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New Biotechnology �Volume 30, Number 6 � September 2013 RESEARCH PAPER

Effect of cell immobilization on theproduction of 1,3-propanediolMine Gungormusler, Cagdas Gonen and Nuri Azbar

Bioengineering Department, Faculty of Engineering, Ege University, 35100 Bornova, Izmir, Turkey

Immobilized cultures of locally isolated Klebsiella pneumoniae (GenBank no: 27F HM063413) were

employed in the continuous production of the high value added biomonomer, 1,3-propanediol from

waste glycerol. The effect of hydraulic retention time (HRT) was tested by increasing the dilution rate

gradually. Three different immobilization materials (stainless steel wire, glass raschig ring and

Vukopor1) were tested. The highest productivity was reported with the reactor filled with stainless steel

wire as 4.8 g/(L hours) and the highest 1,3-propanediol concentration was 17.9 g/L when glass raschig

rings were used as the packing material with the HRTs of 0.5 hours and 1.5 hours, respectively.

Compared to the suspended culture system 1,3-propanediol production was more resistant to shorter

hydraulic retention times that leads to higher 1,3-PDO productivities. All three of the materials are good

candidates for immobilization purpose; however, stainless steel wire and Vukopor1 are better support

materials in terms of productivities. The results reported in this study revealed that continuous

fermentation in a packed-bed bioreactor system is a suitable method to enhance 1,3-propanediol

production.

IntroductionRecently, considering the escalating global energy and environ-

mental problems which have stimulated scientists worldwide to

develop methods for substituting the refineries with biorefineries,

bio-conversion of the by-product of biodiesel production raw

glycerol is reasonable [1]. The production of the high-value added

bio-monomer 1,3-propanediol (1,3-PDO) by wild strains is depen-

dent on the bio-conversion of glycerol. Raw and pure glycerol were

both tested and compared to understand whether raw glycerol has

inhibitory effects on Clostridium butyricum [2] and Klebsiella pneu-

moniae [3]. Ma et al. [3] reported that there were slight differences

on the conversion rate of glycerol to 1,3-PDO (50.1% for raw and

53.5%, w/w, for pure glycerol). The usability of raw glycerol for this

bio-conversion makes this process economically friendly. Waste

glycerol is a by-product of the biodiesel process that contains

different percentages of glycerol, free fatty acids, soap, moisture

impurities and volatiles, methanol and sediment. During

the production of biodiesel, a large amount of raw glycerol is

Corresponding author: Azbar, N. (nuriazbar@gmail.com)

1871-6784/$ - see front matter � 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.nbt.2013.0

generated as a by-product in the range of 10% (w/v) of biodiesel

production [4].

1,3-PDO is mainly used with terephthalic acid to polymerize

polytrimethylene terephthlate which can be used in fibre, auto-

mobile, carpet and apparel industries [1]. Furthermore, because it

is biodegradable, it has higher light stability and solubility [5,6], it

can be used as solvents and it also can be formulated into lami-

nates, solvents, mouldings, adhesives, resins, detergents, cos-

metics, deodorants and other end uses [7]. When considering

these numerous applications of 1,3-PDO, the need for an eco-

nomical production of this high-value bio-based monomer is

obvious. Because immobilization provides several advantages

such as; easier downstream processing, reusability of the bioca-

talyst, operating at high reaction rates that leads to high produc-

tivities, it is an important approach especially for industrial

purposes [1]. Biotechnologically produced 1,3-PDO was mostly

produced in suspended cultures [1,8–10] resulting in higher pro-

cess volumes [11]. By contrast, there are very limited studies with

immobilized cell systems for 1,3-PDO production in the literature

[10,12–18].

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RESEARCH PAPER New Biotechnology � Volume 30, Number 6 � September 2013

FIGURE 1

Sample photographs of VukoporW (a), glass raschig ring (b) and stainless

steel wire (c).

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The continuous fermentation process with K. pneumoniae in

which immobilized cell bioreactors are used have been found to

be resistant to washout at high dilution rates [16]. K. pneumoniae is a

facultative anaerobic mesophilic bacteria mostly used in 1,3-PDO

studies. In fact, this species was also used with immobilized cultures.

However, gel entrapment [13,14] was preferred unlike the method

used in the proposed study. The objective of this work was to

investigate the effects of hydraulic retention time (HRT) (16, 12,

8, 6, 4, 2, 1 and 0.5 hours) and alternative immobilization materials

(stainless steel wire, glass raschig ring and VUK) on cell growth, and

1,3-PDO productivity using packed-bed bioreactor systems.

The current study is an attempt to find out alternative immo-

bilization materials (VUK, glass raschig ring and stainless steel

wire) and enhance the volumetric productivity of 1,3-PDO from

crude glycerol using immobilized cells of K. pneumoniae by upflow

packed-bed bioreactors.

Materials and methodsK. pneumoniae (GenBank no: 27F HM063413) was provided from

the Faculty of Pharmacy, University of Ege, Izmir, Turkey. The

microorganism was activated from agar cultures in Nutrient Broth

(NB) and then incubated at 378C for 6 hours. First activation of the

microorganisms was with an initial inoculum ratio of 1% then this

ratio was increased to 10% before the fermentation process. The

culture media used for the studies were prepared as reported in

Gungormusler et al. [16]. Initial substrate concentration was 40 g/L

of waste glycerol. 1,3-PDO, 2,3-butanediol (2,3-BD), lactic acid,

acetic acid, succinic acid, and ethanol measurements were carried

out as reported in Casali et al. [17]. Total suspended solids (TSS)

measurements were carried out in accordance with standard meth-

ods [19].

VUK (Lanik, Boskovice, CZ), glass raschig ring (Ege University

Glass Atelier, Izmir, TR) and stainless steel wire 316L (Ultra Metal,

Izmir, TR) (Fig. 1) were used as the immobilization supports in the

packed-bed column bioreactors having a height of 30 cm, internal

diameter of 4.5 cm and a total volume of 280 mL [17]. The working

volumes of each bioreactors were 240 mL, 230 mL and 260 mL for

Vukopor1 (w = 0.75 cm, h = 1.1 cm), raschig ring (w = 1.1 cm,

h = 1.0 cm) and steel wire (d = 7.99 g/cm3), respectively. All the

immobilization materials were washed with distilled water and

dried overnight at 378C before use. The temperature in the bior-

eactors was kept at 378C using heating blankets. pH was initially

adjusted to 7.0 using 2 M NaOH and monitored continuously but

not controlled during fermentation. All reactors were kept at

37 � 18C for 504 hours.

Each packed bed bioreactor including immobilization materials

were sterilized via autoclave (1218C, 1 atm, 30 min) before use. The

reactors were allowed to cool down and then inoculated with 1%

(v/v) of stock culture of K. pneumoniae together with sterile nutri-

ent broth medium. To immobilize the microorganisms to the

materials, the stock inoculum solution was continuously recycled

through the packed-bed bioreactor under sterile conditions for

about a week (HRT = 6.25 hours). Following this, the continuous

production of 1,3-PDO was initialized (at 101th hour). The con-

tinuous process were performed at HRTs of 16, 12, 8, 6, 4, 2, 1 and

0.5 hours that corresponds to dilution rates 0.06, 0.08, 0.12, 0.16,

0.25 0.50, 1 and 2 hour�1, respectively. At each dilution rate,

steady states were obtained after six cycles.

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New Biotechnology �Volume 30, Number 6 � September 2013 RESEARCH PAPER

58.5a

b

c

%

78.6%

70.3%

FIGURE 2

Cell immobilization ratios (%) (white area) and suspended cells (striped area)

in VUK (a), glass raschig ring (b) and stainless steel wire (c) packed-bedbioreactors.

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Results and discussionThe remarkable advantages of immobilization in biotechnology

motivated scientists to search for suitable methods to use these

benefits [13]. Immobilization may be achieved both by attachment

of the microorganisms to the inert material and by entrapment in

gel polymers. Despite the fact that attachment of cells to an inert

material is a cost reducing way for immobilization, gel entrapment

was also preferred for this fermentation [13,14]. The advantages of

immobilization by attachment include stability, reusability, con-

venience in continuous operation, and higher volumetric produc-

tivity [20]. Ceramic and porous glass materials have been

commonly used as immobilization supports for various biotech-

nological applications [21,22]. Their porous and hydrophilic char-

acteristics create a suitable environment for immobilization, and

they are especially suitable for microbial colonization. However,

stainless steel wire has never been studied for immobilization

purposes. In a recent review of Kaur et al. [1] the biotechnological

production technologies of 1,3-PDO particularly with respect to

bioprocess engineering methods were thoroughly discussed and

compared. The authors suggest further investigation for immobi-

lized cell cultures apart from fed-batch processes, continuous

processes with/without cell recycling and mixed culture systems.

This paper suggests alternative packing materials for upflow

packed-bed systems. The most successful immobilization was

observed in the bioreactor which was filled with glass raschig rings

(78%). The degree of cell immobilization was measured via the

biomass suspended and attached on the immobilization materials.

The results indicated that successful immobilization between the

range of 70–78% was achieved for ceramic and glass materials;

interestingly, a lower percentage (58%) of immobilization was

observed with the bioreactor filled with stainless steel wire (Fig. 2).

As mentioned before, most of the literature reports on 1,3-PDO

production are based on suspended cell systems. When consider-

ing the maximum 1,3-PDO concentrations obtained from sus-

pended studies, a study with an integrated fed-batch system was

reported to have the highest value of 1,3-PDO (87 g/L) which,

however, had a low value of productivity (1.9 g/(L hours)) [23]. By

contrast, Deckwer et al. [24] presented the data for continuous

fermentation and reported that this process had higher produc-

tivity up to 8.8 g/L at HRT of 4 hours, but the final concentration of

1,3-PDO was lower than fed-batch systems.

Maximum concentrations of 1,3-PDO were achieved at a HRT of

12 hours for the bioreactors filled with stainless steel wire and glass

raschig ring (13 g/L for each) and the highest value was 17.9 g/L

when the bioreactor filled with VUK was employed at an HRT of

4 hours (Fig. 3). In accordance with the 1,3-PDO concentrations

maximum glycerol consumption percentage was 71% when cera-

mic cubes were used. In both too low and too high HRT values,

glycerol could not be consumed and this trend explains the low

concentrations of 1,3-PDO in fermentation broth. Jun et al. [10]

reported that the fed-batch fermentation carried out with immo-

bilized cells resulted in higher concentrations of 1,3-PDO (71.1 g/L);

however, productivities after five cycles (1.51 g/L/hours) were four

times lower compared to the proposed study. In another study with

a two-phased immobilization process Wong et al. [18] proved that

using immobilized cells of Klebsiella sp. HE-operational stability and

reusability of the cells were improved and 1,3-PDO concentrations

were doubled.

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RESEARCH PAPER New Biotechnology � Volume 30, Number 6 � September 2013

HRT (h)

0 2 4 6 8 10 12 14 16

1,3

-PD

O C

on

ce

ntr

atio

n (

g/L

)

0

5

10

15

20

FIGURE 3

1,3-PDO concentrations (g/L) at different HRTs (average values of triplicates).

VUK (filled circle), glass raschig ring (open circle), stainless steel wire (filledtriangle).

HRT (h)

0 2 4 6 8 10 12 14 16

1,3

-PD

O V

olu

metr

ic P

roductivity (

g/L

/h)

0

1

2

3

4

5

6

FIGURE 4

1,3-PDO productivities (g/L/hours) at different HRTs (average values of

triplicates). VUK (filled circle), glass raschig ring (open circle), stainless steel

wire (filled triangle).

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Figure 4 shows the 1,3-PDO productivity under various HRT

conditions for each bioreactor. Higher 1,3-PDO concentrations

were observed with longer HRTs, and by contrast higher produc-

tivities were observed at lower HRT conditions (ANOVA, Tukey’s

test a = 0.05, P < 0.05). A combined process with Zygosacharomyces

rouxii and K. pneumoniae first to produce glycerol and then to

convert into 1,3-PDO was performed and a fermentation period of

30 hours in fed-batch cultures was conducted [3]; however, our

TABLE 1

The by-product concentrations and pH values of the immobilized b

Immobilization material HRT (hours) HSuc (g/L) HLac (g/L

VukoporW 16 0.3 � 0.2 0.4 � 0.6

12 0.3 � 0.2 3.7 � 0.6

8 0.1 � 0.1 3.5 � 0.9

6 0.5 � 0.1 1.6 � 0.5

4 0.3 � 0.1 0.6 � 0.6

2 0.4 � 0.1 0.8 � 0.3

1 0.3 � 0 0.2 � 0.4

0.5 0.3 � 0.1 0

Raschig ring 16 0.3 � 0.3 0.3 � 0.5

12 0.3 � 0 1.6 � 0.1

8 0.3 � 0.3 1.8 � 0.2

6 0.3 � 0.1 2.5 � 0.7

4 0.3 � 0.1 1.5 � 0.2

2 0.3 � 0 0.7 � 0.1

1 0.1 � 0 0.2 � 0.4

0.5 0.3 � 0.2 0

Stainless steel wire 16 0.3 � 0.5 0.3 � 0.4

12 0.2 � 0.3 3.3 � 0.7

8 0.5 � 0.3 3.2 � 0.4

6 0.4 � 0.1 1.8 � 0.2

4 0.4 � 0.2 1.3 � 0.2

2 0.4 � 0.2 1.2 � 0.1

1 0.2 � 0.1 0.4 � 0.4

0.5 0.2 � 0.2 0

HSuc, succinic acid; HAC, acetic acid; HLac, lactic acid; 2,3-BD, 2,3-butanediol; EtOH, ethanol.

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results shorten the fermentation period 60 times with 4.3 times

higher productivities than reported in the literature. In contrary to

the immobilization method of the proposed paper the entrapment

study using NaCS/PDMDAAC microcapsules was carried out with

K. pneumoniae [14], and it was reported that 14.8 g/L of 1,3-PDO

was produced with a productivity of 2.96 g/(L hours) in fed-batch

fermentations. The locally isolated K. pneumoniae in this report

obtained 1.6 times higher productivity values that can be

iroeactors operated under continuous conditions

) HAc (g/L) 2,3-BD (g/L) EtOH (g/L) pH

0.6 � 0.5 0.1 � 0.1 0 7.9 � 0

1.8 � 0.5 1.3 � 1.1 1.2 � 0.3 6.5 � 0.61.9 � 0.8 2.0 � 0.5 1.3 � 1.1 6.5 � 0.3

2.4 � 0.4 3.2 � 0.3 0.3 � 0 6.8 � 0.3

2.3 � 0.5 2.3 � 0.6 0.1 � 0.2 6.7 � 0.31.9 � 0.8 2.4 � 0.7 0 6.7 � 0.5

0.9 � 0.9 2.0 � 0.1 0 7.3 � 0.6

0 1.1 � 1.9 0 8.0 � 0.6

0.3 � 0.5 0.1 � 0.2 0 8.8 � 0.6

1.9 � 0.8 2.6 � 1.4 0.9 � 0.1 6.3 � 0.62.4 � 0.1 1.8 � 0.5 0.8 � 0.9 6.5 � 0

1.8 � 0.6 2.2 � 0.5 0.6 � 0.3 6.5 � 0.3

2.1 � 0.3 2.4 � 0.3 0.3 � 0.2 6.7 � 0.52.0 � 0.3 2.4 � 0.5 0.1 � 0.1 7.0 � 0.5

0.4 � 0.7 3.1 � 0.3 0 7.3 � 0.3

0 1.3 � 2.2 0 7.7 � 0.3

0.8 � 0.8 1.5 � 2.6 0 10 � 0.6

1.7 � 0.7 1.9 � 0.7 0.6 � 0.5 6.0 � 0.1.9 � 0.1 1.8 � 0.1 0.7 � 0.2 6.2 � 0.3

2.4 � 0.2 3.1 � 0.4 0.4 � 0.4 6.7 � 0.3

2.8 � 0.2 2.7 � 0.2 0.9 � 0.8 6.7 � 0.32.1 � 0.1 2.6 � 0.4 0.4 � 0.1 6.8 � 0.3

0.6 � 0.4 3.0 � 0.1 0 7.3 � 0.6

0 0 0 8.3 � 0.8

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explained by the fact that immobilization by attachment is more

stable when compared with the entrapment of cells to the micro-

capsules.

As a result of the immobilization process, the biomass concen-

tration increases on the attached material that increase the reduc-

tion of glycerol and 1,3-PDO yield, consequently [1]. This higher

concentration may eliminate the need for a precultivation. The

biomass calculations for VUK proved that a concentration of

1.2 g/L TSS can be increased up to 7.7 g/L TSS via immobilization

that increased the glycerol consumption percentage from 14%

to 75%.

The main by-products produced out of the reduction reaction

of glycerol are succinic acid, acetic acid, lactic acid, 2,3-BD and

ethanol [25]. To increase the amount of 1,3-PDO production,

the microorganisms should be forced to produce less amounts of

above-mentioned by-products. The data obtained from the

experiments were summarized in Table 1. According to the table

succinic acid was the by-product with the lowest concentrations

(up to 0.5 g/L for VUK) and lactic acid was the by-product which

had the highest concentrations (up to 3.7 g/L for VUK) among

the other by-products. Ethanol, acetic acid and 2,3-BD were also

produced up to 1.75 g/L for VUK, 2.8 g/L for stainless steel wire

and 3.2 g/L for VUK, respectively. The effects of acetate, tem-

perature and vitamin B12 concentrations on 1,3-PDO produc-

tion by Halanaerobium saccharolyticum subsp. saccharolyticum

were studied by Kivisto et al. [26] and the results showed that

the presence of acetate was only negatively effective on 1,3-PDO

production when the concentrations were between 29 and 58 g/L;

by contrast, temperature (between the range of 30 and 408C) did not

have any significant effects on the production; however, H2 was

produced in higher amounts when the temperature was set to 378C;

in addition, vitamin B12 addition (64 mg/L) increased the growth

and thus the production of 1,3-PDO. Nevertheless, 1,3-PDO produc-

tion is only vitamin B12 dependent when the enzyme glycerol

dehydratase is not included in the microorganism. Another

approach with

Klebsiella oxytoca was obtained under conditions without N2 [27]

and 2,3-BD was not produced; in addition, the production yields

were achieved up to 47% (w/w). In comparison, the highest yields

over consumed substrate were obtained for 1 hour of HRT as: 40%,

59% and 81%, for glass raschig ring, VUK and stainless steel wire,

respectively.

ConclusionsIt can be concluded that a novel immobilization bioprocess by

locally isolated K. pneumoniae (GenBank no: 27F HM063413)

was achieved. Among the experimented HRTs a HRT of

0.5 hours is found to be best one in terms of volumetric produc-

tion rates. However, 1,3-PDO concentrations reached the high-

est values when a HRT of 12 hours was used. Furthermore, cell

immobilization had obvious benefits especially for resistance of

the microorganisms to extreme conditions. Immobilization

with the alternative inert materials has proved to be an eco-

nomical and easy method to produce 1,3-PDO because this

process shortens the duration of production. All three materials

are good candidates for immobilization, nevertheless, glass

raschig ring is a better support material than stainless steel wire

and VUK in terms of immobilization ratios. However, VUK was

more successful in terms of glycerol conversion percentages

(50.4%). VUK may be considered to be the best material because

the productivities were comparable and efficient utilization

of raw glycerol could lead to a more economical production

of 1,3-PDO.

AcknowledgementsThe authors wish to thank TUBITAK-CAYDAG under the grant no

109Y150 for the financial support of this study. The authors also

wish to thank to Silvia Casali for technical assistance and to Dr.

Lorenzo Bertin for providing the Vukopor1 material. The data

presented in this article were produced within the projects above;

however it is only the authors of this article who are responsible for

the results and discussions made herein.

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