m inim ized to its lowest

energy level a t pH 7.0 with a dielectric constant of 78.8 (equivalent

of water). Once the molecule was a t its lowest energy levels for the

above conditions, it was solvated in w ater by com puter and heated

to 400°K for a total of 100,000 fem toseconds (10"11 seconds). This

allowed the solvent to a tta in equilibrium . A distance depen d en t

dielectric constan t was used to avoid the conditions of a vacuum .

Energy m easurem ents were m ade a t 250 fem tosecond intervals.

X. Alginate Reductions

Alginates were chemically reduced p rio r to acid hydrolysis of

the polym ers. The m ethod used for the reduction of the uronic

acids in alginates to the corresponding neu tra l sugars was th a t of

Taylor e t al (140). An aqueous solution of alginate containing 100

m icroequivalents of carboxylic acid in 10 ml of deionized w ater was

ad justed to pH 4.75 with 0.1 M NaOH. One millimole of 1-ethyl-3-

(3-d im ethylam inopropyl)carbodiim ide was added to the alginate

solution to convert the uronides to esters. The pH of the reaction

m ixture was m aintained a t 4.75 by titra tion with 0.1 M HC1. The

reaction was allowed to continue until hydrogen ion uptake ceased

(45-60 m inutes). Then, 25 ml of a 3 M NaBH4 solution was added

dropwise over a 1 hour period to reduce the uronides to the m ore

readily hydrolyzable neu tra l polymers. The pH was m ain tained at

54

7.0 by titra tion with 4 M HC1. 1-Propanol was added dropwise, as

necessary to m inim ize foam ing. The reac tion m ixture was th en

m ade slightly acidic to destroy any rem aining sodium borohydride,

an d the solution was dialyzed exhaustively against deionized water.

Each reduced alginate sam ple was then con cen tra ted in a Buchi

Model R110 ro ta ry evaporator (Buchi Lab., Flawil, Switzerland) and

precip itated by addition of th ree volumes of isopropanol and dried

by washing in acetone.

XI. Alginate Compositions

Composition m easurem ents of the alginates from Macrocystis

pyrifera (Sigma Chemical Co., St. Louis, MO), and P. syringae ATCC

19304 were m ade by ion chrom atography of the acid hydrolyzed,

reduced polymers. Each alginate sam ple was p rep ared in the same

m an n er as the D-m annose, and L-gulose sugar stan d ard s for ion

chrom atography described previously. The reduced sam ples were

m ixed a t a concen tration of 1 m g/m l an d 1 m l of solution mixed

w ith 1 ml of acid (1 N HC1 or 1 N H2 9 0 4 ). The sam ples w ere th en

hydrolyzed at 100°Cfor 0.5, 1, 2, 3, and 4 hours. After hydrolysis,

5 0 [xl o f e a c h sam ple was in je c te d in to th e D ionex io n

chrom atography system described above. The resulting peak areas

w ere th en co rre la ted an d ex trap o la ted back to tim e zero to

determ ine the percen tage of m annose an d gulose p resen t in the

reduced polym er. This com position was then directly correlated to

the com position of both Macrocystis pyrifera and P. syringae ATCC

19304 alginates.

55

XII. Properties of Bacterial Alginates and Effects of Acetylation

1. Viscosity

The viscosities of Macrocystis pyrifera alginate and acetylated

an d deacetylated P. syringae ATCC 19304 alginate were m easured

as a fu n c tio n of te m p e ra tu re , co n cen tra tio n , a n d d eg ree of

acetylation. In each case, viscosities w ere d e te rm in ed by the

m eth o d of A llison a n d M atthew s (1) using a sim ple U -shaped

Ostwald capillary viscom eter designed for small volumes. The time

taken for a sam ple to fall a fixed distance und er gravity (N), divided

b y th e tim e taken by w ater to fall th a t same distance (No) was

expressed as a m easure of com parative viscosity (Visc.COm = N/N0).

The effect of tem peratu re on alginate solution viscosity was

m easured at alginate concentrations of 400 tig/m l (w /v) over a

te m p e ra tu re ran g e of 30°C to 85°C. The effects of a lg inate

concentration on viscosity was m easured a t 50°C a t concentrations

ranging from 50 fxg/ml (w/v) to 1000 f.ig/ml (w /v). The effect of

a c e ty la tio n on v iscosity was m e asu re d a t 50°C a t a lg in a te

concentrations of 50 ng/m l (w/v), 100 ng/m l (w /v), an d 200 ng/m l

(w/v) and the values averaged. Deacetylation of bacterial alginate

was as described previously with m inor variations. A 400 ng/m l

sam ple of h ighly acety lated bacterial alginate was deacety lated to

various degrees by varying the concentration of NaOH used in the

reac tio n an d by altering the reac tion tim e. Sodium hydrox ide

concentrations ranged from 0.25 M-1.0 M while reaction times w ere

betw een 5 m inutes and 20 m inutes. A cetylated seaweed alginate

was ob ta ined from Jin W. Lee (72) and p artia lly deace ty la ted as

described previously.

56

2. W ater Holding Capacity

W ater holding capacities of alginate gels w ere m easured by

determ ining the am ount of w ater lost from the gels upon drying.

A lginate gel beads w ere m ade from a 0.6% (w /v) so lu tion of

seaweed, acetylated or deacetylated bacterial alginate. Using a 5 ml

p ipet tip, 4 ml of the alginate solution was d rip p ed slowly in to 30

ml of CaCl2 , FeCl3 , o r PbCl2 solution. Each m etal solution was tested

at concentrations of 0.05 M, 0.1 M, 0.25 M, o r 0.5 M. The beads

w ere allowed to form in each m etal solution for 15 m inutes after

w hich th e y w ere w ashed th o ro u g h ly in d e io n ized w ate r (5

m inutes). A fter a ir d ry ing fo r 5 m inutes, 10 beads from each

sam ple were p laced in to p red ried , ta red alum inum weigh dishes

an d weighed. The weighing dishes were th en p laced in a drying

oven a t 100°C and the sam ple d ried un til a co n stan t w eight was

reached . The dishes were then rew eighed an d the w ater holding

capacity of each bead calculated as g w ater/ g d ry alginate gel.

3. Surface Tension

The relative surface tensions of beads were m easured using

bead d iam eter, the sm aller the bead d iam eter th e h ig h e r the

surface tension on the bead. Gel beads w ere m ade as described

prev iously in 0.5 M CaCl2 . U pon form ation , 1 /3 of each bead

sam ple was w ashed in deionized w ater for 5 m inutes an d allowed

to d ry fo r 5 m inutes p r io r to d iam eter m easurem ent w ith a dial

caliper (L. S tarre tte Co., Athol, MA). The second po rtion of each

sample was incubated a t 4°C for 24 hours in deionized w ater prior

to m easurem ent, and the final th ird of each sam ple was incubated

57

a t 4°C fo r 24 h o u rs in th e 0.5 M CaCl2 so lu tion p r io r to

m easurem ent. W ater holding capacity was d irectly re la ted to the

relative surface tension on each bead. A lower surface tension on

the bead resu lted in a la rger b ead d iam eter an d h ig h e r w ater

holding capacity of the gel.

4. Precipitation by Metal Ions

The p rec ip ita tio n of seaw eed alg inate an d acety la ted and

d ea ce ty la ted b ac te ria l a lg ina te by m eta l ions was com pared .

P u rified a lg in a te s w ere d isso lv ed in d e io n ize d w a te r a t a

concentration of 400 M.g/ml (w /v). M etal salts w ere dissolved in

deionized w ater to p rep are for the solutions with concentrations of

0 to 25 o r 100 mM. The m etal ions tested were: Csl+, Rbl+, Mg2+,

Ca^+, Sr2+, Mn2+, Fe^+, Co2+, Cu2+, Zn2+, Pb2+, an d U ^ . All m etal

salts were ob tained from Sigma Chemical Co., St. Louis, MO, except

fo r u ran y l ace ta te (Eastm an Kodak Co., R ochester, NY). Four

volum es of seaweed alginate solution or acetylated or deacetylated

bacteria l alg inate solution were m ixed w ith one volum e of each

m etal solution, respectively. The m ixtures were incubated for 12

hou rs a t room tem p era tu re an d cen trifuged (17,000 x g fo r 30

m inutes) in a Sorval Superspeed Model RC-5B centrifuge (Du Pont

Co., W ilmington, DE). The su p ern a tan ts were sep ara ted an d the

co n cen tra tio n of a lg inate rem ain ing in each su p e rn a ta n t was

m easu red by th e u ron ic acid assay. The am ounts of a lg inate

separated as a gel were calculated by difference and those values

w ere used to determ ine the rela tive precip ita tion of the alginate

solutions by the m etal ions.

RESULTS

I. Production of Bacterial Alginate

1. Media Compositions and Conditions

In b ro th cu lture , alg inate p roduction by P. syringae ATCC

19304 fo llow ed th e g row th cu rve of th e o rgan ism (Fig. 9).

Maximum cell mass (dry weight) an d alginate yields were obtained

48 hours post inoculation. The type of carbon source used affected

the alginate yield of P. syringae ATCC 19304, as well as the degree

of acetylation within the polym er (Table 5). P. syringae grew well

on glucose, sucrose, glycerol, and gluconic acid, bu t d id no t grow on

fructose. Sucrose grown cells yielded an EPS, only 77% percen t of

which was uronic acid. Gluconic acid grown cells yielded the m ost

alg inate (approxim ately 2 0 0 i-ig/mg cell d ry w eight), w ith th e

h ighest degree of acetylation (approxim ately 100%). Gluconic acid

was the carbon source of choice due to the increased alginate yield

of gluconic acid grown P. syringae.

P. syringae ATCC 19304 u tilized am m onia as a n itro g en

source, b u t was unable to utilize nitrate, n itrite, or urea. There was

an inverse co rre la tio n betw een a lg ina te y ie ld a n d th e in itia l

concen tra tion of am m onia in the m edia. As in itia l am m onium

co n cen tra tio n increased from 2 mM to 15 mM th e cell m ass

increased 2.4 fold, from 0.66 mg cell d ry w eight/m l to 1.58 mg cell

d ry w eight/m l. At the same time, alginate yield decreased 2.4 fold,

from 1200 j.ig/ml to 500 ^g/m l (Fig. 10). This ind ica ted th a t at

h ig h e r in itia l am m onium concen tra tions the ca rbon norm ally

d es tin ed fo r alg inate p roduction was used by the cells for cell

58

Cell

mas

s (m

g dr

y w

eigh

t/

ml

brot

h)

59

10002

- 8 0 0

- 6 0 0

1

- 4 0 0

- 2 0 0

01006040 800 20

do*3ddadod3ddtuod

oH

Tim e (hr)

Figure 9. The re la tionsh ip betw een cell mass (♦), and alginate (uronic acid) accum ulation (D) by P. syringae ATCC 19304 with time in shake flask culture.

tyig

/ml

brot

h)

Table 5. Effects of Carbon Source on Alginate Yield from P. syringae ATCC 19304

CarbonSourcea>b

Cell Yield (m g/m l)c

Yield Total EPS Oig/ml)d

% Alginatee Yield Alginate (ng/m g cell)f

% AcetylationS

Glucose 1.25 175 1 0 0 140 107 (±23)

Sucrose 1.23 211 77 132 4 (± 1)

Glycerol 0.96 134 1 0 0 140 84 (± 17)

Gluconic acid 1.04 213 100 205 97 (± 30)

a Cultured in DF bro th supplem ented with 2% carbon source, and grown for 48 hours a t 30°C with shaking a t 180 rpm .b Fructose was also tested bu t there was no growth. c Cell yield was m easured as mg cell d ry w eight/m l broth. d Yield of total EPS was m easured as ng EPS/ml broth. e The percen t of to tal EPS th a t was alginate.f Yield of alginate was m easured as [ig alginate/m g cell d ry weight.8 The fimolar ratio (%) of acetyl to uronic acid.

61

3©*&

asAtxo'H

b©awwrt2

©L>

1400

- 1200

- 1000

- 80 0

- 6 0 00.8 -

4 0 00.612 15i 9

d©S

P

I sn3 -% a00 ar t 5 "

3©H

In itia l [Am m onium ] (mM)

Figure 10. The effect of the initial am m onium concentration on cell mass (♦), and total alginate (uronic acid) accum ulation (□), by P. syTingae ATCC 19304 at 48 hours.

62

division. As initial am m onium concentrations increased from 2 to 9

mM, the specific yield of alginate decreased from 2100 fxg/mg cell

d ry w eight to 500 ^ig/mg cell d ry weight. In itia l am m onium

concentrations above 9 mM resulted in a consistently lower specific

alginate yield (Fig. 11).

Initial pH of the DF b ro th and tem peratu re bo th affected the

cell mass and alginate yield of P. syringae ATCC 19304. M aximum

growth of P. syringae ATCC 19304 occurred in DF b ro th with initial

pH's betw een 6.4 and 7.2. Alginate yield was greatest in DF b ro th

with initial pH's betw een 6.8 and 7.0. Above pH 7.0 the alginate

y ield declined (Fig. 12). The optim um tem p era tu re fo r alg inate

p roduction was 30°C. T here was little change in cell m ass w ith

tem p era tu res betw een 25°C to 32°C. A lginate p ro d u c tio n was

extrem ely tem pera tu re dependen t, with a sharp optim um at 30°C

(Fig. 13).

2. Agar vs. Broth Culture

The ability of P. syringae ATCC 19304 to grow an d produce

a lg in a te was co m p ared on ag ar an d in b ro th cu ltu re . Upon

successive tran sfe rs in b ro th cu ltu re , a lg inate y ie ld dec reased

dram atically. Initially, alginate yield reached a maxim um of 550 |ng

a lg inate / mg cell d ry weight a t 27 hours. After one tran sfe r back

into b ro th culture, the yield decreased to only 50 (.ig a lg in a te / mg

cell d ry weight. After a second transfer, there was less th an 10 j.ig

alg inate/ mg cell d ry weight. On agar m edia alginate production by

P. syringae ATCC 19304 was consisten t upon tran sfe r from one

Spec

ific

algi

nate

ac

cum

ulat

ion

tyig

/mg

cell

dry

wei

ght)

63

3000

2000

1000

0

In itia l [A m m onium ] (mM)

t '-----------1----------- 1------------'-----------1------------'-----------r

2 5 y 12 15

Figure 11. The effect of the initial am m onium concentration on specific yields of alginate (uronic acid) by P. syringae ATCC 19304 at 48 hours.

Cell

mas

s (m

g dr

y w

eigh

t/m

l br

oth)

64

5 0 0

1 .6 -

4 0 0

- 3 0 0

0.8 -

- 2 0 0

0.6

0 .4 1006.0 6.2 6.4 6.6 6.8 7.0

d3to

h* 2 « &s a.23>CO

3oH

In itia l pH

Figure 12. The effect of initial pH on cell m ass (♦), and alginate(uronic acid) accum ulation (0), by P. syringae ATCC 19304 a t 48hours.

65

f l©PXi

as•pjd&o

pt?&Qa0)CO

a©©

2.75

2 .50

2.25

2.00

1.75

1 . 5 0 -

1.25 -

1.0 0 -

0 .75 -

0 .50 -

0 .25 -

0.00

500

- 4 0 0

300

d©3«iqpag

a sd ©© ps s

d &oS 5 'rt

5©H

24—1"26

—r-28

—r~30

—r~32

20034

Tem p. (°C)

Figure 13. The effect of tem perature on cell mass (♦), and alginate(uronic acid) accum ulation (0), by P. syringae ATCC 19304 at 48hours.

66

culture to another. The organism averaged approxim ately 1870 jug

a lg inate / mg cell d ry weight over 5 successive transfers (Table 6).

Total and specific alginate yields were determ ined in various

m edia. P. syringae was tested in b ro th and on an agar surface. DF

salts, n u trien t m edia, peptone m edia, and beef ex tract m edia were

tested fo r th e ir ability to support bacteria l growth and prom ote

alginate production by P. syringae ATCC 19304 (Table 6). In both

b ro th an d on agar, n u tr ie n t m edia sup p o rted the h ighest to tal

alginate yield in a 48 hour period. The specific alginate y ield was

1.5 to 2 fold g reater in beef extract m edia than in any of the o ther

m edia tested . In all cases, grow th on agar m edia increased the

specific yield of bacterial alginate by abou t 3 fold. In DF b ro th the

specific yield was approxim ately 500 M-g/mg cell d ry w eight an d

was constan t from 24 hours to 96 hours. This ind icated th a t the

to tal yield of alginate increased at a constant ra te over this period.

On agar m edia specific alginate yields reached approxim ately 1850

ng/m g cell d ry weight betw een 24 and 72 hours an d then began to

decrease after 72 hours.

Ferric ion affected alginate production . By increasing the

initial concentrations of ferric ion from 0 mM to 1 mM, the specific

alginate yield by P. syringae ATCC 19304 decreased by 87% (Fig.

14). This indicated tha t iron starvation m ay have a role as a trigger

for alginate production.

Table 6. Alginate Yield by P. syringae ATCC 19304 on Different Media.

Yield in Liquid Media Yield on Solid Media

Mediaa >b Specific (ng/m g cell)c

Total(fig/ml)d

Specific (fig/mg cell)c

Total (fig/cm2 )e

DF Salts 550 480 1870 630

Beef Extract, Peptone

580 620 2030 980

Nutrient 590 640 2040 960

Peptone 680 540 2060 700

Beef Extract 1320 450 3000 550

a Cultures were grown a t 30°C for 48 hours. Liquid cu ltu res w ere shaken a t 180 rpm . All cultures were supplem ented with 2% (w/v) gluconic acid.t> Beef extract was used a t a 3 g/L concentration and Peptone was used a t a 5 g/L concentration. Beef extract, Peptone, and N utrient m edia were from Difco Labs., Detroit, ML c Specific production was m easured as ng alginate/m g cell d ry weight. d Total production in liquid culture was m easured as fig alg inate/m l broth. e Total production on solid culture was m easured as fig alginate/cm 2 of agar surface.

Spec

ific

algi

nate

ac

cum

ulat

ion

(pg/

mg

cell

dry

wei

ght)

68

2000

1000 -

0.2 0.4 0.8 1.0 1.20.G0.0

In itia l [Fe+++] (mM)

Figure 14. Effect of in itial ferric ion (Fe^+) concentration on thespecific yields of alginate (uronic acid) by P. syringae ATCC 19304at 48 hours.

69

II. Characterization of Bacterial Alginate

1. Recovery

Alginate p roduced by P. syringae ATCC 19304 was recovered

from the surface of DF agar plates supplem ented w ith 2% (w/v)

gluconic acid. Several d iffe ren t alcohols w ere tested fo r th e ir

ability to precip itate bacterial alginate. Recoveries were com pared.

All of the alcohols tested: m ethanol, ethanol, n-propanol, and iso­

p ropano l, p rec ip ita ted 100% of th e alg inate a t a fin a l a lcohol

concentration o f 50% (v /v ). In o rd e r to de term ine the effect of

acetylation on alcohol precip itation , equal am ounts of acety lated

a n d d e a c e ty la te d a lg in a te w ere p re c ip ita te d w ith v a r io u s

concentrations of iso-propanol. Acetylation d id no t affect p ro d u ct

recovery.

2. M olecular Weight

The w eight average m olecu lar w eight (Mw ) a n d n u m b e r

av e rag e m o lecu la r w eigh t (Mn) w ere d e te rm in e d fo r b o th

acety lated and deacety lated bacterial alginates by gel perm eation

ch ro m ato g rap h y (GPC). The Mw an d th e Mn of th e seaw eed

alginate were approxim ately 65% sm aller than the native bacterial

alginate. D eacetylation w ith sodium hydroxide d id n o t a lte r the

m olecular weights appreciably. Upon deacetylation, the Mw of the

bacterial alginate decreased by 6%, and the Mn decreased by 11%.

In each case, th e po lyd ispersity was approx im ately 3.00. This

ind ica ted th a t th e re was a wide range of m olecular sizes in each

alginate sam ple (Table 7).

70

Table 7. M olecular Weights of Alginates

Alginate sample Mna (x 104 ) Mw b (x 104 ) Mw /M n c

Macrocystis 1.4(± 1.5 x 103)

4.7(± 2.6 x 103)

3.36

A cetylated P. syringae

4.3(± 8.2 x 103)

12.7 (±7.2 x 103)

2.95

Deacetylated P. syringae

3.8(± 7.7 x 103)

11.9(± 8.9 x 103)

3.13

a Num ber average m olecular weight, b Weight average m olecular weight. c Polydispersity

71

3. Composition

The d e te rm in a tio n of alg inate com position req u ired the

reduction of the uronic acids to their corresponding neu tra l sugars

p rio r to acid hydrolysis of the polym er. This reduction facilitated

the acid hydrolysis of the glycosidic bonds by conversion of the acid

resistan t glycosyluronic acid bonds to the m ore acid labile glycosyl

bonds. The acid sensitivities of D -m annose an d L-gulose (Sigma

Chem ical Co., St. Louis, MO) w ere s tu d ied using th in la y e r

chrom atography (TLC) and ion chrom atography. TLC indicated that

D -m annose and L-gulose have m arked d ifferences in th e ir acid

sensitivity. Acid hydrolyzed (HC1) D-mannose resulted in only one

spot on TLC (Rf=.40). The intensity of this spot rem ained constant

betw een 0 an d 4 h o u rs hyd ro lysis tim e (Fig. 15). L-gulose

produced a second spot (Rf=.56) after m ore than 1 hou r hydrolysis.

The in tensity of this spot increased with hydrolysis tim e. At the

sam e tim e, the in tensity of the gulose spo t (Rf=.37) decreased

p ropo rtiona lly (Fig. 16). Both D-m annose an d L-gulose reac ted

similarly in HC1 and H2 SO4 (Table 8 ).

The relative acid sensitivity of each sugar was m easured by

Dionex ion chrom atography. D-Mannose was acid stable. After 4

hours of HC1 o r H2 SO4 hydrolysis, 98% of the original sugar was

recovered. L-gulose was relatively stable for 1 hour, after which it

began to rapidly breakdown. After 4 hours hydrolysis, only 22% of

the original sugar was recovered (Fig. 17).

The acid breakdow n p roduct of L-gulose was no t definitively

identified. TLC of this com pound showed a m igration (Rf=.58) equal

to 1,6 anhydro p-D-mannopyranose (Rf=.58), and very close to 1,6

72

.4. .... *: ■ MW.",., ?A--' - 4 . ) > M»r-5)-St>i*>•

I

1 2 3 4 5 6 7

Figure 15. Thin layer chrom atograph of HC1 hydrolyzed D-mannose. Lane 1= unhydrolyzed D-mannose in H20, Lane 2= unhydrolyzed D- m annose in HC1, Lanes 3-7= hydrolyzed D-mannose in HC1 for 0 .5 ,1 , 2 ,3 , and 4 hours respectively at 100°C

73

1 2 3 4 5 6 7

Figure 16. Thin layer chrom atograph of HC1 hydro lyzed L-gulose. Lane 1= unhydro lyzed L-gulose in H2 O, Lane 2= unhydro lyzed L- gulose in HC1, Lanes 3-7= hydrolyzed L-gulose in HC1 for 0.5, 1, 2, 3, an d 4 hours respectively a t 100°G

74

Table 8 . Thin Layer Chrom atography (Rf values )a

Sample Spot A Spot B

D-mannose in HC1 .40 (± .03) N/AC

L-gulose in HC1 .37 (± .03) .56 (± .03)

D-mannose in EI2SO4 .37 (± .04) N/A

L-gulose in H2 SO4 .34 (± .03) .50 (± .04)

a Each Rf value was calculated by the distance of the spot from the origin divided by the total distance traveled by the running solvent, b Each sample was hydrolyzed in an equal volum e of 1 N acid and hydrolyzed for 4 hours a t 100°G c N/A= Not Applicable

75

HWkHo>oooPiurtooi/j

120

100

80

GO

40

203 41 7

H y dro lysis Tim e (h r)

Figure 17. The stability of m onom eric D-mannose an d L-gulose in HC1 and H2904 under hydrolysis conditions at 100°C, as determ ined by ion chrom atography. L-gulose in HC1 (□), D-m annose in HC1 (♦), L-gulose in H29D4 (■), D-m annose in H2SO4 (0 ).

76

anhydro p-D-glucopyranose (Rf=.60, Fig. 18). This indicated tha t the

m olecule m ay be a 1,6 anhydride . M olecular m odeling of 1,6

anhydro p-L-gulopyranose (Fig. 19) showed tha t this molecule has a

low total energy (approxim ately -1400 kcal/m ol) and thus is stable

(Table 9). This lended em phasis to the possibility of 1,6 anhydride

form ation upon acid hydrolysis of L-gulose.

D estruction of the reduced sugars in the alginates on acid

hydrolysis paralleled the results seen w ith the D-m annose an d L-

gulose m onom ers. A fter co rrection fo r gulose d es tru c tio n by

extrapolation back to tim e 0, a composition of 60% m annuronic acid

and 40% guluronic acid was obtained for Macrocystis alginate, and

82% m annuronic acid and 18% guluronic acid for P. syringae ATCC

19304 alg inate (Fig. 20). This co rre la ted well to the rep o rted

com position fo r a lg in a te p ro d u ced by P. aeruginosa o f 80%

m annuron ic acid an d 20% guluronic acid, an d fo r Macrocystis

alginate of 60% m annuronic acid and 40% guluronic acid (150).

III. Functional Properties

1. Viscosity

The viscosity of a solution depends on the m olecular weight

an d the rig id ity of the solute, as well as env ironm ental factors,

especially tem perature. The concentration, percent acetylation, and

tem peratu re all con tribu ted to variations in the flow properties of

alginates. At an alginate concentration of 0.1% (w/v) the viscosity

of acetylated bacterial alginate was 2.7 fold greater th an seaweed

alginate. Deacetylated bacterial alginate was 2.0 fold m ore viscous

than seaweed alginate. As alginate concentrations increased from

77

§ •

Figure 18. T hin lay e r ch rom atograph of th e acid hydro lyzed p roduct of L-gulose. Lane 1= unhydrolyzed L-gulose in H2O, Lane 2= standard 1,6 anhydro p-D-mannopyranose, Lane 3= standard 1,6 anhydro p-D-glucopyranose, Lane 4= acid hydrolysis p ro d u ct of L- gulose.

Figure 19. Com puter derived image of 1,6 anhydro p-L-gulopyranose. The image is depicted cross stereo view.

Table 9. The Energetics and Stability of 1,6 Anhydro p-L-Gulopyranosea

Sample # Time(Femtosec)b

PotentialEnergyc

KineticEnergy

TotalEnergy

T em perature(°K)

2 250 -2307.73 861.11 -1446.62 387.77

3 500 -2432.09 886.92 -1545.17 399.39

4 750 -2475.61 855.84 -1619.77 385.40

5 1000 -2512.66 878.87 -1633.79 395.77

7 1500 -2542.10 870.16 -1671.94 391.85

14 3250 -2554.33 865.87 -1688.46 389.92

31 7500 -2512.04 820.92 -1691.11 369.67

33 8000 -2569.74 875.05 -1694.70 394.05

a Values w ere ob ta ined using SYBYL m olecular m odeling software (Tripos Assoc. Inc., St. Louis, MO).b 1 fem tosecond equals 10" 15 seconds. c All energy values are m easured in kcal/m ol.

80

50

■OImo►oo4>P4to©53OM

4 0 -

3 0 -

20 -

10 -

I4

H ydro lysis T im e (h r)

Figure 20. Gulose recovered, expressed as a p ercen t of the to tal sugar p re se n t in the HC1 hydro lyzed reduced alg inates, from M acrocystis pyrifera (□), a n d P. syringae ATCC 19304 (♦), as determ ined by ion chrom atography.

81

0 jig/m l to 1000 (.ig/ml (w /v), the com parative viscosity (N/No) of

each solution increased non linearly . In each case, th is increase

exhibited non-new tonian flow dynam ics (Fig. 21). Seaweed alginate

deviated least from new tonian flow. A cetylated bacteria l alginate

deviated most. The difference in the viscosities of acety lated and

deacety lated alginates ind icated th a t acety lation linearly affected

th e flow dynam ics of a lg inate so lu tions. An average of the

c o m p a ra tiv e v isc o s ity o f seaw eed a lg in a te m e a s u re d a t

c o n c e n tra tio n s of 50, 100, a n d 200 f-ig/ml (w /v) in c reased

approxim ately 8% per a 50% increase in acetylation. An average of

the com parative viscosity of bacteria l a lg inate m easu red a t th e

sam e co n cen tra tio n s , in c reased 16% p e r a 50% in c re a se in

acety la tion (Fig. 22). This d ifference was p ro b ab ly due to the

in c re a se d average m o lecu lar w eight o f th e bac teria l alg inate

polym er, an d its m ore extended structure due to high am ounts of D-

m annuronic acid.

T em perature also affected the viscosity of alginate solutions.

Each alg inate so lu tion re sp o n d e d sim ilarly to th e affects of

te m p e ra tu re . The v iscosity o f each a lg in a te so lu tio n a t a

co n cen tra tio n of 400 f.ig/ml (w /v) was co n s tan t from 30°C to

approxim ately 52°C. At 52°C the viscosities o f each a lg inate

so lu tion decreased d ram atica lly . Between 52°C a n d 85°C th e

viscosities of the seaweed alginate sam ple decreased 3.4%, on the

average, p e r °C. The viscosities of the acety la ted bac teria l an d

deacetylated bacterial samples decreased 9.2% p er °C, and 6.5% per

°C respectively . At 85°C the com parative viscosities of each

alginate solution were approxim ately equal (Fig. 23).

Com

para

tive

Vis

cosi

ty

(N/N

o)

82

10

8

6

4

2

00 200 4 0 0 600 800 1000 1200

A lg ina te C o n cen tra tio n (^ig/m l)

Figure 21. Com parative viscosity, (N/N0), of Macrocystis alginate (□), a c e ty la te d P. syringae a lg ina te (I), an d d e a c e ty la te d P. syringae alginate (♦), as a function of alginate concentration, (w/v).

Com

para

tive

Vis

cosi

ty

(N/N

o)

83

2.0

1 . 6 -

1 .4 -

0 50 100 150

A cety la tion (%)

Figure 22. The effects of acetylation on the com parative viscosity, (N/Nq), of Macrocystis alginate (D), and P. syringae alginate (♦).

Com

para

tive

Vis

cosi

ty

(N/N

o)

84

6

4

3

2

130 32 38 42 4 4 50 52 58 0 4 08 7 4 78 80 84

T em p e ra tu re (°C)

Figure 23. The effects of tem perature on the com parative viscosity,(N/No), of Macrocystis a lg inate (0), acetylated P. syringae alginate(I), and deacetylated P. syringae alginate (♦).

85

2. Physical Effects

The affects of acetylation on gelation, w ater holding capacity,

surface tension, and gel porosity were determ ined. Calcium induced

gelation was altered by bo th acetylation, and calcium concentration.

The gels p ro d u c e d fro m ac e ty la ted b ac te ria l a lg in a te h e ld

approxim ately 100 g w ate r/g d ry alg inate gel com pared to the

deacetylated bacterial alginate gel which held approxim ately 3 2 g

w ate r/ g d ry alginate gel (Table 10). As the concentration of CaCl2

increased from 0.05 M to 0.50 M, the w ater holding capacity of each

Ca-alginate gel decreased linearly. The w ater holding capacities of

bacterial alginate gels m ade with 0.50 M CaCl2 were approxim ately

54% th a t of gels m ade with 0.05 M CaCl2. In contrast, Ca-alginate

gels m ade w ith seaweed alginate showed a 41% decrease in w ater

holding capacity over the same calcium ion increase.

The w ater holding capacities of bo th Fe-alginate (ferric), and

P b-alg inate gels w ere d e te rm in e d fo r seaw eed a lg in a te an d

acety lated and deacety lated bacterial alginates. The resu lts were

com pared to those obtained from Ca-alginate gels. In each case, Fe-

alg inate gels held less w ater th an th e ir Ca-alginate counterparts,

b u t m ore than Pb-alginate gels. The w ater holding capacity of Fe-

a lg inate (0.05 M FeCl3 ) gels m ade w ith seaw eed a lg inate was

approxim ately 82% th a t of Ca-alginate gels. Pb-alginate (0.05 M

PbCl2) gels held w ater a t 67% th a t of Ca-alginate gels (Fig. 24). As

the concen tra tion of m etal (Fe^+ o r Pb2+) increased , the w ater

holding capacity of the resulting gels decreased. By increasing the

m etal concentration from 0.05 M to 0.50 M in increm ents of 0.05 M,

the w ater holding capacity in Fe-alginate gels decreased by an

86

Table 10. Effects of Calcium Concentration on the W ater HoldingCapacitiesa of Alginate Gels.

Alginate sample 0.05 M

Calcium Concentration^

0.10 M 0.25 M 0.50 M

Macrocystis 65 47 36 27(±3) (±4) (±4) (±3)

A cetylated 100 85 69 56P. syringae (±6) (±5) (±5) (±5)

Deacetylated 32 26 24 17P. syringae (±3) (± 2) (±2) (± 1)

a W ater holding capacities are calculated as g w ater/g d ry alginate gel.b Calcium was derived from CaCl2 (Sigma Chemical Co., St. Louis, MO).

Wat

er

Hol

ding

{%

of C

alci

um)

87

90

80 -

70 -

60 ■■

50 -

40.25 M.05 M .1 M .5 M

M etal c o n c e n tra tio n

Figure 24. P ercen t d iffe ren ce in w ater ho ld ing cap ac ity ofMacrocystis alginate gels m ade with ferric iron (D), and lead (♦), ascom pared to calcium alginate gels.

88

average of 2.2% per 0.05 M increase, and in Pb-alginate gels by an

average of 3.7% p er 0.05 M increase. The w ater holding capacities

of Fe-alginate gels and Pb-alginate gels m ade with acety lated and

deacety la ted bacterial alginate were sim ilar to those of seaweed

alginate. The w ater holding capacity of Fe-alginate (0.05 M FeCl3)

gels m ade w ith acety la ted an d deace ty la ted bac teria l a lg inate

averaged approxim ately 79% tha t of the corresponding bacterial Ca-

alginate gels. The w ater holding capacity of Pb-alginate (0.05 M

PbCl2) gels m ade under the same conditions averaged only 46% that

of the corresponding bacterial Ca-alginate gels (Fig. 25, 26). As in

seaw eed a lg in a te gels, th e w ater h o ld in g cap ac ity fo r both

acetylated and deacetylated bacterial alginate gels decreased as the

m etal concentration increased. The w ater holding capacities of Fe-

alg inate gels m ade w ith acety la ted an d d eace ty la ted bac teria l

alginate decreased by an average of 2.0% p e r 0.05 M increase in

iron concentration. The w ater holding capacity of Pb-alginate gels

decreased by an average of 3.0% p e r 0.05 M increase in lead

concentration (Fig. 25, 26).

The bead size is a function of the surface tension of the gel

th a t m akes up the bead, the sm aller the bead the higher the surface

tension . A cetylated bacteria l alginate form s gel beads in 0.5 M

CaCl2 th a t averaged 4.04 millimeters in d iam eter (Table 11). These

beads are 37% larger th an equivalent beads m ade with seaweed

alg inate , a n d 45% la rg e r th a n beads m ade w ith d eace ty la ted

bacterial alginate.

Incubation of the gel beads in deionized w ater or 0.5 M CaCl2

a t 4°C fo r 24 hours a lte red the size of the gel beads. Beads

Wat

er

Hol

ding

(%

of C

alci

um)

89

90

80 -

70 -

60 -

50 -

4 0 -

30.05 M 1 M .25 M .5 M

M etal c o n c e n tra tio n

Figure 25. P ercen t d iffe ren ce in w ater ho ld ing cap ac ity ofacetylated P. syringae alginate gels m ade with ferric iron (D), andlead (♦), as com pared to calcium alginate gels.

Wat

er

Hol

ding

{%

of C

alci

um)

90

80

7 0 -

6 0 -

50 -

4 0 -

3 0.05 M ] M .25 M .5 M

M etal c o n c e n tra tio n

Figure 26. P ercen t d iffe ren ce in w ater ho ld ing cap ac ity ofdeacety lated P. syringae alg inate gels m ade w ith ferric iro n (□),and lead (♦), as com pared to calcium alginate gels.

91

Table 11. Effects of Acetylation on Bead Surface Tensiona

Bead Diameters (mm.)

Time in W aterb Tim einCaCl?c

Alginate sample 5 m inutes 24 hours 24 hours

Macrocystis 2.95 2.69 2.64(± 0.05) (± 0.04) (± 0.06)

Acetylated 4.04 2.82 2.26P. syringae (± 0.06) (± 0.04) (± 0.05)

Deacetylated 2.79 1.96 1.68P. syringae (± 0.05) (± 0.04) (±0.05)

a The rela tive surface tension was m easured by bead d iam eter (mm.), the sm aller the bead the h igher the surface tension on the bead.b W ater values were m easured after 15 m inutes in 0.5 M CaCl2 and incubation in water a t 4°C for the relevent times. c CaCl2 values were m easured after incubation in 0.5 M CaCl2 at 4°C for 24 hours.

92

in cu b a ted in deion ized w ater m ade from ace ty la ted b ac te ria l

alginate h ad diam eters averaging 2.82 m illim eters, a decrease of

42% from the initial size. These beads were still 5% larger th an the

seaweed alginate gel beads and 44% larger th an the deacety la ted

bacterial alginate gel beads. This indicated th a t the relative surface

tension of acetylated gels was less th an deacety lated alginate gels.

Extended incubation (24 hours) of Ca-alginate gel beads in 0.5 M

CaCl2 p roduced d ifferen t results. The Ca-alginate gel beads m ade

from seaweed alginate were the largest with an average d iam eter

o f 2 .64 m illim eters. These beads w ere 17% la rg e r th a n the

acetylated alginate beads and 57% larger than the beads m ade from

d eace ty la ted bac teria l alg inate. This in d ica ted th a t acetylation

d ecreased the surface tension on the Ca-alginate gel beads, an d

extended exposure to calcium decreased the size of the beads and

increased surface tension on the beads.

The re la tiv e p o ro sity of the C a-alginate a n d Fe-alginate

(ferric) gel beads were determ ined from the ra te of w ater loss w ith

time (Fig. 27). A large absolute slope indicated a m ore rap id w ater

loss resulting from larger relative pore sizes. Ca-alginate gels, m ade

from acety lated bacteria l alginate, showed the m ost rap id loss of

w ater (absolute slope= 6.8 x 10 '2). The ra te of w ater loss in the

gels m ade from deacetylated bacterial alginate (absolute slope= 5.2

x 10" 2) was 24% slower th an the acetylated bacterial polym er and

13% faster than seaweed alginate (absolute slope= 4.6 x 10"2). The

ra tes of w ater loss of Fe-alginate gels were also d e te rm in ed and

com pared as a percentage of Ca-alginate gels. Both the gels m ade

from seaw eed a lg inate an d deace ty la ted bac teria l a lg inate lost

93

■N

fed Qa •©isOas« ,£rt£ M

Im&

to

5

4

30 10 20 30

Time (m in.) a t 50 C

Figure 27. Rate of w ater loss of calcium alginate gels m ade fromMacrocystis alginate, I slope l= 4.6 x 10"2 (□), acety lated P. syringaealginate, I slope 1= 6.8 x 10"~ (♦), and deace ty la ted P. syringaealginate, I slope 1= 5.2 x 10"2 (I), as a function of incubation time at 50°C.

94

w a te r ap p ro x im a te ly 2 fo ld fa s te r th a n th e ir C a-a lg in a te

counterparts, indicating a m ore open structure. The gels produced

by the acety lated bacteria l alginate show ed a ra te of w ater loss

approxim ately equal to th a t of Ca-alginate gel beads indicating

com parable pore sizes between these gels (Fig. 28).

3. Cation Precipitation by Alginates

P re c ip ita tio n o f ac e ty la te d a n d d e a c e ty la te d b ac te r ia l

alginates, m easured as gelation, by cations was com pared to th a t of

seaw eed alginate. Twelve m etal ions w ere screened an d th en

classified into 3 groups, depending on the ir ability to p recip ita te

acetylated a n d /o r deacetylated bacterial alginate. The groups were:

1) those cations having the ability to p recip itate half the available

bacterial alginate at a concentration of less than 20 mM m etal, (U6+,

Cu2+, Pb2+,Ca2+, Sr^+, and Fe3+), 2) those cations having the ability

to precipitate half the available bacterial alginate at a concentration

of g reater than 20 mM m etal, (Zn2+, Co^+, and Mn^+), and 3) those

cations unable to precip itate half the available bacterial alginate at

a concentration up to 100 mM metal, (Mg2+, Csl+, and Rbl+).

Of all the cations tested, uranium , copper, lead and ferric ions

precip itated both acetylated and deacetylated bacterial alginate a t

low concentrations. They were able to precipitate m ore than 90% of

the alginate a t m etal concentrations less th an 5 mM. A cetylation

d id n o t significantly affect the ability of these ions to p recip ita te

bac teria l alg inate. U ranium p rec ip ita ted g rea te r th a n 90% of

deacetylated bacterial alginate a t a m etal concen tra tion of 1 mM

an d g rea te r th an 90% of the acety la ted bac teria l a lg inate a t a

95

UiBa § rt dQ °

S 3tt or °s Wi WHS3 4> 12 bo2 do rt2 8 J S SfldEt

110

100 -

9 0 -

80 -

70 -

GO10 20 300

Tim e (m in.)

Figure 28. Comparison of the w ater loss of ferric iron alginate gels of Macrocystis alginate (D), acetylated P. syringae alginate (♦ h a n d deacetylated P. syringae alginate (I), com pared to calcium alginate gels as a function of incubation time at 50°G

96

concentration of 5 mM (Fig. 29). Cupric ion reacted m uch the same

way, precipitating 98% of the deacetylated bacterial alginate a t 2.5

mM copper, and greater than 90% of acetylated bacterial alginate a t

a m etal concentration of 5 mM (Fig. 30). Lead induced gelation of

bacterial alginates was least affected by acetylation. G reater than

90% of b o th ace ty la ted a n d d eace ty la ted b ac te ria l alginates

precipitated in the presence of 1 mM lead chloride (Fig. 31). Ferric

ions p rec ip ita te d b o th ace ty la ted an d d eace ty la ted b ac te ria l

alginate. Acetylation increased the precipitability of the bacterial

po lym er by ferric ion. A pproxim ately 90% of the ace ty la ted

b ac teria l po lym er p rec ip ita ted w ith 1 mM ferric ch loride. In

con trast, 90% of the deacety la ted bacteria l alginate p recip ita ted

with 2 mM ferric chloride (Fig. 32).

A high affinity of calcium ions for polyguluronate residues is

the basis for the cu rren t gelling theory (the "egg box model") for

seaw eed alginates. As expected, alm ost 100% of the seaw eed

alg inate was p rec ip ita ted by 5 mM calcium ch lo ride (Fig. 33).

Calcium p rec ip ita tio n was less efficient for b ac teria l alg inates,

p robab ly due to the absence of extensive polyguluronate blocks.

The am o u n t of bac teria l a lg inate p rec ip ita ted by calcium ions

increased as the calcium concentration increased. A pproxim ately

95% of the deacetylated bacterial alginate and 65% of the acetylated

bacterial alginate precipitated with 60 mM calcium chloride.

As m ight be expected from its chemical sim ilarity to calcium,

stron tium was also an effective precip itan t of bo th acetylated and

deacetylated bacterial alginate. As with calcium, strontium showed

a g re a te r ab ility to p rec ip ita te d eace ty la ted th a n ace ty la ted

97

■0$Bao0kH&3rtabjo<

100

80 -

60 -

4 0 -

20 -

103 84 (> 7 90 1 2

U ran iu m ch lo rid e (mM)

Figure 29. Precipitation of Macrocysns alg inate (D), acety la ted P. syringae alginate (♦), an d deacetylated P. syringae a lg inate (I) by uran ium ions. Relative precipitation is expressed as the % alginate precipitated by U&+- ions.

98

3 Bao

£$rtaao<

120

100 -

80 -

60 -

4 0 -

20 -

0 +0 2 64 8 10

C upric ch lo rid e (mM)

Figure 30. Precipitation of Macrocysns alginate (D), acety la ted P. syringae alginate (♦), and deacetylated P. syringae a lg inate (I) by copper ions. Relative p recip itation is expressed as the % alginate precipitated by Cu2+ ions.

99

’O$B‘8.ooWn&B«aGJO<

120

100 -

80 -

GO -

4 0 -

20 -

0 ■*0 1 2 4

Lead ch lo rid e (mM)

Figure 31. Precipitation of Macrocystis alg inate (D), acety la ted P. syringae alginate (♦), and deacetylated P. syringae a lg inate (I) by lead ions. Relative p rec ip ita tion is expressed as th e % alg inate precip itated by Pb^+ ions.

100

■oB$Gk-o<bu

O h

O4->(0a&0VN

<

100

80 -

6 0 -

4 0 -

20 -

7 4 6 8 10

Ferric ch lo rid e (mM)

Figure 32. Precipitation of Macrocystis alginate (□), acety lated P. syringae alginate (♦), and deacetylated P. syringae a lg inate (I) by ferric ions. Relative p recip ita tion is expressed as the % alginate precip itated by F e ^ ions.

101

*0B&Q.oOb*a,Bcadbo<M

100

80 -

60 -

4 0 -

20 -

0 •#0 20 6040 80

C alcium ch lo rid e (mM)

Figure 33. Precipitation of Macrocystis alg inate (D), acety lated P. syringae alginate (♦), and deacetylated P. syringae a lg inate (I) by calcium ions. Relative precip itation is expressed as the % alginate precipitated by Ca^+ ions.

102

bacterial alginate. S trontium precipitated 100% of the deacetylated

bacterial alginate a t a final concentration of 20 mM. Approxim ately

78% of the acetylated bacterial alginate was p rec ip ita ted w ith 60

mM strontium chloride (Fig. 34).

The second set of cations were those tha t precip itated half the

available bacterial alginate a t a concentration of g rea te r th an 20

mM m etah . Zinc ions fell in to this category. Zinc p rec ip ita ted

d e a c e ty la te d b a c te r ia l a lg in a te b e t te r th a n its a c e ty la te d

counterpart. The precipitation of both acetylated and deacetylated

bacterial alginate d id no t begin until zinc concentrations were above

10 mM. The concen tra tion of p rec ip ita ted b ac teria l a lg inate

increased as the concen tration of zinc increased. A pproxim ately

55% of the acetylated polym er precipitated in 60 mM zinc chloride,

an d 95% of the deace ty la ted polym er p rec ip ita ted a t th e sam e

m etal concentration (Fig. 35). It appears th a t acetylation decreased

the precipitability of alginate by zinc ions.

M anganese an d cobalt ions show ed a lim ited affin ity for

deacety la ted bacteria l alginate an d no affinity fo r the acetylated

po lym er. M anganese p rec ip ita ted 100% of th e d eace ty la ted

bacterial polym er a t a concen tration of 75 mM (Fig. 36). Cobalt

p rec ip ita ted approx im ately 87% of the d eace ty la ted b ac te ria l

polym er a t th a t concen tra tion (Fig. 37). A cetylation com pletely

in h ib ite d th e ab ility of m anganese an d co b a lt to precipitate

bacterial alginate. N either acety lated n o r deacety lated bacteria l

alg inate p rec ip ita ted w ith cesium, rub id ium , o r m agnesium ions

(Fig. 38 ,39).

103

120

100T>$S 80a,o<L>

3rta 40&o<M 20

00 20 40 GO 80

S tro n tiu m ch lo rid e (mM)

Figure 34. Precipitation of Macrocystis alg inate (D), acety la ted P. syTingae alginate (♦), an d deacetylated P. syringae a lg inate (I) by strontium ions. Relative precipitation is expressed as the % alginate precip itated by Sr^+ ions.

104

•o$JS•pN&•HoOlM&

coo&o

pN<

120

100 -

8 0 -

60 -

4 0 -

20 -

200 40 60 80

Zinc ch lo rid e (mM)

Figure 35. Precipitation of Macrocystis alg inate (0), acety la ted P. syiingae alginate (♦), and deacetylated P. syTingae a lg inate (I) by zinc ions. Relative p rec ip ita tio n is expressed as the % alginate precipitated by Zn^+ ions.

105

x?B BoOu&Brta•̂ 4ao<

120

100 -

80 -

6 0 -

4 0 -

20 -

-200 50 100 150 200 25 0

M anganese ch lo rid e (mM)

Figure 36. Precipitation of Macrocystis alginate (0), ace ty la ted P. syringae alginate (♦), and deacetylated P. syringae a lg inate (I) by m anganese ions. Relative p rec ip ita tion is exp ressed as the % alginate precipitated by Mn^+ ions.

106

•0$Bf t•vMo4kn&&rtaeuo<

120

100 -

80 -

GO -

4 0 -

20 -

-200 50 100 150 200

C obalt ch lo rid e (mM)

Figure 37. Precipitation of Macrocystis alginate (□), acety la ted P. syringae alginate (♦), an d deacetylated P. syringae a lg inate (I) by cobalt ions. Relative p recip itation is expressed as the % alginate precipitated by Co2+ ions.

107

■eis5a»HokH6

i s

a&0fN<

3

1

10 20 40 60 80

Cesium ch lo rid e (mM)

Figure 38. Precipitation of Macrocystis alg inate (0), acety lated P. syiringae alginate (♦), and deacetylated P. syTingae alg inate (i) by cesium ions. Relative precip itation is expressed as the % alginate precipitated by Csl+ ions.

108

■o8 8aoOa8rtaCUD

<M

100

90

80

70

60

50

40

30

20

0 10 3020 40 50 60 70

M agnesium ch lo rid e (mM)

Figure 39. Precipitation of Macrocystis a lg inate (□), acety la ted P. syringae alginate (♦), and deacetylated P. syringae a lg inate (I) by m agnesium ions. Relative p rec ip ita tion is expressed as the % alginate precipitated by Mg 2+ ions.

109

The relative ability of these m etal ions to precip itate alginates

w ere com pared to the P i /2 values fo r seaw eed a lg in a te an d

reco rded as the fold difference from those values (Table 12). The

P l /2 value is defined as the concen tra tion of m etal ions (mM)

requ ired to precipitate 50% of a polym er from a 400 iig/m l (w/v)

solution. The re la tive o rd e r of p rec ip ita tion of ace ty la ted an d

deace ty la ted bac teria l alg inate by these ions, as d e te rm in ed by

P l /2 , were as follows:

A cetylated bacterial alginate: Pb2+=Fe3+=u6'1- > Cu2+ > Sr2+ > Ca2+ >

Zn^+ > Mg 2+ =Co 2+ =Cs 1+=Mn 2+ =Rb 1+

Deacetylated bacterial alginate: Pb2+=Fe3+=U(5+ > Cu2+ > Sr2+ > Ca2+ >

Zn2+ > Mn2+ > Cq2+ > Mg 2+ =Cs 1+=Rb 1+

110

Table 12. The Precipitation of Macrocystis Alginate and Acetylated and Deacetylated P. syringae Alginate by Metal Ions

Fold Increase from P i / 2 a of Macrocystis Alginate^

Ions Periodic Macrocystis Acetylated Deacetylated Group Alginate P. syringae P. syringae

(P l/2 )c Alginate Alginate

Csl+Rbl+

I A No affinity No affinity

No affinity No affinity

No affinity No affinity

Mg 2+ Ca2+Sr2+

II A No affinity 3.1 1.8

No affinity 3.4 3.7

No affinity 1.7 1.1

Mn2+ VII A 30.0 No affinity 0

pe3fCb2+

VIII A 1.89.6

-0.7 No affinity

-0.63.7

Cu2+ I B 0.5 1.6 2.0

Zn2+ II B 7.0 4.3 2.3

Pb2+ IV B 0.5 0 0.2

U&f Actinidem etal

0.9 -0.2 -0.4

a P l /2 is the co n cen tra tio n of m etal ions (mM) req u ired to p rec ip ita te 50% (w /v) a lg inate from 400 [.ig/ml (w /v) alg inate solutions.b V alues exp ressed a re th e fo ld d iffe ren ce fro m P l/2 o f Macrocystis alg inate (positive values = less ability to precipitate, Negative values = g reater ability to precipitate, zero values = equal ability to precipitate).c " No affinity" signifies th a t the ion d id no t precipitate 50% of the alginate sample up to 100 mM ion concentration.

DISCUSSION

Alginate biosynthesis is a common characteristic of a m ajority

of pseudom onads in rRNA-DNA hom ology group I (94). This group

in c lu d e s a ll th e f lu o re s c e n t a n d a few n o n f lu o re s c e n t

pseudom onads. Until recently, only certain strains of P. aeruginosa

isolated alm ost exclusively from cystic fibrosis patients, and strains

of A. vinlandii were known to produce O-acetylated alginate as EPS

(67). The fluorescent pseudom onads, P. fluorescens , P. mendocina,

an d P. pu tida , w ere subsequently found to p roduce alginate, bu t

o n ly u n d e r co n d itio n s o f stress (bac terioc in , b ac te rio p h ag e ,

an tib io tic , 53, 58). W ithin the p a s t seven years, m any p la n t

pathogenic fluorescen t pseudom onads have been recogn ized as

alginate producers u n d er the appropriate conditions. This strongly

in d ica tes th a t th e ab ility to syn thesize a lg inate m ay have an

im portan t evolutionary role.

L ittle is know n ab o u t th e syn thesis of a lg in a te by th e

p h y to p a th o g en ic p seu d o m o n ad s. P seu d o m o n a s syringae p v

phaseolicola ATCC 19304 produces high am ounts of O -acetylated

alginate in vitro. The am ount of alginate, as well as the degree of

acetylation of the polym er, varied with carbon source. P. syringae

ATCC 19304 grown on sucrose produced a mixed population of EPS.

A lthough alginate was the p redom inan t EPS produced, P. syringae

ATCC 19304 also produced levan (C-2 -» C-6 fructan backbone) on

sucrose (57). Levan production indicates th a t the bacterium also

has the ability to synthesize levansucrase. Sucrose was no t utilized

as the carbon source in this research since the presence of levan in

111

112

the exopolym er extracts m ade purification of the alginate m ore

difficult.

Gluconic acid was chosen as the carbon source in this work

because cells grown on gluconic acid produced approxim ately 21%

m ore alginate th an cells grown on glucose. It is well estab lished

th a t the p rim ary rou te of glucose catabolism in pseudom onads is

th rough the Entner-D oudoroff pathway, which produces pyruvate

an d glyceraldehyde 3-phosphate (52). Glucose conversion to 6-

ph o sphog luconate is re q u ire d p rio r to en try in to th e E ntner-

Doudoroff pathway. This conversion can be accom plished in one of

two ways. Glucose can be phosphorylated to glucose 6-phosphate

by a hexokinase and subsequently oxidized to 6-phosphogluconate

by a glucose 6-phosphate dehydrogenase (148), o r glucose can be

converted to gluconate a t the surface of the cell by an NAD(P)

d ep en d en t glucose dehydrogenase followed by gluconolactonase.

G luconate is th en oxidized to 2-ketogluconate by a m em brane

bound gluconate dehydrogenase during transport of the sugar into

the cell. Once inside the cell the 2-ketogluconate is phosphorylated

to 6-phosphogluconate (47). Most pseudom onads, i.e., P. aeruginosa,

P. fluorescens, an d P. putida oxidize glucose to gluconate using the

second m echanism prior to transport of the sugar into the cell (52).

The increased production of alginate by gluconate grown cells over

glucose grow n cells p ro b ab ly resu lts from a decreased energy

req u irem en t fo r the enzym atic oxidation of glucose to gluconate

p rio r to transport into the cell.

In P. aeruginosa, the C-6 of g luconate in co rp o ra te s into

alginate (13). Carbon atom s 1, 2, and 3 of gluconate are converted

113

to pyruvate through the 2-keto 3-deoxyphosphogluconate aldolase

reaction an d are eventually lost as CO2 and acetyl CoA. Carbon

a to m s 4, 5, a n d 6 a re ch a n n e le d in to a lg in a te th ro u g h

g lyceraldehyde 3-phosphate. G lyceraldehyde 3 -phosphate can

condense w ith d ihydroxyacetone phosphate to p roduce fructose

1,6-diphosphate and ultim ately fructose 6-phosphate, w hich is the

starting m aterial for alginate biosynthesis (7, 78). Besides being a

source of fructose 6-phosphate, gluconic acid can also be oxidized to

acetyl CoA (7). Acetyl CoA is reported ly the source of the O-acetyl

groups on the m annosyl residues of xan than gum (66), and is the

probable source of acetyl in bacterial alginates (136).

P. syringae ATCC 19304 utilized am m onia as a n itrogen source

fo r grow th. It was unab le to use n itra te , n itr ite , o r u rea. All

pseudom onads p ro d u ce energy by resp ira tion . In som e cases,

n itra te can be used as an a lte rn a te e lec tron accep to r allowing

grow th to occur anaerobically . Those pseudom onads th a t can

conduct n itra te resp ira tio n can reduce n itra te beyond the toxic

n itrite stage to m olecular n itrogen by "denitrification." P. syringae

was unab le to utilize n itra te as a n itrogen source and therefore

lacks the cellular m akeup to conduct n itra te respiration. This m ay

be due to the absence of cytochrom e C in the electron tran sp o rt

chain of P. syringae (94). In p lan ts , free am m onium ions are

p re se n t a t v ery low levels. A m m onium is toxic to the p lan ts,

because it inhibits the production of ATP in the m itochondrial and

photosynthetic electron tran sp o rt systems. Most n itrogen presen t

in plants is found associated w ith organic com pounds. In plants,

am m onium is converted in to organic com pounds prim arily through

114

three d ifferent reactions: 1) form ation of glutamic acid by reaction

w ith a-ketoglutarate, 2) form ation of g lutam ine by reac tion w ith

glutamic acid, and 3) form ation of carbam yl phosphate in arginine

b io syn thesis an d p y rim id in e b iosyn thesis . P. syringae is a

proteolytic bacterium (94). This bacterium probably provides itself

w ith am m onia through deam ination of the com ponent am ino acids

allowing the survival of the bacterium on the host leaf surface.

Polysaccharide production by m icroorganism s is enhanced in

n itrogen lim ited, high carbon conten t m edia (147). The changing

c a rb o n /n itro g e n ra tio ex h ib ited th e sam e effect on a lg ina te

p ro d u c tio n in P. syringae th a t has b een r e p o r te d fo r o th e r

p seu d o m o n ad s. H ig h er y ie ld s w ere o b ta in e d w ith h ig h

carbon /n itrogen ratios (low n itrogen content, high gluconate). It

has been proposed tha t lim itation of essential nu trien ts, in this case

n itro g e n , in h ib its g row th a n d d ire c ts th e co u rse o f to ta l

po lysaccharide b iosynthesis from cell w all m a te ria l (LPS a n d

peptidoglycan) to extracellular polysaccharide synthesis (133). It

was fu rth e r postu lated th a t during active growth the same pool of

isoprenoid lipid carriers involved in polysaccharide synthesis are

u tilized by cell wall an d LPS precursors (133). These conditions

d irec t a lg inate b iosynthesis tow ard p ro d u c tio n as a secondary

m etab o lite . In the case o f P. syringae ATCC 19304, a lg inate

biosynthesis d id no t fully fit this hypothesis. A lginate p roduction

by P. syringae is necessary for successful colonization of p lan t host

tissue (113). A lthough alginate p roduction d id inc rease as the

ca rb o n /n itro g en ratio increased , a lg in a te was p ro d u ced by P.

syringae coincidental with cellular reproduction.

115

Bacteria secrete EPS for m any different reasons. Bacterial EPS

m ay aid colonization (16, 50), prevent desiccation, store energy, or

help concentrate and take up charged molecules, particu larly m etal

ions (11, 17, 46). In cystic fibrosis patien ts P. aeruginosa secretes

alg inate to help colonize the lungs an d to p ro tec t itself against

phagocytosis. P. syringae produces alginate to aid in colonization

and parasitization of leaf surfaces (30, 138).

The ab ility of P. syringae to p roduce alg inate decreased

ra p id ly u p o n successive tra n sfe rs in liqu id m edia. A lginate

p ro d u ctio n rem ain ed stable upon tran sfe r on solid m edia. The

pathogenicity of P. syringae is dependen t upon its ability to produce

alginate (113). Colonization of a leaf surface by P. syringae causes

halo blight (leaf spots) o f the leaf. Alginate m ust be p re se n t to

p roduce leaf spots (34). These leaf spots are p robab ly due to an

in te rru p tio n of th e p h o to sy n th e tic pathw ay in th e leaf. All

pseudom onads respire, and thus m ust have a source of ferric iron

fo r synthesis of cytochrom es. On the leaf surface, the sources of

ferric iron are lim ited to ferredoxin and the cytochrom e b f complex.

The b reak d o w n of fe rred o x in o r the cy tochrom e b f com plex

in te rrup ts photosynthesis by stopping electron tran sp o rt an d aids

in leaf spotting. A cetylated P. syringae a lg inates have a h igh

affinity for ferric iron. By increasing the ferric iron concentration in

the growth m edia alginate production decreased. The parasitization

o f P. syringae ATCC 19304 m ay be due to th e ab ility of the

acetylated alginate to scavenge the ferric iron from ferredoxin or

the cytochrom e bf complex. By scavenging the iron, photosynthesis

116

w ould be in te rru p te d a n d leaf spots w ould occur due to the

inability of the p lant to make energy for chlorophyll biosynthesis.

The n a tiv e a lg in a tes of P. syringae ATCC 19304 w ere

polydisperse. This in d ica ted th a t th e re was a h igh deg ree of

variability in size of the alginate p roduced by the biological system.

The Mw for the native bac teria l polym er was 1.3 x 1 0 $. Upon

deacetylation, the Mw dropped approxim ately 6 % to 1.2 x 105. This

d rop was due to the loss of the acetyl groups at C-2 a n d /o r C-3 of

the m annuron ic acid residues w ithin the polym er. By monitoring

the difference in the acid stability of D-mannose an d L-gulose from

the acid hydro lyzed , red u ced alginates, m ore accu ra te a lg inate

com positions were obtained for the bacterial polym er. The alginate

p roduced by P. syringae ATCC 19304 had a final com position of 82%

m annuronic acid and 18% guluronic acid. This com position was in

con trast w ith the previously rep o rted com position of g rea ter th an

95% m annuronic acid an d less th an 5% guluronic acid w here the

relative acid sensitivity of each sugar was no t considered (38).

Both the solution an d gelling properties of bacterial alginate

w ere a lte red by the degree of acety lation on the polym er. The

v isc o s ity o f th e a c e ty la te d p o ly m e r was h ig h e r th a n th e

d e a c e ty la te d b a c te r ia l o r seaw eed a lg in a te s a t e q u iv a le n t

concentrations. The native bacterial alginates were acety lated with

an average of 1.2 acety l un its p e r u ron ic acid resid u e . This

p ro d u ced a 6 p e rcen t increase in Mw an d a 26% increase in

viscosity a t a concen tra tion of 1 m g/m l. A cetylation rep o rted ly

increases the viscosity of alginate solutions (129). These increases

m ay be d u e to th e increase in to ta l m olecu lar w eight of the

117

polym er, o r a lte red conform ations of the po lym er in aqueous

solution. Alginates high in m annuronic acid exhibit a flat ribbon­

like conform ation. High am ounts of gu lu ronate p roduce a m ore

puckered structu re . It is possible th a t the in troduction of acetyl

groups shifts the conform ational energy w ithout increasing the

accessible surface of the molecules, eventually producing a flexible

polym er with an increased num ber of conform ations. Because the

acety la ted po lym er show ed a 4 fold increase in v iscosity over

m olecular weight, it is probable tha t both factors play a role.

A cetylation a lte red the affin ity of th e po lym er fo r m any

cations, including calcium. Except for ferric an d to som e ex ten t

copper ions, the ability of m ultivalent cations, particularly calcium,

to induce gelation of th e alginate was reduced in the acety lated

polym er. In parallel, there was a m arked decrease in the strength

of the gels. The effects of acetylation on the gelling p roperties of

bacteria l alginates were seen in the w ater holding capacities, the

surface tensions, and the relative porosities of the Ca-alginate gels.

Acetylation profoundly affected the rigidity of the Ca-alginate

gels and the ir ability to hold w ater. Generally, the volum e of an

ionic gel is d ep e n d en t upon a positive osm otic p ressu re . The

osmotic pressure of the gel is due m ainly to the positive en tropy of

the mixing of counterions with water, which is counterbalanced at

equilib rium by a negative p ressu re due to th e elasticity of the

netw ork (139). For Ca-alginate gels, which are en thalp ic ra th e r

th a n en tro p ic (3), the elastic ity d ep en d s on the n u m b er an d

strength of the crosslinks. Since the in troduction of acetyl groups

im pairs the cooperative b inding of calcium ions, the num b er of

118

dissociated counterions p er polymer chain increases with increasing

degree of acetylation. The high num ber of dissociated counterions

enhances the positive osm otic p ressu re . At th e sam e tim e, it

weakens the forces holding the network together. Reduction in the

cooperative binding of calcium ions reduces bo th the streng th and

the num ber of crosslinks in the network. Fewer crosslinks lessen

the surface tension on the gel and as a resu lt allows h igher w ater

holding capacities w ithin the gel network. This was observed w ith

th e P. syringae alginate. A cetylated bacterial alg inate gels h ad a

m uch low er surface tension , an d as a re su lt a w ate r ho ld in g

cap ac ity th a t was ap p ro x im ate ly 68 p e rc e n t h ig h e r th a n its

deace ty la ted co u n terp art. Fe-alginate gels an d Pb-alginate gels

showed the same general properties, however neither the ferric no r

lead alginate gels held as m uch water as the Ca-alginate gels. These

differences are attribu tab le to the relative affinities of each cation

to the alginate. Ferric an d lead ions h ad a h igher affin ity th an

calcium ions for both acetylated and deacetylated alginates.

Because of its h igher viscosity and lower affin ity for m any

polyvalent cations, acetylated bacterial alginate m ay be a possible

substitu te for seaweed alginate in m any applications. More viscous

so lu tions can be m ade w ith low er co n cen tra tio n s of bacterial

a lg in a te th a n w ith seaw eed a lg inate , red u c in g th e p o ly m er

requ irem en ts in a given application. By varying the degree of

acetylation, it should be possible to produce alginates with designer

properties.

As a polyelectrolyte, alginate has the potential to concentrate

toxic, heavy a n d /o r valuable m etals from the environm ent. The

119

rem o v al of m etals by m icroorganism s rep o rted ly d ep en d s on

p hysicochem ica l ad so rp tio n to ce llu la r co m p o n en ts such as

polysaccharides and p ro te ins (84). Because of the h igh negative

c h a rg e on th e ce ll w alls o f b a c te r ia , fu n g i, a n d a lgae,

m icroorganism s have been used to concentrate m etal ions including

uran ium , copper, m anganese, cadm ium , m olybdenum , gold, and

m ercu ry (20, 56, 65). The polyanionic n a tu re of alg inates also

allows the binding and concentration of heavy m etals through m etal

induced gelation of the polym ers. The form ation of alginate gels in

the presence of polyvalent cations is a ltered by acetylation of the

alginate, as well as the native conform ation of the polym er. The

physical binding of calcium ions to guluronate residues in seaweed

alginate is due to charge charge in teractions betw een the positive

charges from calcium an d the negative charges from the carboxyl

groups. The size of calcium ions is such th a t they fit in to the space

form ed by the polyguluronate stretches of seaweed alginates (83).

Randomly organized bacterial alginate also gels in the presence of

m any polyvalent cations. This casts some dou b t on the principal

tenan t of the "egg-box" theory; tha t only polyguluronate residues in

a lg inate p lay a key ro le in gelation . The ab ility of bac te ria l

alginates, w ithout polyguluronate blocks, to gel in the presence of

calcium indicates a reevaluation of this theory is necessary.

M ultivalent cations in te rac t d iffe ren tly w ith alg inates (59,

60). Each ca tion exam ined show ed varia tions in its ab ility to

p recip ita te seaweed and bacterial alginate. With the exception of

fe r r ic a n d u ra n iu m ions, p o ly v a len t ca tio n s m ore re a d ily

p rec ip ita ted seaweed alginate over its bacteria l co u n te rp art. In

120

m ost instances, deacetylation of the bacterial polym er enhanced its

ca tion p rec ip itab ility . This ab ility to ad ju s t th e affin ities of

alginates for specific polyvalent cations increases the feasibility of

using th is polym er to rem ove toxic m etals, i.e., lead o r u ran ium

from drinking water.

Acid h y d ro ly s is is u sed to d ep o ly m erize po ly m ers for

com positional analysis. Ideally, hydro lysis goes to com pletion

w ithout loss of the com ponents. Practically, however, some sugar

loss occurs an d m ust be accounted fo r to accurate ly determ ine

polysaccharide compositions. Com positional analysis of alginates

re q u ire s th e red u c tio n of th e u ro n ic ac id res id u es to th e ir

co rresp o n d in g n e u tra l sugars p rio r to acid hydro lysis. This

reduction facilitates the acid hydrolysis of the glycosidic bonds by

converting the acid resistan t glycosyluronic acid bonds to the m ore

acid labile glycosyl bonds. Reduction of alginate polym ers liberates

D-mannose and its C-5 epim er L-gulose after acid hydrolysis. Both

D-mannose and L-gulose react differently u n d er acid conditions. D-

M annose is re la tive ly acid stab le. L-gulose is m ark ed ly acid

sen sitiv e . In general, aldose con ta in ing p o ly saccharides a re

com pletely hydrolyzed w ith m inim al sugar loss by 1 M H2SD4 a t

100°C for 5 hours (119).

At equ ilib rium D -m annose resid u es favor th e ch a ir

confo rm ation while the L-gulose residues ad o p t th e IC4 ch a ir

conform ation because th e bu lky g ro u p a t C-5 o rien ts to an

eq u a to ria l position . T hese sugars a re n o t locked in to these

conform ations. In so lu tion these m onom ers exist in a dynam ic

equilibrium . This equilibrium allows alternation betw een both ^C \

121

an d IC4 conform ations, as well as the boat, half-chair, an d open

conform ations. The open chain conform ation allows anom erization

around C-l producing both the a an d p anom er. This equilibrium

allows num erous conform ational possibilities for each m onom er

including the possible form ation of 1,6 anhydrohexopyranoses in

acid solutions.

Sugars of the gulo., ido., an d altro . configurations undergo

spontaneous conversion to 1,6 anhydrides in acids. Sugars of the

gluco., m anno., an d galacto. configurations produce very little 1,6

anhydride a t equilibrium and are alm ost com pletely hydrolyzed to

the free aldoses under acid conditions (4, 81). This is because of the

o rien ta tion of the hydroxyl groups a t C-2, C-3, an d C-4. Axially

o rien ted hydroxyl groups, particu larly a t C-3, destabilize anhydride

fo rm a tio n . E quato ria lly o rien ted hyd roxy l groups fav o r the

form ation of 1,6 anhydrides. The equilibrium is d ep en d en t on the

steric arrangem en t of hydroxyl groups which do n o t take p a r t in

th e re a c tio n . Once a 1,6 a n h y d rid e b o n d is fo rm ed , th e

conform ational equilibrium is shifted so the sugar no longer exists

in a dynam ic state. The form ation of a second ring w ith in the

molecule locks it into th a t particular conformation.

D -M annose, w hose fre e e n e rg y fav o rs th e 4 q l c h a ir

conform ation, does no t readily form the 1,6 anhydride bond when it

is in the I-C4 chair conform ation and the hydroxyl group attached to

th e an o m eric ca rb o n is in th e p p o sitio n . A lth o u g h th is

conform ation brings C-6 and the anom eric hydroxyl group into axial

positions around the ring and into proximity, this conform ation also

brings the hydroxyl groups a t C-2 and C-3 into axial positions which

122

destabilize an d inh ib it anhydride form ation. The axial hydroxyl

group a t C-3 in teracts w ith the potential anhydride bond blocking

its form ation. In the p conform ation D-mannose, D-glucose, and D-

galactose all have axial hydroxyl groups a t C-3 and therefore do no t

readily form 1,6 anhydrides.

T h e f re e e n e rg y o f L -gulose fav o rs th e 1Gj. c h a ir

conform ation. At dynam ic equilibrium it can also exist in the 4 c i

conform ation , w hich places the bulky group a t C-5 in an axial

orientation. If the hydroxyl group a t the anom eric carbon is in the

p position the C-6 and the anom eric hydroxyl group come into close

enough proxim ity to allow 1,6 anhydride form ation. An exception

is th a t the anhydride bond form s below the plane of the ring w ith

L-gulose ra th e r than above it. This orientation places the hydroxyl

groups a t C-3, and C-4 in equatorial positions a round the ring and

the hydroxyl group a t C-2 axial. In this position the free energy

favors 1,6 anhydride form ation due to the absence of a destabilizing

axial hydroxyl group a t C-3.

U pon ac id h y d ro ly s is , L-gulose lo ck ed in to a n e w

conform ation . This was seen by the increase in in ten sity of a

second spot on TLC over time. Although the acid hydrolysis product

o f L-gulose was n o t defin itive ly id en tified , the m olecule was

ten tatively iden tified as a 1,6 anhydride because it h ad the same

m igration as 1,6 anhydro p-D-m annopyranose, and 1,6 anhydro p-

D-glucopyranose on TLC. Com puterized m olecular m odeling of 1,6

anhydro p-L-gulopyranose using SYBYL software ind icated th a t the

m olecule has a low total energy, favorable for its p roduction and

stability.

123

Existing m ethods fo r de term ination of the com position of alginates recom m end hydrolysis in 1 M H2 SO4 for 90 m inutes at

100°C (38). Since L-gulose is m ore susceptible to conform ational

m odification u n d er these conditions than is D -m annose, lack of

accounting for gulose destruction has resulted in inaccurate reports

of composition.

Analysis of acid hydrolyzed reduced alginates paralleled those

o b ta in ed w ith the D -m annose and L-gulose m onom ers. W hen

correcting fo r gulose destruction, by extrapolation back to tim e 0 ,

the results of the seaweed alginate analysis correlated exactly with

the pub lished results of 60% m annuronic acid and 40% guluronic

acid as determ ined by the reductive cleavage m ethod of Zeller and

G ray (150). The a lg inates p roduced by th e phy topa thogen ic

p seu d o m o n ad s ap p a re n tly have a h ig h e r co n cen tra tio n of L-

guluronic acid than h ad been previously reported . The rep o rted

com position of the P. syringae ATCC 19304 alginate is g reater than

95% D -m annuronic acid an d less than 5% L-guluronic acid (38).

A fter co rrec tin g fo r th e re la tiv e acid sen sitiv itie s o f each

corresponding neutral sugar, the actual com position of the bacterial

a lg inate was 82% D -m annuronic acid and 18% L-guluronic acid.

This ra tio co rre la tes well to the re p o rte d com position of P.

aeruginosa alginate of 80% D-m annuronic acid and 20% L-guluronic

acid (150).

P. syringae ATCC 19304 produced large am ounts of acetylated

alginate w hen grown on gluconic acid. While p roduction of this

po lym er decreased dram atica lly on subculture in liquid m edia,

p roduction was stable on solid m edia. Maximum p roduction was

124

d ep e n d en t on the pH, tem pera tu re , an d a high carbon/nitrogen

ra tio w ithin the growth m edia. The Mw of the bacteria l polym er

was approxim ately 1.7 tim es larger th an the seaweed coun terpart

with a composition of 82% m annuronic acid and 18% guluronic acid,

and an average of 1.2 acetyl groups p er m onom er. These physical

characteristics allowed for m uch higher solution viscosities a t equal

concentrations, an d ca tion in d u ced gels w ith in c reased w ater

holding capacity. By controlling the degree of acety lation it was

possib le to con tro l th e so lu tion an d gelling p ro p e rtie s of the

polym er to some extent. The polyanionic n a tu re of the polym er

allows for the binding of m any toxic and heavy metals. Ultimately,

it should be possible to create inexpensive designer alginates whose

p ro p ertie s are co n tro lled by the degree of ace ty la tion on the

polymer.

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VITA

Richard David Ashby was b o m in Kansas City, Kansas on May

6, 1962. After graduating from Frem ont High School in Sunnyvale,

California in 1980, he a ttended Brigham Young U niversity w here he

obtained his Bachelor of Science degree in Microbiology in 1987. In

August 1988, he began his g raduate tra in ing a t Louisiana State

University in Baton Rouge, Louisiana.

141

DOCTORAL EXAMINATION AND DISSERTATION REPORT

candidate: Richard David Ashby

Major Field: M ic r o b io lo g y

Title of Dissertation: j h e P ro d u c t io n and C h a r a c t e r i z a t i o n o f A l g i n a t eProduced by Pseudomonas s y r i n q a e

Appro?

f % K._Major Professor and Chaxrman

Dean of the Grac^iate School

EXAMINING COMMITTEE:

y g.-yr , . M■ v/

^.u f H ' L Jkl

j2/^ P u ,

Date of Examination:

June 2 1 , 1994