International Journal of Hydrogen Energy 32 (2007) 3284–3292www.elsevier.com/locate/ijhydene

Enhancing biohydrogen production from chemical wastewater treatment inanaerobic sequencing batch biofilm reactor (AnSBBR) by bioaugmentingwith selectively enriched kanamycin resistant anaerobic mixed consortia�

S. Venkata Mohan∗, G. Mohanakrishna, S. Veer Raghavulu, P.N. SarmaBioengineering and Environmental Centre, Indian Institute of Chemical Technology, Hyderabad 500 007, India

Received 1 December 2006; received in revised form 5 April 2007; accepted 18 April 2007Available online 15 June 2007

Abstract

The basic aim of this study was to investigate the feasibility of bioaugmentation strategy in the process of enhancing biohydrogen (H2)production from chemical wastewater treatment (organic loading rate (OLR)—6.3 kg COD/m3-day) in anaerobic sequencing batch biofilmreactor (AnSBBR) operated at room temperature (28±2 ◦C) under acidophilic microenvironment (pH 6) with a total cycle period of 24 h. Parentaugmented inoculum (kanamycin resistant) was acquired from an operating upflow anaerobic sludge blanket (UASB) reactor treating chemicalwastewater and subjected to selective enrichment by applying repetitive/cyclic pre-treatment methods [altering between heat-shock treatment(100 ◦C; 2 h) and acid treatment (pH 3; 24 h)] to eliminate non-spore forming bacteria and to inhibit the growth of methanogenic bacteria(MB). Experimental data revealed the positive influence of bioaugmentation strategy on the overall H2 production. Specific H2 productionalmost doubled after augmentation from 0.297 to 0.483 mol H2/kg CODR-day. Chemical wastewater acted as primary carbon source in themetabolic reactions involving molecular H2 generation leading to substrate degradation. The augmented culture persisted in the system till thetermination of the experiments. The survival and retention of the augmented inoculum and its positive effect on process enhancement may beattributed to the adopted reactor configuration and operating conditions. Scanning electron microscope (SEM) images documented the selectiveenrichment of morphologically similar group of bacteria capable of producing H2 under acidophilic conditions in anaerobic microenvironment.This depicted work corroborated successful application of bioaugmentation strategy to improve H2 production rate from anaerobic chemicalwastewater treatment.� 2007 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved.

Keywords: Biohydrogen; Bioaugmentation; Wastewater treatment; Anaerobic sequencing batch biofilm reactor (AnSBBR); Mixed anaerobic culture; Acidophilic

1. Introduction

Energy is vital to global prosperity, yet dependence on fossilfuels as primary energy source has been contributing to globalclimatic change and environmental degradation [1]. Hydrogen(H2) gas had been deemed to be the green fuel of the future,and it was reported that a H2 fuel-based economy would be lesspolluting than a fossil fuel-based economy. H2 is consideredto be the promising green alternative to fossil fuels as a sus-tainable energy source with minimal or zero use of hydrocar-bons along with high-energy yield (122 kJ/g) [2]. H2 is of high

� IICT Communication No. 061201.∗ Corresponding author.

E-mail addresses: vmohan_s@yahoo.com, svmohan@iictnet.org(S. Venkata Mohan).

0360-3199/$ - see front matter � 2007 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved.doi:10.1016/j.ijhydene.2007.04.043

commercial value as it can be used as a raw material in avariety of industrial applications [3], as well as a clean energysource for fuel cells [4]. H2 production from biological route isone of the alternative methods which is gaining importance inrecent times. Biological H2 production processes can be clas-sified as biophotolysis of water using algae and cyanobacteria,photodecomposition of organic compounds by photosyntheticbacteria and fermentative production from organic compounds[5–15]. These processes were mostly operated at ambient tem-peratures and pressures, which were less energy intensive andmore environmental friendly and open new avenues for the uti-lization of renewable and inexhaustible energy sources [5,10].Biological H2 production is a promising and sustainable pro-cess where renewable organic waste can be used as energygenerating source. Availability of huge quantities of wastewater

S. Venkata Mohan et al. / International Journal of Hydrogen Energy 32 (2007) 3284–3292 3285

coupled with anaerobic treatment can be considered to be auseful methodology to reduce pollution load along with H2generation. At present a practical and efficient H2 generationprocess is a growing concern among the research fraternity[10] and in this direction various strategies have been reported[16–19].

Bioaugmentation is a process of application of indigenous orallochthonous wild type or genetically modified organisms topolluted hazardous waste sites or bioreactors in order to accel-erate the removal of undesired compounds [20]. Bioaugmen-tation was generally used to improve the start up of a reactor[21], to enhance reactor performance [22–25], to protect theexisting microbial community against adverse effects [25–31],or to compensate for organic or hydraulic overloading [32].The success of bioaugmentation strategy was more evidentwhen applied to contaminated soils [33–42]. In some cases,bioaugmented species often failed to compete with indigenouspopulation and thus failed to survive [43–46]. In some casesprocess inhibition was also reported [47,48]. Much work wasnot reported so far on the application of bioaugmentation asstrategy to enhance biological H2 production employing selec-tively enriched anaerobic mixed culture.

The aim of the present study was therefore to investigate thebioaugmentation strategy for enhancing H2 production usingselective enriched anaerobic mixed consortia. The investigationwas also focused on to study the feasibility of H2 production inconjugation with wastewater treatment in anaerobic sequencingbatch biofilm reactor (AnSBBR).

2. Experimental methodology

2.1. Enrichment of bioaugmented culture

Anaerobic mixed consortia acquired from laboratory scaleupflow anaerobic sludge blanket (UASB) reactor operating withchemical wastewater for the past five years was used as parentaugmented inocula. Dewatered inoculum sample from UASBafter washing in phosphate buffer (50 mM; pH 7.4) was incu-bated in Wilkins–Chalgren anaerobic broth [(g/l) casein enzy-matic hydrolase—10, peptic digest of animal tissue—10, yeastextract—5, dextrose—1, sodium chloride—5, L-alginate—1,sodium pyruvate—1, hemin—0.005 and menadione—0.0005]overnight at 30 ◦C. The resulting harvested mixed culture wassubjected to repetitive pre-treatment sequences altering be-tween heat-shock treatment (100 ◦C, 2 h) and acid treatment(pH 3 adjusted with orthophosphoric acid, 24 h) four timesto eliminate the non-spore forming anaerobic bacteria as wellas to inhibit the growth of methanogenic bacteria (MB) tofacilitate enrichment of spore forming acidogenic H2 pro-ducing microflora. The resulting enriched mixed culture wasfurther selectively enriched (24 h; 30 ◦C) for antibiotic resis-tant in Wilkins–Chalgren anaerobic broth supplemented withkanamycin (10 mg/l) for five times. The resulting broth waswashed twice in phosphate buffer and incubated in 100 mlof chemical wastewater at 30 ◦C overnight and was used foraugmentation through feed (0.59 g VSS).

2.2. Wastewater composition

Composite chemical wastewater collected from a com-mon effluent treatment plant (CETP), Hyderabad, Indiawas used as substrate for H2 production. Characteristi-cally (pH 7.8; TDS—11,000 mg/l, oil and grease—14 mg/l;chemical oxygen demand (COD)—7680 mg/l; biologicaloxygen demand (BOD5)—2350 mg/l, sulfates—1740 mg/l;phosphates—360 mg/l and total nitrogen—125 mg/l), thewastewater can be considered as complex in nature due to itscomposite nature, low-biodegradability [BOD5/COD ∼ 0.3]and high sulfate concentration. The wastewater was compos-ite/combined chemical wastewater collected from about 100chemical processing industries aggregated from bulk drugs,chemical intermediates, dye and dye intermediates, pharma-ceuticals, pesticides and various chemical process units.

2.3. Reactor design and operation

Bench scale anaerobic biofilm reactor (AnSBBR) was fab-ricated in the laboratory using ‘perplex’ material with a totaldesigned volume of 4 l (liquid volume 2 l) and gas holdingcapacity of 0.35 l (L/D ratio∼6) (Fig. 1). Inert stone chips

PPR

PPF

AnSBBR

FL

RL

DL

FST

GCT

GL

DST

T

T

T

HP

Fig. 1. Schematic details of AnSBBR (HP—hydrogen monitoringprobe; PPF—feeding peristaltic pump; PPR—recirculation peristalticpump; FL—feed line; RL—recirculation line; GL—gas collection line;T—preprogrammed timer; PPD—decanting peristaltic pump; FST—feed stor-age tank; GCT—gas collection container; DST—decant storage tank).

3286 S. Venkata Mohan et al. / International Journal of Hydrogen Energy 32 (2007) 3284–3292

(0.02 cm × 0.05 cm; void ratio ∼0.54) were used as fixedbed packing material to support the biofilm growth.Reactor was operated in the upflow mode at room tem-perature (28 ± 2 ◦C) employing sequencing batch modeoperation consisting of a total cycle period of 24 h [FILLphase—15 min, REACT (anaerobic)—23 h, SETTLE—30 minand DECANT—15 min]. At the beginning of each cycle, im-mediately after withdrawal/decanting, a pre-defined volume(1.4 l) was fed to the reactor during FILL phase. Through-out the REACT phase the reactor volume was continuouslymixed by employing recirculation [recirculation volume tofeed volume ratio of 3] to achieve homogeneous and uniformdistribution of the substrate as well as augmented consortiaalong the reactor depth. Peristaltic pumps controlled by pre-programmed electronic timer (ETTS, Germany) were used toregulate the FEED, recirculation, and DECANT operations.The controller was programmed to operate on a repeating 24 hcycle with a sub-program and output dedicated to the oper-ation of each controllable element. The reactor was initiallyfed with designed synthetic wastewater [(g/l) glucose—3.0,NH4Cl—0.5, KH2PO4—0.25, K2HPO4—0.25, MgCl2·6H2O—0.3, FeCl3—0.025, NiSO4—0.016, CoCl2—0.025,ZnCl2—0.0115, CuCl2—0.0105, CaCl2—0.005 and MnCl2—0.015; COD—5890 mg/l] to support biofilm formation onthe stone chips at an organic loading rate (OLR) of 2.4 kgCOD/m3-day by adjusting feed pH to 6 (29 days). ConstantCOD removal and gas production (±5% variation) were con-sidered as indicators for successful formation of the biofilm.Subsequently, the reactor was evaluated with chemical wastew-ater as substrate at operating OLR of 6.3 kg COD/m3-day.Prior to feeding the wastewater, the influent pH was adjustedto 6.0 using orthophosphoric acid to maintain acidophilicmicroenvironment.

2.4. Analysis

H2 produced during the reactor operation was monitored un-der closed conditions to avoid external environmental contam-ination using a microprocessor based pre-calibrated H2 sensor(electrochemical H2 sensor, FMK satellite 4–20 mA version,ATMI GmbH Inc., Germany). The sensor had a measuring rangeof 0.01–10% H2 with 5 s response time in a temperature rangeof 20–80 ◦C. The output signal displays the % volume of H2in the head space of the bioreactor. The system was calibratedonce in two days. Wastewater sampled during reactor operationat pre-defined time intervals was filtered (0.22 �m; Millipore)and stored at 0 ◦C prior to analysis. Samples were analyzedfor COD (closed refluxing method), oxidation–reduction po-tential (ORP), pH, volatile suspended solids (VSS), and BOD5according to the methods prescribed in standard methods [49].Reactor outlet was also monitored periodically for the presenceof kanamycin resistant strains in conjugation with total anaero-bic count in terms of colony forming units (CFU) by spreading100 �l of sample suspension on Wilkins–Chalgren anaerobic-kanamycin agar plates [agar—10 g/l,g/l; kanamycin—10 mg/land incubated at 37 ◦C for 24 h prior to colony counting (CFU)].Control plates in the absence of kanamycin were also studied

separately to track total anaerobic count. The augmented cul-ture and biofilm formed on the supporting material in the re-actor were subjected to scanning electron microscopy (SEM;Hitachi S-3000N). Prior to imaging the samples were trans-ferred to vials and fixed in glutaraldehyde (2.5%) in 0.05 Mphosphate buffer (pH 7.2) for 24 h at 4 ◦C and postfixed inaqueous osmium tetroxide (2%) in the same buffer for 2 h.Samples after fixation were dehydrated in a series of gradedalcohol and scanned in SEM. The separation and quantitativedetermination of volatile fatty acids (VFAs) was carried outby high performance liquid chromatography (HPLC; ShimadzuLC10A) employing optimized conditions (UV–vis detector;C18 column—reverse phase column—250 × 4.6 mm and 5 �particle size; flow rate—0.5 ml/h; wave length—210 nm; mo-bile phase—40% of acetonitrile in 1 mN H2SO4 (pH 2.5–3.0);sample injection—20 �l).

3. Results and discussion

Prior to bioaugmentation, the reactor was operated withchemical wastewater as feed at OLR of 6.3 kg COD/m3-dayat room temperature (28 ± 2 ◦C) in sequencing batch modeoperation (total cycle period of 24 h) by adjusting the influ-ent pH to 6 (acidophilic conditions) to assess the potential ofnative anaerobic mixed microflora with respect to H2 yield.After achieving stable performance with respect to H2 yieldand substrate reduction efficiency, the reactor was augmentedwith selectively enriched kanamycin resistant mixed consor-tia and operated at the same operating conditions until stableperformance was achieved.

3.1. Performance of non-augmented AnSBBR

The H2 production pattern before and after augmentationwith the function of total period of reactor operation are de-picted in Fig. 2a and b. Prior to augmentation, the reactor de-picted a maximum H2 production of 0.395 mmol within 2 h ofthe cycle operation along with a cumulative H2 production of0.979 mmol/day at the end of the cycle period. This accountedfor a volumetric H2 production rate of 697.4 mmol H2/m3-day.H2 production showed a gradual rise up to 120 min of the cycleoperation and thereafter reduced significantly and approachedzero at the end of the cycle period. Fig. 2c shows specific H2yield along with the substrate removal efficiency (in terms ofCOD reduction) with the function of single cycle operation.A steady decrease in the substrate removal was observed withthe function of the cycle period. Maximum substrate degra-dation rate (SDR) of 4.84 kg COD/m3-day accounting for aCOD removal efficiency of 76.68% was registered in the thirdcycle of operation. However, even though higher COD re-moval efficiency was observed in the third cycle of operation(from 51.85% to 78.66%), a marked decrease in the H2 pro-duction rate was documented (0.29–0.276 mol/kg CODR-day)(Fig. 2c), which may be attributed to the non-conducive mi-croenvironment for H2 production in the reactor. The substratedegradation suggests that the chemical wastewater was uti-lized as primary carbon source in the process of molecular

S. Venkata Mohan et al. / International Journal of Hydrogen Energy 32 (2007) 3284–3292 3287

0

0.1

0.2

0.3

0.4

0.5

0.6

0 50 100 150 200 250 300

Time (h)

Hydro

gen (

mm

ol)

Augmentation

0

0.5

1

1.5

2

2.5

72 96 120 140 164 187 210 232 256 278

Time (h)

Cum

mula

tive H

ydro

gen

Pro

duction (

mm

ol/day)

Augmentation

0

20

40

60

80

72 96 120 140 164 187 210 232 256 278

Time (h)

CO

D R

em

oval effic

iency (

%)

0

0.1

0.2

0.3

0.4

0.5

0.6

Specific

Hydro

gen Y

ield

(m

ol/kgC

OD

R-d

ay)

Augmentation

Fig. 2. (a) Hydrogen production (mmol) and (b) cumulative hydrogen production (mmol/day) with the function of reactor operation (before and afteraugmentation with kanamycin resistant mixed culture); (c) specific hydrogen production (mol H2/kg CODR-day) and COD removal efficiency (%) with thefunction of reactor operation.

H2 generation. The relatively low concentration of H2 pro-duction and discrepancy with respect to substrate removalefficiency (R2 < 0.5) observed during operation revealedthe inability of the resident anaerobic microflora present inthe reactor. The inferior performance observed may also beattributed to the complex nature of the substrate, due toits low-biodegradability. (BOD/COD∼0.3) and compositenature.

3.2. Performance of augmented AnSBBR

The reactor was subjected to augmentation with selectivelyenriched kanamycin resistant anaerobic mixed culture afterthe third cycle of operation. Immediately after augmentation,the reactor showed a sharp increase in H2 production rate andstabilized after three cycles of feeding operations (Fig. 2a–c).Maximum H2 production of 0.579 mmol was registered afterfive feeding cycles of augmentation. Prior to augmentation, the

H2 production was only observed during the initial few hoursafter feeding. On the contrary, after augmentation, the H2production was documented through out the cycle operationcontributing for higher cumulative H2 yield. Maximum cumu-lative H2 production of 2.05 mmol/day was observed at the endof the 5th cycle accounting for a volumetric H2 production rateof 1464.3 mmol H2/m3-day. Specific H2 production rate almostdoubled after augmentation from 0.297 to 0.483 mol H2/kgCODR-day. A significant enhancement in H2 evolution ratewas evidenced after augmentation. COD removal efficiencyof 55.2% accounting for an SDR of 4.62 kg COD/m3-day wasobserved during the fifth cycle of operation. After augmenta-tion, the reactor showed relatively good correlation (R2 < 0.9)

between H2 production and SDR which apparently indicatedthe favorable microenvironment and suggested the fact thatthe metabolic activity is key for effective H2 generation. Theexperimental observation depicted above indicates the success-ful application of augmentation strategy in enhancing the H2yield. The success of augmentation may be attributed to the

3288 S. Venkata Mohan et al. / International Journal of Hydrogen Energy 32 (2007) 3284–3292

0

1

2

3

4

5

6

7

0 20 40 60 80 100 120 140 160 180

Time (h)

CF

U-

Kan/A

naer(

T)

0

1

2

3

4

0 0.5 1 24

Time (h)

CF

U- K

an/A

naero

b(T

)

2 3 4 6 8

Fig. 3. Variation of kanamycin/total anaerobic (CFU) ratio with the functionof: (a) total period of reactor operation and (b) single cycle operation.

parent inoculum used and applied pre-treatment procedureson the parent inoculum apart from the operating conditionsand reactor configuration. The parent augmented culture wasadopted to chemical wastewater for a period of three yearsin the UASB reactor and was further enriched selectively forH2 producing spore forming acidogenic group of bacteria byrepetitive pre-treatments prior to augmentation.

The pre-treated culture was further enriched for antibioticresistance in order to track its survival and persistence in the re-actor environment after augmentation. To visualize the survivaland persistence of culture after augmentation, periodic platingof reactor outlet samples was performed on Wilkins–Chalgrenanaerobic agar (with and without kanamycin) and the CFU wasquantified in terms of kanamycin resistant microflora and totalanaerobic microflora. The variation in CFU (kanamycin/totalanaerobic) ratio with the function of the reactor operation (af-ter augmentation) and single cycle operation (immediately af-ter augmentation) are shown in Fig. 3a and b, respectively. Thekanamycin resistant to total anaerobic ratio showed a steady in-crease with time, which presumes the survival and subsequentproliferation of the augmented culture in the system. The ratioof kanamycin resistant microflora to total anaerobic microfloraratio was 1.21 (immediately after augmentation) and showed amarked increase up to 40 h. Subsequently a drop in ratio wasobserved which maintained up to 120 h. Further a gradual risein the ratio was registered up to 144 h (6.54) and thereafter

Fig. 4. Scanning electron microscopy (SEM) images (X5 K). (a) selectivelyenriched kanamycin resistant anaerobic mixed culture (bioaugmented culture).(b) Biofilm taken after five cycles of feeding after bioaugmentation.

stabilized (168 h—4.23; 192 h—4.23). CFU ratio variation dur-ing a single cycle operation (immediately after augmentation;Fig. 3) showed a continual growth up to the end of the cy-cle period. Initial adaptation problem due to competition withthe resident bacterial community was not evidenced after aug-mentation. The augmented culture persisted in the system upto the termination of the experiments (eight cycles; Fig. 3).The survival and maintenance of the augmented consortia sug-gests that the growth rate of the organism might be higher thanwashout and the activity of the grazers was negligible. SEM(X5 K; Fig. 4) image of augmented mixed consortia visualizedslightly bent, rod shaped and thick fluorescent capsid bacteria(∼ 10 �m length). It can be presumed from the image visibil-ity that the adopted selective enrichment procedure might haveresulted in enrichment of morphologically similar group of rodshaped bacteria capable of producing H2. SEM image (X 5K)of the biofilm samples acquired from the bioreactor after 5thcycle of augmentation showed morphologically similar shortbend rods (streptobacilli) with fluorescence (Fig. 4b). Images

S. Venkata Mohan et al. / International Journal of Hydrogen Energy 32 (2007) 3284–3292 3289

0

400

800

1200

1600

0 49 120 123.8 146 187 211 235 278

Time (h)

VF

A (

mg/l)

Augmentation

0

0.4

0.8

1.2

1.6

0 20

Time (h)

Cum

ula

tive H

ydro

gen

Yie

ld (

mm

ol/da

y)

0

200

400

600

800

1000

Tota

l V

FA

(m

g/l)

1 3 6

Fig. 5. (a) VFA production with the function of total period of reactoroperation. (b) Cumulative hydrogen and VFA production with the functionof single cycle operation.

of both the augmented inoculum and mixed consortia visual-ized morphological similarities indicating the proliferation ofrelated group of bacteria in the bioreactor.

Along with COD, VFA (represented as the total of all acidsgenerated during acidogenic fermentation step), pH and ORPwere also monitored during the cycle operation to assess thevariation in the bioprocess mechanism. H2 production is gen-erally accompanied by acid and solvent production due tothe acidogenic metabolism. Generation of the acidic inter-mediates reflected changes in the metabolic pathway of themicroorganisms involved and provided a better knowledgewhich can be used to improve the conditions favorable for H2production. VFA production was always associated with theconversion of organic fraction to acid intermediates in theanaerobic microenvironment with the help of specific group ofbacteria. Fig. 5a illustrates VFA produced during reactor oper-ation. Relatively higher production of VFA was observed priorto augmentation which might be due to accumulation in thesystem. However, after augmentation, relatively low concen-tration of VFA was observed with less accumulation. Duringnon-augmentation phase of operation, VFA concentration var-ied between 606 and 1478 mg/l. The VFA concentration variedbetween 1026 and 216 mg/l after augmentation. VFA produc-tion varied consistently with H2 production (R2 > 0.9) duringcycle operation (Fig. 5b; 3rd cycle of feeding after augmenta-tion). When substrate (COD) conversion was correlated with

quantity of VFA generated, a reasonably good correlation wasobserved after augmentation (R2—0.924) compared to non-augmentation operation (R2—0.719). Reactor samples duringthe course of experiments were collected and analyzed for thecomposition of VFA. The distribution of metabolites formedduring H2 fermentation was often crucial in assessing the ef-ficiency of H2-producing cultures [50,51]. The quantities ofacetate acid (HAc), butyric acid (HBu), propionic acid (HPa)and ethanol (HEt) were monitored by chromatography to havea better understanding of the change in metabolic pathway.Results obtained by chromatography documented the presenceof only butyric acid during non-augmentation operation andacetic acid along with relatively lower concentrations of butyricacid after augmentation. In both the cases formation of propi-onate and ethanol was not observed. The metabolic phenomenamight be associated with the acid-forming pathway (acido-genesis) instead of solventogenesis, which was considered asoptimum environment for effective H2 generation. High H2yields were reported to be associated with a mixture of acetateand butyrate fermentation products, and low H2 yields withpropionate and reduced end products (alcohols and lactic acid)[7]. The pH drop in the bioreactor system especially in anaer-obic microenvironment was considered as an index of volatileacid generation in alliance with the existing buffering capacityof the system. pH drop studied with and without augmentationwas comparable and showed a distinct trend toward the acidifi-cation. Prior to augmentation, pH values moved towards basicenvironment with time (Fig. 6). During this phase of opera-tion, pH varied between 6 and 6.8. The reactor registered pHvariation between 6 and 7.3 after bioaugmentation operation.

Bioaugmentation is presumed to be successful if there is sur-vival and retention of the augmented/inoculated strain therebyspreading the information to native biofilm populations associ-ation with enhancement in the performance. The survival andretention of the inoculated strain in the system for long periodfacilitates transmission of the requisite property to the indige-nous microorganisms. After augmentation, persistence of aug-mented inoculum over a prolonged period and resulting stableperformance with respect to H2 production and substrate degra-dation suggested that bioaugumentation was effective and notephemeral. Improvement in H2 production rate after augmen-tation is considered to be one important aspect while evaluat-ing the success of bioaugmentation. Typical anaerobic mixedcultures could not produce H2 as it was an intermediate formethane formation, and was rapidly consumed by the methane-producing bacteria [52,53]. One of the most effective ways toenhance H2 production using anaerobic culture was to restrictor terminate the process of methanogenesis by allowing H2 tobecome an end product in the metabolic flow. The sequentiallycoupled heat-shock (100 ◦C; 2 h) and acid (pH 3.0; 24 h) treat-ment applied to augmented culture prior to inoculation showedpositive influence on the H2 generation. Heat-shock treatmentfacilitated removal of non-spore forming methanogenic bac-terial groups and maintenance of acidogenic pH during theculture growth inhibited the methane-forming bacteria. Num-ber of studies were reported in the literature pertaining todifferent pre-treatment studies performed on diverse type of

3290 S. Venkata Mohan et al. / International Journal of Hydrogen Energy 32 (2007) 3284–3292

5.5

6.5

7.5

8.5

0 48 96 121 124 141 165 187 192 214 234 257 279 302

Time (h)

pH

-250

-150

-50

50

OR

P (

mV

)

150

pH

ORP (mV)Augmentation

Fig. 6. pH and ORP variation with the function of total period of reactor operation.

inoculums [11–13,54–59]. Concurrent control of pH of the feed(pH 6.0) during the reactor operation further helped to limit themethanogenic growth. The pH range of 5.5–6 was consideredto be ideal to avoid both methanogenesis and solventogenesis[11–13,60,61].

Integration of biofilm configuration with sequencing batchmode operation in this study had great potential to providethe possibilities of influencing the microbial system by selec-tively enriching the specific group of microflora. The adoptedoperation mode of reactor and reactor configuration seems tohave great potential and considerable influence to provide thepossibilities of influencing the microbial system by selectivelyenriching the specific group of microflora which is quite usefulin survival and persistence of augmented culture. Sequenc-ing batch reactor operated in periodic discontinuous batchmode process induced controlled unsteady state conditionsoscillating between alternative feast and famine conditionsduring reactor operation resulting in high biological activity[25,31,62–67]. Recent research indicated that the sequentialfeast and famine conditions enhanced the overall performanceof the biological system [62,68]. Watanabe and coworkers [68]reported the effectiveness of short starvation treatment of cellsbefore inoculation which resulted in high survivability result-ing in the improved efficacy of the inoculated microbial strainin the augmented microenvironment. It was also suggested thatsurvival of the microorganisms after introduction into the newenvironment was affected by the physiological status of cellsto be introduced. Enforced short-term unsteady state condi-tions coupled with periodic exposure of the microorganisms todefined process conditions by controlling their physiologicalstate (incorporating required metabolic conditions) might havepositive influence on the persistence and subsequent dissem-ination of requisite property of augmented inoculum to theresident bacteria. Under transient conditions, growth was

unbalanced, and the population’s physiological state adaptedto the imposed conditions and the organisms experienced highgrowth rates that increased cellular RNA content resulting inmicroflora with high reactivity [69]. The sequencing batchsystems had high retention capacity of biomass due to batchmode operation, which also favored the persistence of theaugmented strain. Biofilm reactor configuration coupled withsequencing batch operation, maintained high biomass concen-tration, encouraged the enrichment of slow growing organismsand can obtain homogeneous biomass distribution throughoutthe reactor [25,65,66,70], which is the essential pre-requisite ofthe augmented culture to persist and survive in the inoculatedenvironment and subsequent dissemination of the requisiteproperties to resident species.

4. Conclusions

This work corroborated successful application of bioaug-mentation strategy to improve H2 production rate from chem-ical wastewater treatment in an anaerobic sequencing batchbiofilm reactor (AnSBBR). The survival and retention of theaugmented inoculum resulted in enhancing the process ef-ficiency. Chemical wastewater was used as primary carbonsource in metabolic reactions involving molecular H2 gener-ation and this was evident from substrate reduction and H2production. The study showed selective enrichment of a groupof morphologically similar bacteria capable of proliferating inbioreactor on stone chips producing H2. The selected reac-tor operating conditions, biofilm configuration coupled withperiodic discontinuous/sequencing batch operation seems tohave great potential to provide the possibilities of influenc-ing the microbial system in survival of the augmented strainand dissemination of the requisite properties to the residentspecies.

S. Venkata Mohan et al. / International Journal of Hydrogen Energy 32 (2007) 3284–3292 3291

Acknowledgments

The authors gratefully acknowledge the financial support ofDepartment of Biotechnology (DBT) [BT/PR/4405/BCE/08/312/2003], Government of India in carrying out this researchwork. One of the authors (SVR) also acknowledges IndianCouncil for Medical Research (ICMR), New Delhi for provid-ing research fellowship.

References

[1] Bokris JO’ M. The origin of ideas on a hydrogen economy and its solutionto the decay of the environment. Int J Hydrogen Energy 2002;27:731–40.

[2] Yu H-Q, Mu Y, Wang G. Kinetic modeling of batch hydrogen productionprocess by mixed anaerobic cultures. Bioresource Technol 2006;97:1302–7.

[3] Kirk RE, Othmer DF, Grayson M, Eckroth D. Concise encyclopedia ofchemical technology XIII. New York; Wiley: 1985. p. 838–93.

[4] Hart D. Hydrogen power: the commercial future of the ultimate fuel.London: Financial times energy publishing; 1997.

[5] Das D, Verziroglu TN. Hydrogen production by biological process: asurvey of literature. Int J Hydrogen Energy 2001;26:13–28.

[6] Gavala HN, Skiadas IV, Ahring BK. Biological hydrogen production insuspended and attached growth anaerobic reactor systems. Int J HydrogenEnergy 2006;31:1164–75.

[7] Hawkes FR, Dinsdale R, Hawkes DL, Hussy I. Sustainable fermentativehydrogen production: challenges for process optimisation. Int J HydrogenEnergy 2002;27:1339–47.

[8] Lin CY, Lay C. Effects of carbonate and phosphate concentrations onhydrogen production using anaerobic sewage sludge microflora. Int JHydrogen Energy 2004;29:275–81.

[9] Lin C, Cheng C. Fermentative hydrogen production from xylose usinganaerobic mixed microflora. Int J Hydrogen Energy 2005;31:832–40.

[10] Logan BE. Biologically extracting energy from wastewater: biohydrogenproduction and microbial fuel cells. Environ Sci Technol 2004;38:160A–7A.

[11] Venkata Mohan S, Vijaya Bhaskar Y, Murali Krishna T, Rao NC,Lalit V, Sarma PN. Hydrogen production from chemical wastewater assubstrate by selectively enriched anaerobic mixed consortia: influenceof fermentation pH and substrate composition. Int J Hydrogen Energy;2007, in press, doi:10.1016/j.ijhydene.2007.03.015.

[12] Venkata Mohan S, Lalit Babu V, Sarma PN. Effect of various pretreatmentmethods on anaerobic mixed microflora to enhance biohydrogenproduction utilizing dairy wastewater as substrate. Bioresource Technol.doi:10.1016/j.biortech.2006.12.004.

[13] Venkata Mohan S, Vijaya Bhaskar Y, Sarma PN. Biohydrogen productionfrom chemical wastewater treatment by selectively enriched anaerobicmixed consortia in biofilm configured reactor operated in periodicdiscontinuous batch mode. Water Res. doi:10.1016/j.watres.2007.02.015.

[14] Venkata Mohan S, Saravanan R, Veer Raghuvulu S, Mohan KrishnaG, Sarma PN. Bioelectricity production from wastewater treatmentin dual chambered microbial fuel cell (MFC) using selectivelyenriched mixed microflora: effect of catholyte. Bioresource Technol.doi:10.1016/j.biortech.2006.12.026.

[15] Vijayaraghavan K, Ahmad D, Ibrahim MKB, Herman HNB. Isolation ofhydrogen generating microflora from cow dung for seeding anaerobicdigester. Int J Hydrogen Energy 2006;31:708–20.

[16] Kovács KL, Kovács ÁT, Maróti G, Bagi Z, Csanádi G, Perei K, BálintB. et al. Improvement of biohydrogen production and intensification ofbiogas formation. Rev Environ Sci Biotechnol 2004;3:321–30.

[17] Kovácsa KL, Marótia G, Rákhelya G. A novel approach for biohydrogenproduction. Int J Hydrogen Energy 2006;31:1460–8.

[18] Lee K, Lo Y, Lin P, Chang J. Improving biohydrogen production in acarrier-induced granular sludge bed by altering physical configurationand agitation pattern of the bioreactor. Int J Hydrogen Energy 2006;31:1648–57.

[19] Vignaisa PM, Magninb Jwillisona JC. Increasing biohydrogen productionby metabolic engineering. Int J Hydrogen Energy 2006;31:1478–83.

[20] Van Limbergen H, Top EM, Verstraete W. Bioaugmentation in activatedsludge: current features and future perspectives. Appl MicrobiolBiotechnol 1998;50:16–23.

[21] Wilderer PA, Rubio MA, Davids L. Impact of the addition of purecultures on the performance of mixed culture reactor. Water Res1991;25:1307–13.

[22] Ahring BK, Christiansen N, Mathrani I, Hendriksen HV, Macario AJL,deMacario EC. Introduction of a de novo bioremediation ability, arylreductive dechlorination, into anaerobic granular sludge by inoculationof sludge with Desulfomonile tiedjei. Appl Environ Microbiol 1992;58:3677–82.

[23] Jianlong W, Xianghun Q, Libo W, Yi Q, Hegemann W. Bioaumentationas a tool to enhance the removal refractory compound in coke plantwastewater. Proc Biochem 2002;38:777–81.

[24] Stephenson D, Stephenson T. Bioaugmentation for enhancing biologicalwastewater treatment. Biotech Adv 1992;10:549–59.

[25] Venkata Mohan S, Chandrasekhara Rao N, Krishna Prasad K, SarmaPN. Bioaugmentation of anaerobic sequencing batch biofilm reactor(ASBBR) with immobilized sulphate reducing bacteria (SRB) for treatingsulfate bearing chemical wastewater. Proc Biochem 2005;40(8):2849–57.

[26] Bathe S, Schwarzenbeck N, Hausner M. Plasmid-mediatedbioaugmentation of activated sludge bacteria in a sequencing batchmoving bed reactor using pNB2. Lett Appl Microbiol 2005;41(3):242–7.

[27] Bathe S. Conjugal transfer of plasmid pNB2 to activated sludge bacterialeads to 3-chloroaniline degradation in enrichment cultures. Lett ApplMicrobiol 2004;38:527–31.

[28] Boon N, Goris J, De Vos P, Verstraete W, Top E. Bioaugumentation ofactivated sludge by an indigenous 3-chloroaniline-degrading Comamonastestosteroni strain 12gfp. Appl Environ Microbiol 2000;66:2906–13.

[29] Hajji KT, Lepine F, Bisaillon JG, Beaudet R, Hawari J, Guiot SR. Effectsof bioaugmentation strategies in UASB reactors with a methanogenicconsortium for removal of phenolic compounds. Biotechnol Bioeng2000;67:418–23.

[30] Quan X, Shi H, Wang J, Qian Y. Biodegradation of 24-dichlorophenol insequencing batch reactors augmented with immobilized mixed culture.Chemosphere 2003;50:1069–74.

[31] Venkata Mohan S, Nancharaiah YV, Flankentof FC, Wattiau P, Wuertz S,Wilderer PA. Monitoring the conjugal transfer of plasmid PWWO fromPseudomonas putida in sequencing batch biofilm reactor. In: Proceedingsof VAAM conference; 2002.

[32] Chong N, Pai S, Chen C. Bioaugmentation of an activated sludgereceiving pH shock loadings. Bioresource Technol 1997;59:235–40.

[33] Brandon CG, Nasser AA, Ronald FT. Removal of atrazine contaminationin soil and liquid systems using bioaugmentation. Pest Sci1999;50(3):211–20.

[34] Halden RU, Tepp SM, Halden BG, Dwyer DF. Degradation of 3-phenoxybenzoic acid in soil by Pseudomonas pseudoalcaligenes POB 310(pPOB) and two modified Pseudomonas strains. Appl Environ Microbiol1999;65(8):3354–9.

[35] Newby DT, Gentry TJ, Pepper IL. Comparison of 2,4-dichlorophenoxyacetic acid degradation and plasmid transfer in soilresulting from bioaugmentation with two different pJP 4 donors. ApplEnviron Microbiol 2000;66:3399–407.

[36] Roane TM, Josephson KL, Pepper IL. Dual-bioaugmentation strategyto enhance remediation of contaminated soil. Appl Environ Microbiol2001;67:3208–15.

[37] Rojas-Avelizapa NG, Martinez-Cruz J, Zermeno-Egvia Lis JA,Rodriguez-Vazquez R. Levels of poly-chlorinated biphenyls in Mexicansoils and their biodegradation using bioaugmentation. Bull EnvironContam Toxicol 2003;70:63–70.

[38] Sarma PN, Venkata Mohan S, Rama Krishna M, Shailaja S.Bioremediation of pendimethalin contaminated soil by augmentedbioslurry phase reactor operated in sequential batch (SBR) mode: Effectof substrate concentration. Indian J Biotechnol 2006;5:169–74.

[39] Top EM, Maila P, Clerinx M, Goris J, Vos PD, Verstraete W. Methanceoxidation as a method to evaluate the removal of 2,4-dihlorophenoxyactic

3292 S. Venkata Mohan et al. / International Journal of Hydrogen Energy 32 (2007) 3284–3292

acid (2,4-D) from soil by plasmid mediated bioaugmentation. FEMSMicrobiol Ecol 1999;28:203–13.

[40] Top EM, Pringael D, Boon N. Catabolic mobile genetic elements andtheir potential use in bioaugmentation of polluted soils and water. FEMSMicrobiol Ecol 2002;42:199–208.

[41] Venkata Mohan S, Shailaja S, Ramakrishna M, Reddy KB, SarmaPN. Bioslurry phase degradation of DEP contaminated soil in periodicdiscontinuous mode operation: influence of augmentation and substratepartition. Proc Biochem 2006;41(3):644–52.

[42] Zhang C, Hughes JB, Nishino SF, Spain JC. Slurry-phase biologicaltreatment of 2,4-dinitrotoluene and 2,6-dinitrotoluene: role ofbioaugmentation and effects of high dinitrotoluene concentrations. Environ Sci Technol 2000;34:2810–6.

[43] Eberl L, Schulze R, Ammendola A, Geisenberger O, Erhart R, SternbergC, Molin S. et al. Use of green fluorescent protein as a marker forecological studies of activated sludge communities. FEMS MicrobiolLett 1997;149:77–83.

[44] Goldstein MG, Mallory LM, Alexander M. Reasons for possible failureof inoculation to enhance biodegradation. Appl Environ Microbiol1985;50:977–83.

[45] Leung KT, So J-S, Kostrzynska M, Lee M, Trevors JT. Using a greenfluorescent protein gene-labelled p-nitrophenol-degrading Moraxellastrain to examine the protective effect of alginate encapsulation againstprotozoan grazing. J Microbiol Methods 2000;39:205–11.

[46] Watanabe K, Yamamoto S, Hino S, Harayama S. Population dynamicsof phenol degrading bacteria in activated sludge determined by gyrß-targeted quantitative PCR. Appl Environ Microbiol 1998;64:1203–9.

[47] Bouchez T, Patureau D, Dabert P, Juretschko S, Dore J, Deigenes P,Moletta R. et al. Ecological study of a bioaugmentation failure. EnvironMicrobiol 2000;2:179–90.

[48] Bouchez T, Patureau D, Dabert P, Wagner M, Deigenes P, Moletta R.Successful and unsuccessful bioaugumentation experiments monitoredby fluorescent in situ hybridization. Water Sci Technol 2000;41:61–8.

[49] APHA. Standard methods for the examination of water and wastewater.20th, American Public Health Association, American Water WorksAssociation, Water Pollution Control Federation, Washington, DC, 1998.

[50] Cha GC, Noike T. Effect of rapid temperature change and HRT onanaerobic acidogenesis. Water Sci Technol 1997;36(6–7):247–53.

[51] Dinopoulou G, Sterritt RM, Lester JN. Anaerobic acidogenesis of acomplex wastewater 2. kinetics of growth, inhibition, and productformation. Biotechnol Bioeng 1988;31:969–78.

[52] Nandi R, Sengupta S. Microbial production of hydrogen: an overview.Crit Rev Microbiol 1998;24:61–84.

[53] Sparling R, Risbey D, Poggi-Varaldo HM. Hydrogen production frominhibited anaerobic composters. Int J Hydrogen Energy 1997;22:563–6.

[54] Ferchichi M, Crabbe E, Gwang-Hoon G, Hintz W, Almadidy A. Influenceof initial pH on hydrogen production from cheese whey. J Biotechnol2005;120:402–9.

[55] Ginkel V, Sung S, Lay SJJ. Biohydrogen production as a function ofpH and substrate concentration. Environ Sci Technol 2001;35:4726–30.

[56] Kim J, Park C, Kim T-H, Lee M, Kim S, Kim S-W. et al. Effectsof various pretreatments for enhanced anaerobic digestion with wasteactivated sludge. J Biosci Bioeng 2003;95:271–5.

[57] Logan BE, Oh SE, Kim IS, Ginkel SV. Biological hydrogen productionmeasured in batch anaerobic respirometers. Environ Sci Technol2002;36:2530–5.

[58] Oh SE, Ginkel SV, Logan BE. The relative effectiveness of pH controland heat treatment for enhancing biohydrogen gas production. EnvironSci Technol 2003;37:5186–90.

[59] Zhu H, Béland M, Evaluation of alternative methods of preparinghydrogen producing seeds from digested wastewater sludge. Int JHydrogen Energy; 2006. doi:10.1016/j.ijhydene.2006.01.019.

[60] Fang HHP, Liu H, Zhang T. Characterization of a hydrogenproductiongranular sludge. Biotechnol Bioeng 2002;78:44–52.

[61] Rogers O. Genetics and biochemistry of clostridium relevant todevelopment of fermentation process. Appl Microbiol 1984;31:1–60.

[62] Chiesa SC, Irvine RL, Manning Jr. JF. Feast/famine growthenvironment and activated sludge population selection. BiotechnolBioeng 1985;27:562–9.

[63] Irvine RL, Moe WM. Periodic biofilter operation for enhanceperformance during unsteady state loading condition. Water Sci Technol2001;45:231–9.

[64] Ramaswami A, Luthym RG. Mass transfer and bioavailability of PAHcompounds in coal tar NAPL-slurry systems. 1. Model development.Environ Sci Technol 1997;31(8):2260–7.

[65] Venkata Mohan S, Chandrasekhar Rao N, Sarma PN. Low-biodegradable composite chemical wastewater treatment by biofilmconfigured sequencing batch reactor (SBBR). J Hazardous Mater 2007;144(1–2):108–17.

[66] Venkata Mohan S, Chandrasekhar Rao N, Sarma PN. Composite chemicalwastewater treatment by biofilm configured periodic discontinuous batchreactor operated in anaerobic metabolic function. Enzyme MicrobialTechnol 2007;40(5):1398–406.

[67] Wilderer PA, Irvine RL, Goronszy MC. Sequencing batch reactortechnology. Scientific and technical report no. 10, IWA Publishing; 2001.

[68] Watanabe K, Miyashita M, Harayama S. Starvation improves survivalof bacteria introduced into activated sludge. Appl Environ Microbiol2000;66:3905–10.

[69] Woolard CR, Irvine RL. Treatment of hypersaline wastewater in thesequencing batch reactor. Water Res 1995;29(4):1159–68.

[70] Kaballo HP, Zhao Y, Wilderer PA. Elimination of p-chlorophenol inbiofilm reactors: a comparative study of continuous flow and sequencedbatch operation. Water Sci Technol 1995;31(1):51–60.