Journal of Scientitlc & Industrial Research

Vol. 61. April 2002, pp 302-308

Treatment of Pulp and Paper Mill Effluent by 4-Chlorosalicylic Acid­degrading Microbial Comnlunity for Analysis of Toxicity of Seedlings

V Venkat Ramanan and Indu S hekhar Thakur*

Environmental Biotechnology Laboratory , Department of Environmental Sciences, College of Bas ic Sciences and Humanities, Govind Ballabh Pant University of Agriculture and Technology, Pantnagar 263 145, Uttaranchal, India

Received:07 November 200 I; accepted: 04 February 2002

A microbial community utili zing 4-ch lorosalicylic acid as sole source of carbon and energy was enriched in the chemostat. The bacterial community contained four different isolates characterized on the basis of morpholog ica l properties of the colon ies. The iso lates were iden ti fied as Klebsiella sp. (one isolate) and Pseudolllonas sp. (three isolates) . The bacterial community was appl ied for the treatment of pulp and paper mill effluent in fixed film bioreaetor containing soi l. sand, and gravel at the boltom of the reactor for immobili za tion of bacterial biomass. The community exhi bited significant reducti on in colour (80 per cent ). phenol (67 per cent). Chemical Oxygen Demand (COD) (70 per cent ) and lignin content (41 per cent ) 3rter treatment of 15 d. The d iluent treated by microbial community was appl ied for the germin ati on of seeds of addy and wheat and growth of seedling, exh ibi ted sign ili cant reducti on in tox icit y of contaminant presen t in d iluent.

Introduction Environment is affected by the ever-growing

population coupled with technologica l deve lopments in the indu strial and agricultural sector, both havi ng positive growth rate and deve lopment as we ll industrialization has brought in the hazards of environmental polluti on a long with benefits . Owing to the manufacture of goods, liquid waste has been traditi onally discharged on open land or in wate r body is major cause o f vary ing degree of pollution . For many decades, paper was considered important and used by the print media and for other various purposes. The demand fo r wood by pulp and paper indu stry has bright prospects in near future. But unfortunately, it pollutes the environment with its e ffluvnt. Since, we a re harvesting the bene ficial e ffects, it is des irable to manage the e fflu ent let out from pulp industry by way of treatment to the possi ble ex tent. Pulp and paper mill indu stry has been categorized as one of twe nty mos t polluting industries

in Indi a t•

A lthough the phys ical and chemical methods are on the track of treatment , they are not on par with biological treatment because of cos t- ineffect iveness

*Author for correspondence: Phone: 0594433393: Fax: 05944 33473; c-mai l: isth akur@hotmai l.com

and resi dual effec ts. The biol ogica l treatment is known to be effec ti ve in reduc in g the organic load and toxic e ffect s o f kraft mill e ffluent. The practicabi I ity and appl icabi I ity of biotreatment w i II become inte resting onl y when it uses minimum inpu t and indi genous mi croorgani sms ha vin g the feature to be used in the field conditi ons. M ost o f the synthe tic chemical s present in the environme nt are degraded by microorgani sm, others are onl y degraded slowly. Due to continuous presence of mic roorgani sms with recalc itrant compounds, genetic potency develops to degrade the compounds. But these isolates are present in low numbers and a re not effective in bi oremediation. Such type of indi genous microbia l community can be enriched in the presence of inte rmedi ary metabo lites of toxic co mpounds and s ign ificant stra ins wil l be evo lved w ith the process o f adaptation ' . The metabol ic act i vity leads to the change in the structure and fun" tion of the community'A. The pragmatic approach of bi otreatment is analyzed by way of us ing chemostat for enrichment , and bio reactor for testing the re levance of the mi croorgani sms in the treatment process. Mixed communities can overcome the problems faced by 11l0noculture in the environment such as nutriti onal limitati on and toxic substrate.l . The microbial growth in the bioreactor ascerta ins the

.-+ .

RAMANAN el at.: ANALYSIS OF TOXICITY OF SEEDLINGS 303

utilization of compound in the effluent as carbon and energy source. The physicochemical characterization of the industrial effluent is one of the important tools for examining the presence of contaminants and other major chemicals in the effluent. The field survey and toxicity analysis conducted so far derived the conclusion that the crops grown in and around the pulp and paper mill factory were affected at the reproductive stage. Though the vegetative stage of the crops look much greener because of the higher concentration of nitrogen in the pulp and paper mill effluentS. The symptoms expressed by the crop plants paved way for this pre liminary germination test so as to find the impact of effluent on field crops grown in industria l effluent of pulp and paper mill (Lalkua, Nainital, Uttaranchal). Therefore the present study aims at the enrichment of microbial community fer treatment of e ffluent and analysis of detoxification by conducting the germination test for the utilizati on of the treated effluent in the field condition.

Materials and Methods

--1 Source of Seeds

Paddy seeds (Sarj u-S2) and wheat seeds (PBW-373) were procured from the Department of Agronomy, G B Pant University of Agriculture and Technology, Pantnagar, Uttaranchal, India.

Sampling Site

The study was conducted on the effluent released from the Century Pulp and Paper Mill (Ghanshyamdham, La lkua, Nainital , Uttaranchal) . The effluent flows out off factory premises through a kaccha nala for about 15 km, passing through the villages like Bindukhatta, Jawaharnagar and Kichha and join the river Gola, Ram Ganga and finally into Ganga. The effluent samples were collected at the main outlet point, where the combined effluent from pulp and paper mill are being disposed into Kaccha nala , and stored in a refrigerator at 4°C.

Microbial Community

Microbial community was made ava ilable from the Department , which was developed by continuous

+- enrichment of microbial population of a sediment core in the Chemos tat, as described ear lier4

.

Chemostat Culture The microbial community was enriched in g lass

vesse l, dimension 22x 12 cm, served as C hemostat by providing an inlet (10 mUh) fo r the entry of fres h

medium and an outlet (10 mUh) for the removal of spent medium. An inlet was provided for the entry of alkaline solution, the culture medium was maintained at the pH of 7 .2-7.4 . Sterile air was made to pass into the culture vessel by way of using aeration pump and filter. The culture vessel kept on a magnetic stirrer was capable of maintaining the stirring at 200 rpm and temperature at 28 to 30°C. The mineral sa lt medium supplemented with 4-chlorosalicylic acid was used for the enrichment of microbial communit/ . The growth of the community was determined by measuring absorbance (A,6o), The utilization of 4-chlorosalicylic acid was determined by high performance liquid chromatography, as described earlier4

. After continuous enrichment , bacterial cells were removed and cultured on nutrient­agar plates for structural and chemo-taxonomica l analysis of the colonies6

.

Bioreactor

The principle of bioreactor analysis and design was fabricated on laboratory scale using glass column (s ize Sx 100cm). It has an inlet, present in the upper most part of the reactor, and an outlet at lower part of the column. Upper most part "vas provided with three openings for stirring, aeration and inocculation and removal of bacterial community . The s~ irring of the effluent was made poss ibl e by fixing a motor. To the motor, a plastic hollow tube was fixed. The termi na l end of the tube was thickened and it was quite wide enough ( 1.5 cm in diam) capable of sw irling the effluent present in the reactor. The effl uent was loaded with oxygen by pass ing the sterile air through an air pump. The lower portion contained a layer of gravel ( IOOg), sand (SOg) and so il (SOg), a rep lica of natural soi I layer fOf i mmobil ization of microbial community over a plastic plate so that effluent passes long way through the sand substratum to the outlet.

Colour Estimation

The colour content 111 the effluent was measured, as described by Bajpai ef at.7. In thi s method, sample was centrifuged at 10000 rpm for 30 min and pH was adjusted to 7.6 . Absorbance was measured at 465 nm and was transformed into colour unie.

Chemical Oxygen Demand (COD)

COD was determined by dichromate re flux methodS. In thi s method , sample is reflux ed with potassium dichromate and sulphuric acid. and titrated

304 J SCII ND RES VOL 61 APRIL 2002

with fe rrous ammonIUm sulphate, as described In

APHA8.

Phenol Estimation

Total phenol was extrac ted from the efflu ent9. 4-

Aminoantipyrine ' method was used for es timation of phenol, as described in APHA

g

Lignin Content

Lignin , in efflu ent, was estimated by reacti on of effluent with acetic ac id and sodium nitrite and ammonium hydroxide and measuring absorbance at 430 nm 1o

.

Seed Germination Inhibition alld Seed Vigor Index Test

The paddy and wheat seeds were soaked overni ght in test so lu tion (HN01, 0. 1 per cent ). The study was conducted by soaking the seeds with four treatment of 25,50,75 and lOOper cent v/v of the effluent. Seeds were kept on the petri plates containing filter paper soaked with respecti ve test so lution at the rate of 50 seeds per plate in triplicate for each treatment. The plates were incubated at 28°C fo r 48 h and germ inat ion percentage was calcul ated by counting the number of germinated seeds on the sixth day. The effect of the efflu ent on the shoot length and root length were studied by measuring the length . The percentage seed germinati on inhibition and seed vigor index were analysed 1 I. 12. The biomass of the seedlings was measured by fresh weigh of seed lings and after drying seedlings in an oven at 50°C for 48 h.

Results and Discussion

The lignocellul os ic raw materi als and chemi cal s used during manufacture of the pulp and paper are major contaminants in the efflu ent of the paper industry. The chemica ls in the effluent can be removed by biotreatment and bi odegradat ion. In the present in vesti gati on, mi crobial community was applied, instead of monoculture, because of their possible interacti ons in the metabolism of the chemi cal compounds hav ing di versified structural fo rmul ati on. Chloroli gnins, chl orosul phonic ac id , chl orores ins, chl orophenols, and chl orocateco l are major tox ic compounds present in the effluent of pulp and paper mill )1. Chl orinated dibenzo-p-di ox in and dibenzofurans are also formed unintenti onall y in pulp and paper mill efflu ent l4

. 4-chl orosa licy li c ac id is released during co-metabolic turn over of dibenzo-p-

di oxin and dibenzofuran l 4. There are few reports of

microorgani sms degrade dibenzo-p-diox in, dibenzo­furan and related pol ychlorinated phenols . Therefore, mixed microbial community was developed in the presence of 4-chorosalicylic ac id in the chemostat and appl ied for the treatment of effluent.

The enrichment of microbi al community was performed in glass vesse l containing mineral sa lt medium and 4-c hl orosa licy lic ac id (5 mM) as sole carbon and energy source. The growth of the bacteri al cell s was determined by taking absorbance at 560 nm and biomass, and also carbon source utilizati on determined by High Performance Liquid Chromato­graphy (HPLC). It was observed that initi all y there was significant increase in growth of the cell s, but after day 11 the turbidity of the culture medium went down. However, there was increase in turbidity after day 33, which was reached to the maxi mum on day 4 1. The data indicates fluctuati on in uti lizati on of 4-chl orosa licy li c ac id initi all y, but after day 27 the per cent utili zati on of sa li cy li c ac id was inc reased and it reached to 94 per cent on day 4 1 by the mi xed microbial community. During the initi al and subsequent enrichments, bacterial strains utili ze 4-chl orosa licylic ac id as sole carbon source indicated by the increase in turbidity accompanied by utili zati on of carbon source(Fi gure I ).

The liquid culture removed from the glass vessel was diluted, in 10-fold seri al diluti on. The liquid culture (0.1 mL) was spread on nutri ent-agar plates . Four different types of co lonies were observed whi ch were morphologicall y characteri zed, based on di ameter, colour, opac ity, fo rm, elevation, margin , smoothness and tex ture of mi crobi al co lonies. The biochemical tests fo r all the four isolates were performed. The isolates appeared as ye ll ow co lour gave Voges proskauer, S i mmOl1" s citrate, glucose and sucrose fermentati on, catalase, urease and nitrate reducti on test pos it ive. However, indole producti on, methyl red starch hydro lys is, case in utili zation, gelat in liquefaction, lac tose fermentat ion, oxidase test, and hydrogen sulfide producti on tes ts were negati ve. Based on the data of biochemical test and earlier report of Biolog test method. one of the isolate was identi fied as Klebsiella Sp4. Other three isolates indicated Simmon's c itrate. catalase. ox idase, nitrate reducti ons, and Glucose fe rmentati on tes t was pos itive, but indole producti on, methyl red, starch hydrol ys is, Voges proskauer, casein utili zat ion, gelatin liquefac ti on, urease and hydrogen sulph ide

RAMA AN el at .: ANA LYSI S OF TOXICITY OF SEEDLI NGS 305

production tests were negati ve. The isolates were identified as Pseudomonas sp. The data of the study were similar to earlier studies, which indicated the presence of Pseudomonas fluorescens, P. mendocina and P. cichorii identifi ed by Biolog test method,as described earlier4

.

The pulp and paper mill effluent is co loured due to chlrolignin compounds di scharged from the bleaching step of the manufac turing process. The effluent treated by microbial community indicates that significant reducti on in colour was observed for different peri od and is presented in Figure 2a. The colour unit was found to decrease fo r both the control and treated. The per cent colour removal during IS d period for control and treated were 32 and 80 per cent. More than SO per cent of the colour. wa c; removed in the treatment reactor after 3 d of incubati on period and 7 1 per cent after day 7 of the retention peri od.

The li gnin removal by the mi xed community was determined. Data of the study indicated 25 per cent removal of lignin on day 3 which reached to 4 1 per cent after IS d of the retention peri od (Figure 2b). The removal of colom by biological treatment could

be correlated for the minerali zati on of' chl orolignin compounds. The chlorolignins are not onl y huge in amount in pulp and paper industry but also fo und to be recalcitrant and are cause of hi gher chemica l oxygen demand. COO, a major indicator of organi c and inorganic load of the effluent is presented in Figure 2c. The COD removal in control and treated reactor were found to be 38 and 70 per cent, respecti vely. This showed the ab ility of bacterial consortium to reduce the pollutant load of the effl uent by microbi al treatment.

The phenol content of untreated and treated sample at different interva ls was measured. As shown in Figure 2d, percent reducti on of phenol content was 67 per cent on day IS after treatment by bacterial community. However, onl y 22 .5 per cent phenol was removed in control reactor hav in g indigenous populati on of efflu ent. Vari ous chl orinated phenols are present in pulp and paper mill efflu ent which was transformed into hi ghly recalcitrant compound . The polychlorinated phenols are not degraded eas il y. However the reducti on in phenol content may be due to the ability of bacteri al community to cleave t"lenzene ring.

100 I --------------------------------- 0.6

90

« 80 -C/)

U '<:t 70 --o c: 60 o ~ C\) 50

.!::! :g 40

~ 30 I ~ 20 J

10

o

- 0.5

0.4

0.3

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T"--r-"'-+ 0

o 3 5 7 9 11 13 15 17 19 21 23 25 27 30 33 35 37 39 41

Time in Days

E I c: o u;) It) -C\)

CI) ()

c: C\)

.!l ... o en .!l «

Figure I-Growth curve of microbial communi ty and uti l ization of 4-chlorosalicy l ic ac id in mineral salt mcdium : upplemented with 4-chlorosalicyl ic acid as sole carbon source. e - e . absorbance at 560 nm, and per cent uti l iza ti on of 4-chlorosalicyli c ac id (5 mA-n

306 J SCI (NO RES VOL 6 1 APRIL 2002

The effect of the untreated and treated effluent at the concentration of 0, 25, 50. 75 and 100 per cent v/v on seed germination (per cent), seedling length, shoot length, root length, total seedling fresh weight and total seedling dry weigh t was determi ned , and is presented in Table I . The germination of paddy at J 00 per cent untreated and treated effluent are 76 and 92 per cent, respectively, whereas the germination for the control was 99 per cent. The untreated effluent at concentrati on of 25, 50 and 75 per cent has more or less similar effect on germination of paddy. The germination of wheat at 100 per cent untreated and treated effluent concentration are 58 and 8 1 per cent, respectively, whereas germination for the contro l was 90 per cent. The increase in concentrat ion decreases the germination and seed ling length of both paddy

c: .2 '0 :l '0 Q)

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40

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Time in Days

a

45 ,---·----­c: 40 -o 35 -

30 25 -

~ :l '0 Q)

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---I I

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, I

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and wheat. The seed vigour index (SVI) for paddy and wheat tested with treated effluent was more than the SVI tested with untreated effluent. The data indicate that the seed germination inhibit ion in the case of treated effluent was comparati vely lower than the seed germination inhibition ca lculated for untreated effluent. Somashekar et 01.' :1 have studied the effect of various industria l effluent and observed that paper mil l wastewater has inhibitory effect on seed germination of crops . The seedling growth is also affected by toxic effect of ch lorolignin and phenolics, increase in the concentration of calcium and magnesium and heavy meta ls ' 6. The reduction in lignin and total phenol result in significant germination of paddy and wheat seeds.

80 ,..-------------c 0

~ :l '0 Q)

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70

60

50 40

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Time in Days

c

-------------

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___________ ~il L ______ 3 ____ 7 ____ 1_5_~ I Time in Days Time in Days ~---------------

b d

Figure 2- Per cent reduct ion in colour (a). l ignin (b). Chcmical Oxygen [Jemand (COD) (c), and phenol content (d). in pulp and paper mill emuelll after Ireatment with microbi al commuility enriched in chemostat in presence of 4-chlorosali cyl ic ac id. . and cOlllrol

containing indigenous microorganisms only

;..

RAMANAN el al.: ANALYSIS OF TOXICITY OF SEEDLINGS 307

Table 1- Shoot length. root length . total seed ling fres h weight and total seedling dry weight of paddy and wheat treated with various concentration of effl uent (± SE)

Treatment Shoot length Root length Total seed lings fresh Total seedling dry in cm in cm weight in g weight in g

Paddy Wheat Paddy

Effl uent (per cent)

100 2.61 ± 0.028 3.9 0. 11 6 1.7 ±0. 152

75 2.82 ± 0.27 4.51±0.211 2.1 ±0.115

50 3.0 1 ±0.12 5.24 ± 0.1 54 2.3±0.1 2

25 3.6 1 ± 0.27 5.56±0. 18 2.8 ± 0. 14

Treated effluent (per cent)

100 5.7±0.176 6.1 2 ± 0.11 4.8 ± 0.1 2

75 5.9 ± 0.017 6.24 ± 0. 13 5.1 ± 0.08

50 6.1 ±O.!::! 6.41 ±O. IO 5.3 ±0. 13

25 6.5 ± 0.08 6.61 ± 0. 13 5.6±0.15

Cont rol (per ccnt)

6.9 ± 0.08 7.15±0.02 5.8 ±0.15

The shoot length of paddy at 100 per cent untreated and treated effluent concentration were 2.6 and 5.7 cm, respecti ve ly, whereas for the control the shoot length was 6.9 cm. However the shoot lengths of wheat at 100 per cent untreated and treated effluent were 3.96 and 6.12 cm, respec ti ve ly. However, for the control the shoot length was 7. 15 cm. There is a significant difference between the affect of treated effluent on the shoot length. The seedling weight (fresh and dry) for paddy and wheat was ca lcu lated and it was observed that ch:mge in seed ling weight was affected by the pulp and paper mill effluent. The results from germination and growth of seedlings conducted with the treated e fflu ent, showed beneficial effects of the microbial community in the treatment of pulp and paper mill effluent.

Acknowledgement

The authors are grateful to Department of Biotechnology, Government of India. for providing f inancia l support in the form of research project.

References MinislIT of Planning alld Prog ramme ImplemenJalion. DepJrtment of Statis tics. Central Statis tical Organisation. Compendium of En vironmental Statisti cs. 1998. 21 R.

Wheat Paddy Wheat Paddy Wheat

3.4 ± 0. 12 1.6 ± 0.103 4.39 ± 0. 109 0.83 ± 0.035 1.86 ± 0.03

3.9 1 ±0.1 9 2.0 ± 0.0 18 4.76±0. 11 3 0.97 ±0. 17 1.96 ± 0.02

4.6 ± 0.06 2.01 ±0.0 17 5. 1 ± 0.086 1.02 ± 0.038 2.01 ± 0.049

4.9 ± 0.27 2.05±0.0 15 5.48±0.1 1.08 ± 0.0 15 2.03 ± 0.069

5. 1 ±0.2 1 2.13 ±0.05 5.79 ± 0.09 1.1 1 ± 0.04 2. 11 ± 0.01

5.3 ± 0.26 2.35 ± 0.28 5.9 1 ± 0. 11 1.1 2 ± 0.02 2.22 ± 0.02

6.8 ± 0.25 2.49 ± 0.06 6. 19 ±0.06 1.22 ± 0.03 2.35 ± 0.03

7.2 ± 0.32 2.62 ± 0.29 6.42 ± 0.11 1.26 ± 0.02 2.4 ± 0.04

8.0 ± 0.29 2.65 ± 0.02 6.58 ± 0.28 1.3 ± 0.1 0 2.5 ± 0.04

2 Singh B S. Marwaha S S & Kh an na P K, Characteristi cs of pulp and paper mill eriluents , } In d Pol/III COlllrol, 12 (1996) 163.

3 Senior E, Bull A T & Sl ater J H. Enzyme evolution in a microbi al community growing on the herbicide dalapoll , Natllre, 263 (1976) 479.

4 Thakur I S, Structural and functional charac teri zat ion of 4-ch lorosalicylic acid degrading, stab le mi crobial community in chemustat, World} Mir.robiol Biotechllol. 11 ( 1995) 643 .

5 Dutta S K & Boissya C L. Effect of paper mill effluent on germination of rice seed (Oryza sati va L. var Masuri) and growth behaviour of it s secdlings, } II/ d POI/lit COlllml . 13 ( 1997) 4 1.

6 Pall croni N J. PselldOmOI/ClS, in B('I"gey-s lIIal/llal of systematic BaC/eriolog.\'. edited by N R Kreig and J G Holt (Willi am and Wilkins . Balt imore) 1984. 141 .

7 Bajpai P. Mehra A & Baj pai P K. Deco louri sation of kraft bleach plant diluent with white rot fungu s. Trameles versicolor. Pmc Biochelll . 2/l ( 1993) 377.

8 APHA. SIC/lldard mel hoc", for Ihe eXCIm il/ation of \Wller alld H'aSlell'OI.: r. 14'h cd (American Public Health AssocIation. Washington. D C) 1989.

9 Thakur I S, Verma P K & Upad haY:l K C, In volvement of pl asmid in degradati on of Pentachl orophenol hy Pseudomonas sp. from a chemos tat. Biochl'm 13iophrs Res COIIIIIIIIII. 286 (200 I ) 109.

10 Pearl I A & Benson H K. The determinat ion of li gllln in sulphide pulping li quor. Paper Trade .l. 111 ( 1940) 35 .

308 J SCII ND RES VOL 6 1 APR IL 2002

II Abdul-S ak i A A & Anderson J D, Vigour determination in soyabean by multipl e criteri a, Crop Sci, 13 ( 1973) 630.

12 Rao J V S, Rama M R K & Samba M S, Allelopathic effect of some weed of vegetable crops on the germin ation anu earl y seedling growth of vegetab le crops on the germin ati on anu early seedling growth of bajra, Trap Ecol, 20 (1979) S.

13 Somashekhar R K, Gowda M T G, Shettigar S C N & Srinath K P, Effect of industrial emuents on crop plants, Indiall J Enviroll !-Iealth , 26 (1984) 136.

14 Wittich R M, Wilkes H, Sinwell V, Franke W & Fortnagel P. Metaboli sms of dibenzo-p-dioxin by Sphingomonas sp. strain RW I, Appl Ell viroll Microbiol, 58 ( 1992) 1005.

IS Harms H, Wil kes H, Sinnwell V, Witti ch R M, Figge K, Franke W & Fortnagel P, Transformati on of 3-chlorodibenzofuran by Pseudomol/as sp HH69 , FEMS Microbiol Lett , 81 (199 1) 25.

16 Rajamani G & Oblisami G, Effect of paper factory ertluent on soil and crop plant , II/dial/ J Ellviro!l !-Iealth , 21 (1979) 121 .