Bioaugmentation of potent acidogenic isolates: A strategy for enhancing biohydrogen production at elevated organic load

Bioresource Technology xxx (2014) xxx–xxx

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Bioaugmentation of potent acidogenic isolates: A strategy for enhancing biohydrogen production at elevated organic load R. Kannaiah Goud, Omprakash Sarkar, P. Chiranjeevi, S. Venkata Mohan ⇑ Bioengineering and Environmental Centre (BEEC), CSIR-Indian Institute of Chemical Technology (CSIR-IICT), Hyderabad 500 007, India

h i g h l i g h t s  Augmentation with potent acidogenic isolates enhances biohydrogen production.  Bacillus subtilis strain promoted a significant increase of the H2 yield.  Highest COD removal efficiency was observed with Pseudomonas stutzeri.  Application of bioaugmentation strategy at higher loading was successfully evaluated.

a r t i c l e

i n f o

Article history: Received 10 January 2014 Received in revised form 8 March 2014 Accepted 12 March 2014 Available online xxxx Keywords: Anaerobic sequencing batch reactor (AnSBR) Wastewater treatment Volatile fatty acids Dehydrogenase activity FISH

a b s t r a c t The efficiency of bioaugmentation strategy for enhancing biohydrogenesis at elevated organic load was successfully evaluated by augmenting native acidogenic microflora with three acidogenic bacterial isolates viz., Bacillus subtilis, Pseudomonas stutzeri and Lysinibacillus fusiformis related to phyla Firmicutes and Proteobacteria separately. Hydrogen production ceased at 50 g COD/l operation due to feed-back inhibition. B. subtilis augmented system showed higher H2 production followed by L. fusiformis, P. stutzeri and control operations, indicating the efficacy of Firmicutes as bioaugmentation biocatalyst. Higher VFA production with acetic acid as a major fraction was specifically observed with B. subtilis augmented system. Shift in metabolic pathway towards acidogenesis favoured higher H2 production. FISH analysis confirmed survivability and persistence of augmented strains apart from improvement in process performance. Bio-electrochemical analysis depicted specific changes in the metabolic activity after augmentation which also facilitated enhanced electron transfer. P. stutzeri augmented system documented relatively higher COD removal. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction Biological route of hydrogen (H2) production is a potential alternative to fossil based production process. Biological process involves utilization of waste as major feed-stock for production of H2 through acidogenic fermentative pathway along with simultaneous remediation of the waste (Venkata Mohan et al., 2007a Wang et al., 2008). This process has several advantages like lower cost, ease of control and simultaneous waste remediation associated with clean bioenergy generation in a unified and sustainable approach (Ren et al., 2010; Marone et al., 2012; Venkata Mohan et al., 2009a,b; Wang et al., 2008; Venkata Mohan and Pandey, 2013). The efficacy of acidogenic H2 production depends on many biotic and abiotic factors. Among them, the nature of biocatalyst as well as composition and load of substrate (wastewater) plays a ⇑ Corresponding author. Tel./fax: +91 40 27191664. E-mail address: [email protected] (S. Venkata Mohan).

critical role in overall process efficiency (Vazquez and Valardo, 2009; Venkata Mohan 2008a,b). Wastewater has a significant quantity of organic fraction, which can be metabolically converted to H2 by acidogenesis (Venkata Mohan, 2009). However, the composition of wastewater and the concentration of organic load have a direct influence on the fermentation output. Increment in the organic load effects H2 production positively to certain extent and after that acidification of system microenvironment by production of excess VFA and consequent reduction system buffer capacity gives negative impact (Mohanakrishna et al., 2010). The resulting drop in pH (<4.0) inhibits acidogenic bacteria (Venkata Mohan et al., 2008c; Wang et al., 2008). Thus to operate the bioreactor at higher organic load without undergoing processes inhibition bioaugmentation appears to be a feasible strategy operation. Bioaugmentation implies addition of actively growing, desired and specialized microbial strains to native microbial community of the bioreactor in an effort to enhance the ability of the microbial

http://dx.doi.org/10.1016/j.biortech.2014.03.049 0960-8524/Ó 2014 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Goud, R.K., et al. Bioaugmentation of potent acidogenic isolates: A strategy for enhancing biohydrogen production at elevated organic load. Bioresour. Technol. (2014), http://dx.doi.org/10.1016/j.biortech.2014.03.049

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R.K. Goud et al. / Bioresource Technology xxx (2014) xxx–xxx

community to withstand the process fluctuations and accomplish enhanced and improved treatment waste degradation. Bioaugmentation strategy has been used to improve the start up of a reactor (Ma et al., 2009), to enhance reactor performance (Venkata Mohan et al., 2005, 2009c; Raghavulu et al., 2013) and to protect the existing microbial community against adverse effects (Hajji et al., 2000; Venkata Mohan et al., 2005) or to compensate for organic or hydraulic overloading (Chong et al., 1997). Studies were performed to enhance H2 production by means of bioaugmentation with different degrees of successes (Li and Fang, 2007; Venkata Mohan et al., 2007b; Wang et al., 2008; Ma et al., 2009; Kuo et al., 2012; Marone et al., 2012). The application of bioaugmentation strategy to enhance biological H2 production by augmentation of native microflora by selectively enriched acidogenic consortia has been reported earlier (Venkata Mohan et al., 2007a). Recent studies demonstrated the effectiveness of bioaugmentation in improving H2 production by Clostridium acetobutylicum (Wang et al., 2008). Fermentative bacteria capable of producing H2 were added to the native mixed microflora to improve the yield (Marone et al., 2012). However in some cases, bioaugmented species failed to compete with indigenous population (Goldstein et al., 1985; Bouchez et al., 2000). In the present communication, three potent acidogenic bacterial isolates viz., Bacillus subtilis, Pseudomonas stutzeri and Lysinibacillus fusiformis belonging to phylum Firmicutes and Proteobacteria were augmented to bioreactors inhibited due to operation at elevated organic load for enhancing process performance in terms of biohydrogen production. The viability of bioaugmentation strategy on process performance was studied by process monitoring in terms of H2 production, substrate degradation, VFA production/ composition, dehydrogenase (DH) activity along with bioelectrochemical and FISH analysis. 2. Methods 2.1. Biocatalyst 2.1.1. Anaerobic consortia Anaerobic consortium collected from a full scale anaerobic reactor treating composite wastewater was used as parent inoculum. After sampling, the parent anaerobic consortium was sieved to separate the grit by nylon filter, resulting in thick sludge (3.6 g VSS/l) which then centrifuged (1500 g; 20 °C) and washed thrice with saline buffer. The resultant pellet was used for the inoculum preparation by applying different pretreatment methods separately. The performance of the bioreactors operated with augmented inoculums was evaluated with the reactors operating with untreated inoculums served as control. 2.1.2. Bioaugmentation strains and identification using 16S rRNA gene sequence Three strains isolated from long term operated acidogenic bioreactors producing biohydrogen were used as biocatalyst for bioaugmentation. Genomic DNA of the three isolates was extracted as described earlier (Venkata Mohan et al., 2010). Total extracted genomic DNA was used as a template to amplify 16S rRNA by using universal primers with PCR (Thermal cycler, Eppendorf). The nucleotide sequences of the universal primers were as follows: primer 28F, 50 -AGA GTT TGA TCC TGG CTC AG-30 ; primer 1542R, 50 AAG GAG GTG ATC CAG CCG CA-30 (Ueno et al., 2001). PCR reaction mixture (50 ll) consisted of Taq polymerase (5 U), 1.2 ml, 28F and 1542R (10 pmol), 1 ml DNA template (1 ng), 4 ml of PCR buffer (10), 5 ml of MgCl2 (25 mmol/l) and 37.6 ml of sterile water. PCR was operated for 30 cycles at 95 °C (50 s), 52 °C (40 s) and 72 °C (40 s). The amplified product was identified based on

molecular mass by agarose gel electrophoresis using 1.2% agarose gel. The nucleotide sequences of 16S rRNA gene of the isolates were compared with other related sequences available in GenBank by BLAST programme. Further, the nucleotide sequence of isolates was aligned with closely related sequences (that were downloaded from GenBank) using ClustalW and pair wise evolutionary distances were computed. BLAST analysis confirms isolates belong to B. subtilis (Strain IICTSVMH13), P. stutzeri (strain IICTSVMHi) and L. fusiformis (strain IICTSVMH10) and the sequences of these strains is deposited with in the public domain (GenBank). The GenBank accession numbers of these strains are B. subtilis (FR849706), P. stutzeri (HE586324) and L. fusiformis (HE586325). Phylogenetic analysis was performed using MEGA version 4 and the phylogenetic trees were constructed using neighbourhood joining method of analysis. Confidence in the tree topologies was evaluated by re-sampling 100 bootstrap trees. Phylogenetic tree of three augmented strains depicts 100% sequence similarity with B. subtilis, P. stutzeri and L. fusiformis clusters with the type strains (Supplementary Fig. 1). Based on BLAST search programme and phylogenetic analysis revealed that all the isolates belonged to two major phyla Firmicutes and Proteobacteria. Prior to augmentation, the strains were cultured overnight in specific growth media (LB broth; yeast extract-5 g/l, casein peptone-10 g/l, sodium chloride-10 g/l; pH 7). A loop full of overnight grown culture was inoculated into nutrient broth. After 12 h of incubation at 37 °C, the culture was centrifuged and washed twice in saline buffer (6500g, 20 °C), resuscitated in nutrient solution and augmented through wastewater feeding. 2.2. Experimental methodology Four identical bench-scale anaerobic batch reactors (AnSBR) were fabricated using borosilicate-glass with total/working volume of 1.2/0.84 l and gas holding capacity of 0.36 l. The reactors were operated in suspended growth mode configuration continuously for 50 cycles. Each cycle comprised 48 h (hydraulic retention time, HRT) accounted by 20 min of FILL, 47 h of REACT (anaerobic), 20 min of SETTLE and 20 min of DECANT phases. The reactors were kept in suspension mode during the REACT phase. Predefined volume (0.72 l) of real field food wastewater was fed during the FILL phase at the beginning of each cycle and contents were subjected to continuous mixing (100 rpm). Appropriate measures were taken to maintain the reactors under anaerobic conditions. All the bioreactors were operated under similar conditions. Prior to bioaugmentation, the reactors were operated with real field food wastewater (the characteristics of the food waste prior to adjusting OLR are summarized in Supplementary Table 1) with COD load of 5 g/l, 10 g/l, 20 g/l and 30 g/l at room temperature (28 ± 2 °C) in sequencing batch mode operation (total cycle period of 48 h) and adjusting the influent pH to 6 (acidophilic conditions). At higher load (50 g/l COD) operation the system showed process inhibition and cessation of H2 production. The bioreactors were then augmented with H2 producing strains [Reactor 1 (Control, without augmentation), Reactor 2 (B. subtilis), Reactor 3 (P. stutzeri) and Reactor 4 (L. fusiformis)] through wastewater during feeding and operated under the same conditions until a stable performance was achieved. The four bioreactors were operated simultaneously for 50 cycles accounting for 100 days. 2.3. Analysis Quantification of H2 gas was carried out using a microprocessor based pre-calibrated H2 sensor (ATMI GmbH Inc.).The fraction of H2 in the biogas was determined by a gas chromatograph (Nucon) equipped with a thermal conductivity detector (TCD) and a 2.1/ 800  2 m SS column with a molecular sieve (size 60/80 mesh)

Please cite this article in press as: Goud, R.K., et al. Bioaugmentation of potent acidogenic isolates: A strategy for enhancing biohydrogen production at elevated organic load. Bioresour. Technol. (2014), http://dx.doi.org/10.1016/j.biortech.2014.03.049

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stationary phase. Nitrogen (20 ml/min; 1 kg/cm2) was used as the carrier gas. The operating temperatures of the column, detector and injector were 40, 80 and 50 °C, respectively. VFA concentration was quantified by high performance liquid chromatography [HPLC LC20AD; C18 reverse phase column 250/4.6 mm; UV detector; wavelength 210 nm; 5 m particle size; flow rate-0.6 ml/h; mobile phase acetonitrile (40%) in 1 N H2SO4 (pH, 2.5–3.0); sample injection 20 ll]. The performance of bioreactors was assessed by monitoring chemical oxygen demand (COD), Volatile fatty acids (VFA)

and pH as per the standard procedures (APHA, 1998). Dehydrogenase (DH) enzyme activity was estimated by colorimetric procedure based on the reduction of 2,3,5-triphenyltetrazolium chloride (TTC) (Goud and Venkata Mohan, 2013). Cyclic voltammetry (CV) was performed using potentiostat–galvanostat system (Autolab-PGSTAT12) [counter electrode, graphite rod; working electrode, platinum wire; reference electrode, Ag/AgCl (S)] using a potential ramp between +0.5 and 0.5 V at a scan rate of 30 mV/s.

(a)

Bacillus subtillus Lysinibacillus

Control

Pseudomonas

2.0

CHP (l)

1.5

1.0

0.5

5 g/l

0.0

After augmentation

10 g/l

20 g/l

30 g/l

Before augmentation

0

5

10

50g/l 15

20

25

30

35

40

45

50

Cycle

(b)

Bacillus subtillis

Pseudomonas stutzeri

Lysinibacillus fusiformis

Control

30

H2 (%)

25 20 15 10 5 0 25

CH4 (%)

20 15 10 5 0 12 h

18 h

24 h

30 h

48 h

Time (h) Fig. 1. a) Cumulative H2 production during operation of bioreactors before and after bioaugmentation [cycle 1–5 (5 g/l), cycle 6–10 (10 g/l), cycle 11–15 (20 g/l), cycle 16–20 (30 g/l), cycle, 31–50 (50 g/l)]; b) biogas (H2 and CH4) composition.

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2.3.1. Fluorescent in situ hybridization (FISH) Prevalence of augmented strain was confirmed using fluorescent in situ hybridization (FISH) technique employing modified procedure (Amann et al., 1995; Sarkar et al., 2013). 50 -Cy3 modified oligonucleotide probes derived from sequence of 16S rRNA and 23S rRNA were used for Bacillus (pB-01036: 50 -CTT CAG CAC TCA GGT TCG-30 ) (Salzman et al., 2002), Pseudomonas (pB-00375: 50 -GCT GGC CTA ACC TTC-30 ) (Schleifer et al., 1992) and Firmicutes (pB00195: 50 -TGG AAG ATT CCC TAC TGC-30 ) (Meier et al., 1999). The probes were procured from Eurofins genomics. 6 ml of sample was subjected to three repeated washings (7200 rpm; 6 min; 28 °C) with distilled water for removing the dead cells, contaminants and subsequently suspended in phosphate-buffered saline (PBS; pH 7.2) for additional washing. The resulting cells were fixed with 4% paraformaldehyde and kept for incubation at 4 °C for 4 h.

(a)

10500

10 g/l

5 g/l

The fixed cells were washed twice with PBS and suspended in Ethanol:PBS (1:1) solution and stored at 20 °C. This was followed by smear preparation, washing and hybridization according to previous reports (Sarkar et al., 2013). The hybridized bacterial cells were counterstained with 40 ,6-diamidino-2-phenylindole (DAPI) to observe total bacterial population. DAPI binds to DNA at A-T rich regions in DNA. The fluorescent stained smears were visualized with epifluorescent microscope (Nikon Eclipse-80i) at 400 magnification. Photomicrographs were captured by attached digital camera (YIM-smt, 5.5 mega pixels) and analyzed using NISelements software (D3.0). Counting of Cy3-stained bacterial cells was done in a single microscopic field using a Cy3 filter followed by DAPI-stain cell counting (DAPI-filter). The bacterial cell count was done randomly on the selected fields (more than 10) of the smears.

30 g/l

20 g/l

Before augmentation 9000

VFA production (mg/l)

7500 Control

6000

Bacillus subtilis Pseudomonas

4500

Lysinibacillus

3000

50 g/l After augmentation

1500 0 0

5

10

15

20

25

30

35

40

45

50

Cycles

(b)

6.0 Control Pseudomonas

Bacillus Lysinibacillus

5.5

Outlet pH

5.0

4.5

4.0

3.5 20 g/l

20 g/l 10 g/l 5 g/l Before augmentation

50 g/l After augmentation

3.0 0

5

10

15

20

25

30

35

40

45

50

Cycles Fig. 2. VFA and pH profiles recorded before and after bioaugmentation [cycle 1–5 (5 g/l), cycle 6–10 (10 g/l), cycle 11–15 (20 g/l), cycle 16–20 (30 g/l), cycle, 31–50 (50 g/l)].

Please cite this article in press as: Goud, R.K., et al. Bioaugmentation of potent acidogenic isolates: A strategy for enhancing biohydrogen production at elevated organic load. Bioresour. Technol. (2014), http://dx.doi.org/10.1016/j.biortech.2014.03.049

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R.K. Goud et al. / Bioresource Technology xxx (2014) xxx–xxx

3. Results and discussion 3.1. Biohydrogen production All the bioreactors were initially operated for twenty cycles using composite canteen based food waste employing similar conditions (Fig. 1). H2 production varied as a function of increasing organic load of food waste. Increment in organic load showed corresponding increment in H2 production [0.35 l (5 g COD/l); 0.5 l (10 g COD/l); 1.5 l (20 g COD/l); 1.8 l (30 g COD/l)], which indicated tolerance of native bacteria in acidogenic condition. All the bioreactors showed more or less similar performance with ±0.5 l variation. Further increment in substrate load (50 g COD/l) showed a marked drop in H2 production in all the bioreactors. The system inhibition observed at higher substrate load (over-load) might be attributed to inhibition of acidogenic bacteria. pH and VFA profiles were in good agreement with each other and with H2 production as a function of organic load (Fig. 2). A gradual drop in outlet pH was observed from 4.2 to 3.6 with increment in organic load from 5 g COD/l to 20 g COD/l. H2 production is generally accompanied by acid production due to the acidogenic metabolism (Venkata Mohan et al., 2007a). Corresponding to pH drop, VFA production showed a concomitant rise at every additional increment in organic load. VFA concentration varied from 606 mg/l (5 g COD/l) to 1478 mg/l (20 g/l). VFA production attributed to conversion of organic fraction to short chain acid intermediates in the anaerobic microenvironment. Bio-electrochemical behaviour of the biocatalyst showed prominent reduction reactions with increase in organic load from 5 to 30 g COD/l. The peak shifted from 0 to 0.3 V with increase in reduction catalytic current (-75 nA to -0.7 lA) (Supplementary Fig. 4) which supports the VFA generation and drop in pH. On the contrarily, increment in the organic load to 50 g COD/l showed a marked drop in VFA production which correlates with the process inhibition observed. Outlet pH showed a

Bacillus subtilis Pseudomonas Lysinibacillus fusiformis Control

3.1.1. Bioaugmentation vs biohydrogenesis In order to regain the performance at 50 g COD/l load, bioaugmentation strategy was employed. All the three bioaugmented systems [Reactor 1 (Control, non-augmented system), Reactor 2 (B. subtilis), Reactor 3 (P. stutzeri) and Reactor 4 (L. fusiformis)] were operated under the same operating conditions at organic load of 50 g COD/l for 30 cycles. After augmentation, a significant rise in H2 production was observed compared to pre-augmentation and control operation (Fig. 1; Supplementary Fig. 2). However, the increment in H2 production was dependent on the bioaugmented biocatalyst. P. stutzeri showed comparatively higher H2 production than L. fusiformis and B. subtilis for initial three cycles. After three cycles of operation, B. subtilis started showing higher H2 (2.1 l) production followed by L. fusiformis (1.0 l), P. stutzeri (0.8 l) and control (0.2 l). The same trend continued for the next 30 cycles. Many Bacillus species like B. subtilis, B. licheniformis, and B. coagulans were reported to be capable of producing H2 effectively (Li and Fang, 2007). B. subtilis is metabolically versatile belonging to Firmicutes (gram-positive, endospore forming) and is most studied in terms of physiology, biochemistry and genetics after Escherichia coli. It has several industrial applications (Kunst et al., 1997) and can utilize a wide range of carbon sources. P. stutzeri is a gram-negative bacterium belonging to phylum proteobacteria. L. fusiformis (previously called as Bacillus fusiformis) is a gram-positive bacteria belonging to phylum Firmicutes (Ahmed et al., 2007). Many Bacillus species including B. licheniformis, B. subtilis and B. coagulans were reported to produce H2 effectively (Li and Fang, 2007; Goud et al., 2012).

20

Acetic acid VFA Composition (%)

VFA Composition (%)

30

sharp raise towards neutral pH, as a consequence of low VFA synthesis not interfering much with the in situ system buffering microenvironment. Thus it can be concluded that organic shock-load lead to feed-back inhibition and consequent reticence to H2 production.

20

10

Butyric acid

15

10

5

0

0 12

24

30

48

12

Time (h)

30

48

Time (h) 4

Propionic acid VFA Composition (%)

VFA Composition (%)

1.75

24

1.7

1.65

1.6

Iso valaric acid

3

2

1

0

1.55 12

24

30

Time (h)

48

12

24

30

48

Time (h)

Fig. 3. VFA profile with the function of cycle period and experimental variations studied.

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The success of bioaugmentation basically depends on three factors viz., survival of augmented biocatalyst in new environment, fortifying the requisite function of the native community and most important one is improving the targeted process performance. In this study, all the augmented systems showed significant increment in process performance with different degrees compared to the control operation, which reiterates the success of the strategy. Bioaugmented species succeeded to compete with the native population and were able to overcome process inhibition and resumed the H2 production.

After augmentation, a distinct metabolic pattern signifying the change in the metabolic behaviour of native microflora in the reactor was observed due to persistence of augmented strain, when compared to control operation. This was specifically evident from change in biogas composition, outlet pH, VFA concentration and composition. Biogas composition documented dominance of H2 over CH4 specifically after augmentation with B. subtilis (Fig. 1b). B. subtilis augmented system documented higher fraction of H2 at 24th h (28–30%) followed by L. fusiformis (19%) and P. stutzeri (8–10%). On the contrary, control operation showed higher fraction

80 Control

Pseudomonas stutzeri

Bacillus subtillus Lysinibacillus

COD removal (%)

60

40

20

Before augmentation

5 g/l

10 g/l

After augmentation

50 g/l

30 g/l

20 g/l

0 0

5

10

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50

Cycle

Dehydrogenase (µg/ml of Toulene)

3.5

Bacillus subtilis Lysinibacillus fusiformis

Pseudomonas Control

3.0 2.5 2.0 1.5 1.0 0.5 0.0 5 g/l

10 g/l

20 g/l

30 g/l

50 g/l

COD Fig. 4. Substrate (COD) degradation and dehydrogenase variation during the experimental studied [cycle 1–5 (5 g/l), cycle 6–10 (10 g/l), cycle 11–15 (20 g/l), cycle 16–20 (30 g/l), cycle, 31–50 (50 g/l)] and dehydrogenase variation profile at different organic loading rates at different cycles: 5 g COD/l (cycle: 3), 10 g COD/l (cycle: 8), 20 g COD/l (cycle: 14), 30 g COD/l (cycle: 18) for before augmentation and 50 g COD/l (cycle: 45) is for after augmentation.

Please cite this article in press as: Goud, R.K., et al. Bioaugmentation of potent acidogenic isolates: A strategy for enhancing biohydrogen production at elevated organic load. Bioresour. Technol. (2014), http://dx.doi.org/10.1016/j.biortech.2014.03.049

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of CH4 (26%) than H2 (3%) due to prominence of methanogenesis due to the dominance of methanogenic bacteria (MB). The change in biogas composition after augmentation clearly depicts a metabolic shift from methanogenesis to acidogenesis. Augmented systems evidenced peak shift towards negative potential (control: 0 V, P. stuzeri: -0.1 V, L. fusiformis and B. subtilis: -0.3 V) with increase in reduced catalytic current (Control: -0.5 lA; Pseudomonas: -3.7 lA; Bacillus: -0.40 lA; Lysini Bacillus: -3.5 lA) (Supplementary Fig. 4). Among three isolates, B. subtilis augmented system showed more reduced reaction favoring higher fraction of H2.

augmented system recorded higher fraction of acetate (27.8%) followed by L. fusiformis (14.4%), P. stutzeri (7.8%) and control (3.6%) operation. Highest butyric acid composition was also observed in B. subtilis augmented operation (20.8%) but followed by P. stutzeri (16.2%), L. fusiformis (10.4%) and Control (3.5%). However, higher isovalaric acid was observed in control reactor (3.8%) followed by L. fusiformis augmented (2.7%) and P. stutzeri (16.2%) augmented reactors. Propionic acid was observed in control operation alone. The co-production of propionic acid is detrimental as it decreases overall H2 production due to demand of the additional two moles of H2 for propionic acid pathway. H2 yields and end products are variable based on the metabolic pathway chosen by the bacteria (Mohanakrishna and Venkata Mohan, 2013). Absence of propionic acid in the augmented reactors provides a microenvironment which is favorable for H2 generation rather than consumption. A gradual increment in the fatty acid concentration was observed up to 30 h of the cycle period enumerating the accumulation, followed by drop indicating their utilization. The high VFA synthesis achieved in bioaugmented systems indicates favourable conditions for acidogenic H2 production. System pH also plays a crucial role in regulation of metabolic reactions in all know living systems. Initially during the startup (cycle 1–5) with low organic load of 5 g COD/l, all the reactors showed outlet pH in the range of 4.2–4.25 (Fig. 2b). When load was increased to 10 g COD/l (cycle 6–10), pH varied between 4.3 and 4.5. At 20 g COD/l operation, pH drop was significant in all the reactors with almost similar H2 production levels. Gradually

3.2. VFA and pH variation

c

3.0u 2.0u 1.0u 0 Control -1.0u Lysinibacillus -2.0u Pseudomonas -3.0u Bacillus -4.0u -0.75 -0.50 -0.25 0 0.25 0.50 0.75 E/V vs Ag/AgCl

Current (A)

0.2u 0.1u 0 -0.1u -0.2u R2... -0.3u -0.4u R3... -0.5u R1... -0.6u R4... -0.7u -0.75 -0.50 -0.25 0 0.25 0.50 0.75 E/V vs Ag/AgCl

e

g

0.0u -0.0u -0.1u R1... -0.1u R2... R4... -0.2u R3... -0.2u -0.75 -0.50 -0.25 0 0.25 0.50 0.75 E/V vs Ag/AgCl

b

0.1u 0 -0.1u R4... -0.2u -0.3u R3... -0.4u R2... -0.5u R1... -0.6u -0.7u -0.75 -0.50 -0.25 0 0.25 0.50 0.75 E/V vs Ag/AgCl

d

0.75u 0.50u Lysinibacillus 0.25u 0 Pseudomonas -0.25u Bacillus Control -0.50u -0.75u -0.750-0.500 -0.250 0 0.250 0.500 0.750 E/V vs Ag/AgCl

f

Current (A)

Current (A)

Current (A)

Current (A)

a

Current (A)

Marked variation in VFA production was observed after augmentation (Fig. 2). Higher VFA production was observed in B. subtilis (7 g/l) augmented reactor followed by L. fusiformis (6.5 g/l), P. stutzeri (6 g/l) and control (1.2 g/l) which correlated well with H2 production and pH profiles. Composition of VFA also showed distinct variation based on the organism used for augmentation (Fig. 3). Composition of acidic intermediates reflects changes in the acidogenic metabolic pathway and provides a better insight into process monitoring and control. After augmentation, all the experimental variations documented the presence of acetic acid, butyric acid, iso-valeric acid and propionic acid with variable concentrations. Acetate composition showed a gradual increment with time in all the experimental conditions except control. B. subtilis

75.0n ...R2 50.0n 25.0n 0 -25.0n R4... -50.0n R1... -75.0n ...R3 -100.0n -0.75 -0.50 -0.25 0 0.25 0.50 0.75 E/V vs Ag/AgCl

7

h

Fig. 5. Voltammograms profiles recorded during differential experimental variations studied [(a) 5 g COD/l; (b) 10 g COD/l; (c) 20 g COD/l; (d) 30 g COD/l; (e) 50 g COD/l (20th cycle), (f) 50 g COD/l (40th cycle)] and (g and h) Tafel slopes at different organic load and after augmentation.

Please cite this article in press as: Goud, R.K., et al. Bioaugmentation of potent acidogenic isolates: A strategy for enhancing biohydrogen production at elevated organic load. Bioresour. Technol. (2014), http://dx.doi.org/10.1016/j.biortech.2014.03.049

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Table 1 Total count of the bacterial population with DAPI and Cy3. Bioreactors

DAPI

Bacillus subtilis Pseudomonas stutzeri Lysinibacillus fusiformis Control (non-augmented operation)

0.89  105 0.90  105 0.81  105 0.58  105

Cy3 Firmicutes (per ml)

Bacillus (per ml)

Pseudomonas (per ml)

0.74  105 0.10  105 0.67  105 0.02  105

0.58  105 0.08  105 0.47  105 0.03  105

0.04  105 0.39  105 0.06  105 0.05  105

when load increased to 30 g COD/l, pH variation was observed between 4.6 and 4.63 with good H2 production. After augmentation (30 cycles of operation), marked variation in pH was observed depending on the substrate utilization, VFA production and also the type of strain used for augmentation. B. subtilis augmented system showed a good performance in terms of H2 production with pH variation between 5 and 3.28. P. stuzeri augmented reactor showed good substrate degradation efficiency (Fig. 4) compared to other two strains and control with pH variation range from 4.9 to 4.0. L. fusiformis showed good H2 production after B. subtilis with operational pH range between 5.08 and 4.8. Control reactor showed pH variation between 5.4 and 4.8 at 50 g COD/l operation. Metabolic activity was relatively low yielding less H2 production associated with less VFA generation due to shock-load. 3.3. Substrate degradation Substrate degradation rate (SDR) varied as a function of organic load of food waste (Fig. 4). Increment in organic load showed increment in H2 production along with SDR [2.75 kg COD/m3-day (5 g COD/l); 4.9 kg COD/m3-day (10 g COD/l); 10.3 kg COD/m3-day (20 g COD/l); 14.9 kg COD/m3-day (30 g COD/l)], which indicated effective utilization of the substrate by the biocatalyst with increasing organic load. Further increment in substrate load (50 g COD/l) caused a distinct drop in SDR along with H2 production in all the bioreactors. After augmentation, SDR increased gradually in all the reactors. Initially substrate degradation was higher in L. fusiformis augmented system compared to P. stutzeri, B. subtilis and control (Fig. 4). While at the end of the experiment, higher SDR was observed with P. stutzeri (23.9 kg COD/m3-day; 55.7%) followed by B. subtilis (22.8 kg COD/m3-day; 53.3%), L. fusiformis (21.8 kg COD/m3-day; 50.8%) and control (21.4 kg COD/m3-day; 50%) systems. Specifically after augmentation, COD removal efficiency dropped due to metabolic shift towards acidogenesis rather than methanogenesis. 3.4. Dehydrogenase activity (DH) Dehydrogenase (DH) enzyme activity correlated well with the H2 production profiles (Fig. 4b). Before augmentation, DH activity increased with increase in organic load [5 g COD/l (2.15 lg/ml of Toluene); 10 g COD/l (2.41 lg/ml of Toluene); 20 g COD/l (3.11 lg/ml of Toluene); 30 g COD/l (3.24 lg/ml of Toluene); 50 g COD/l (1.06 lg/ml of Toluene)]. After augmentation, B. subtilis augmented system showed relatively higher DH activity (3.41 lg/ml of Toluene) followed by L. fusiformis (2.33 lg/ml of Toluene), P. stutzeri (1.06 lg/ml of Toluene) and control (0.98 lg/ml of Toluene) operations. Higher DH activity indicates the increased redox inter-conversion reactions which in turn lead to higher proton gradient within the cell that may result in increased H2 production. Redox reactions are catalyzed by the DH enzyme using several mediators (FAD+, NAD+, etc.). On the whole higher DH activity in B. subtilis augmented system corroborated well with microbial community suggesting selective enrichment of Bacillus species where higher process performance was registered.

3.5. Bio-electrochemical analysis Bio-electro kinetic behavior of biocatalyst in terms of redox catalytic currents, redox Tafel slopes, electrons carrier (mediators) during the e transfer process was evaluated employing cyclic voltammetry (CV) (Raghavulu et al., 2012; Velvizhi et al., 2014). Voltammograms (Vs Ag/AgCl (S) visualized significant variation in the biocatalyst behavior prior and after augmentation (Fig. 5). Increment in redox (oxidation currents (OC); reduction currents (RC)) catalytic currents along with decrement in redox slopes ((oxidation slope (ba); reduction slope (bc)) was noticed in all the four reactors with increment in organic load till 30 g/l followed by a decrement at higher load (50 g/l). Lower redox slopes were noticed at 30 g/l (ba/bc: 0.09/0.29 V/dec) followed by 20 g/l (ba/bc: 0.11/ 0.38 V/ dec), 10 g/l (ba/bc: 0.17/0.57 V/dec) and 5 g/l (ba/bc: 0.19/0.62 V/ dec). Similarly, an increment in the redox catalytic currents was noticed till 30 g/l (10 g/l, 0.1/0.2 lA; 20 g/l, 0.1/0.4 lA; 30 g/l, 0.2/0.55 lA). OC were more or less similar in all the reactors at all the load rates. Marked difference/increment in RC with decrement in redox slopes indicates the shift in biocatalyst behavior towards reduction which is in good correlation to the observed H2 production where the redox equivalents are effectively reduced towards H2 formation. Reduction behavior of biocatalyst is also in concurrence with the dehydrogenase enzyme (participates in the inter-conversion of metabolites), which influence H2 production based on protons reduction machinery. Contrary to the above observation, at higher load (50 g/l) a decrement in redox catalytic currents with simultaneous increment in redox Tafel slopes (0.1/0.35 ± 0.15 lA; ba/bc: 0.24/0.87 V/dec) was recorded and noticed in all the four reactors (Supplementary Table 2; Fig. 5). After augmentation, a marginal increment in redox catalytic currents was observed initially (B. subtilis: 0.5/ 0.60 lA > L. fusiformis: 0.3/0.62 lA > P. stutzeri: 0.1/0.30 lA). However, post augmentation (30 cycle) resulted in significant increment in redox catalytic currents (B. subtilis: 2.5/0.40 lA > P. stutzeri: 1.5/3.7 lA > L. fusiformis: 1.5/3.5 lA > Control: 0.5/0.5 lA) which are three folds higher prior to augmentation. Though the RC was marginally higher, lesser H2 production was observed with P. stutzeri, might be due to electron/proton losses. These losses would have drifted the liberated redox equivalents to get scavenged by MB or shift the pathway towards methane. A rapid decrement in redox slopes (P. stutzeri; ba/bc: 0.14/0.32 V/ dec; B. subtilis: ba/bc: 0.09/0.21 V/dec; L. fusiformis: ba/bc: 0.10/ 0.27 V/dec; Control: ba/bc, 0.19/0.41 V/dec) was also noticed indicating the requirement of less activation energy. Polarization resistance (Rp) refers to the resistance in electron transfer from the microorganism to the solution electrode interface. Higher the Rp, lesser will be the electron transfer making the process less favorable. Rp was found to be less in augmented reactors (P. stutzeri: 1.01E+7 O; B. subtilis: 9.53E+6 O; L. fusiformis: 9.93E+6 O) than the control (1.18E+7 O) reactors signifying the impact of bioaugmentation in boosting the biocatalyst capabilities towards electron delivery. B. subtilis augmented reactor showed less Rp, lower slopes and higher redox currents in comparison to other augmented reactors and control that is in good agreement

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with the observed higher H2 production. Tafel plots also visualized a marked shift towards negative potential in comparison to control which is a good sign of reduction behavior/reactions due to the altered metabolic activities of bacteria attributing to the affirmative influence of augmentation strategy (Fig. 5). DCV analysis facilitates the interpretation of the rate of change in voltammetric current (i) with respect to the electrode potential E (di/dE) (Murthy et al., 2012). Extracellular electron transfer (EET) sites are detected in DCV as peak potentials. The presence of peak corresponds to the involvement of redox mediator/shuttler in the electron transfer during redox reactions. Various peaks were identified during DCV analysis prior and after augmentation. At 5 and 10 g/l, a quasi reversible peak with potential of 0.211 V was detected that might correspond to the involvement of cytochrome-C during the electron transfer. At 20 g/l, two quasi reversible peaks with peak potentials of 0.11 V and 0.22 V was detected corresponding to the involvement of cytochrome-bc1 and cytochrome-C, whereas at 30 g/l, two different quasi reversible peaks were detected with 0.171 V and 0.06 V potential corresponding to the involvement of cytochrome bc1 and quinone respectively. At 50 g/l, no peaks were detected. However after augmentation, more number of redox species in the form of peaks was found to be involved in the electron transfer that might aided in the H2 production process. B. subtilis augmented reactor depicted three quasi reversible peaks with potential of 0.091 V, 0.332 V and 0.101 V that might correspond to the involvement of cytochrome-bc1, cytochrome-C and Fe-S proteins respectively. With the reactors augmented with L. fusiformis and P. stutzeri two peaks were detected with peak potentials of 0.10 V and 0.08 V which might attribute to the participation of cytochrome-bc1 and quinines. The visualized redox species (RS) are the membrane bound proteins that serve as electron carriers in transferring the electrons. After augmentation, B. subtilis documented more number of RS than the other two acidogenic isolates as well as control illustrating the capability in enhancing the H2 production. RS such as Fe-S and cytochromes proteins with tightly bound prosthetic groups undergo reversible oxidation and reduction. The involvement of RS is a clear indication of the enhanced electron transfer by the minimization of losses which also corroborates with the observed H2 production. Bio-electrochemical analysis clearly depicted the specific changes in the metabolic activity after augmentation towards H2 production. 3.6. FISH Along with specific bacterial group (Bacillus and Pseudomonas), bacteria belonging to phylum Firmicutes were evaluated using phylum and genus specific Cy3 labeled probes targeted to 16S rRNA gene sequences (Supplementary Fig. 3). Total bacterial population count (DAPI counter staining) was also monitored to differentiate the total bacteria among the specific bacterial population. The distribution of the bacterial community showed marked variation among the augmented systems and correlated well with the H2 production (Table 1). FISH analysis reveals that the specific group of bacteria used for augmentation showed marked influence on the community structure which directly influenced the H2 production. Compared to control, specific bacterial group used for augmentation has persisted and sustained in respective system indicating the primary success of bioaugmentation. Persisted Firmicutes/Bacillus after augmentation in the system facilitated marked improvement in the performance for which it was anticipated. Change in the diversity of microorganisms also infers change in the size and composition of the population. Firmicutes have diverse metabolic activity and can withstand diverse environmental conditions. Bacteria belonging to phylum Firmicutes, especially Bacillus sp. and Clostridia sp. are potential H2 producers

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(Karakashev et al., 2009; Venkata Mohan et al., 2011), which have the ability to survive the harsh acidic conditions by forming endospores. C. acetobutylicum (Esteso et al., 1996), C. butyricum, C. kluyveri and C. pasteurianum (Brosseau et al., 2007) were reported to produce H2. Bacillus sp. viz., B. licheniformis, B. subtilis and B. coagulans were reported to produce H2 effectively (Goud et al., 2012). 4. Conclusions Bioaugmentation strategy was successfully applied by addition of potent acidogenic isolates in enhancing fermentative H2 production at elevated organic load. B. subtilis augmented system showed higher H2 production compared to L. fusiformis, P. stutzeri and control operations. Higher acetate concentration observed with B. subtilis augmented system depicted acidogenic metabolic pathway. Microbial community structure indicated selective enrichment of Firmicutes/Bacillus sp. after augmentation with Bacillus sp. B. subtilis augmented reactor documented lower redox slopes with high redox reactions along with lower polarization resistance. Acknowledgements The authors wish to thank the Director, CSIR-IICT for encouragement in carrying out this work. Research was supported by Council of Scientific and Industrial Research (CSIR; 12th five year plan projects - WUM (ESC-0108) and SETCA (CSC-0113) and Ministry of New and Renewable Energy (MNRE, Project. No. 103/131/ 2008-NT). R.K.G. acknowledge CSIR for the research fellowship. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.biortech.2014. 03.049. References Ahmed, I., Yokota, A., Yamazoe, A., Fujiwara, T., 2007. Proposal of Lysinibacillus boronitolerans gen. nov. sp. nov., and transfer of Bacillus fusiformis to Lysinibacillus fusiformis comb. nov. and Bacillus sphaericus to Lysinibacillus sphaericus comb. nov.. Int. J. Syst. Evol. Microbiol. 57 (5), 1117–1125. Amann, R.I., Ludwig, W., Schleifer, K.H., 1995. Phylogenetic identification and In situ detection of individual microbial cells without cultivation. Microbial reviews. 59 (1), 143–169. APHA, 1998. Standard Methods for the Examination of Water and Wastewater, 20th ed. American Public Health Association/American Water Works Association/ Water Environment Federation, Washington, DC. Bouchez, T., Patureau, D., Dabert, P., Wagner, M., Deigenes, P., Moletta, R., 2000. Successful and unsuccessful bioaugumentation experiments monitored by fluorescent in situ hybridization. Water Sci. Technol. 41, 61–68. Brosseau, J.D., Zajic, J.E., 2007. Hydrogen-gas production with Citrobacter intermedim and Clostridium pasteurianum. J Chem Technol Biotechnol. 32, 496–502. Chong, N.M., Pai, S.L., Chen, C.H., 1997. Bioaugmentation of an activated sludge receiving pH shock loadings. Bioresour. Technol. 59, 235–240. Esteso, M.A., Estrella, C.N., Podesta, J.J., 1996. Evaluation of the absorption on mild steel of hydrogen evolved in glucose fermentation by pure cultures of Clostridium acetobutylicum and Enterobacter. Sens. Act. B: Chem. 32, 27–31. Goldstein, M.G., Mallory, L.M., Alexander, M., 1985. Reasons for possible failure of inoculation to enhance biodegradation. Appl. Environ. Microbiol. 50, 977–983. Goud, R.K., Venkata Mohan, S., 2013. Prolonged applied potential to anode facilitate selective enrichment of bio-electrochemically active Proteobacteria for mediating electron transfer: microbial dynamics and bio-catalytic analysis. Bioresour. Technol. 137, 160–170. Goud, R.K., Raghavulu, S.V., Mohanakrishna, G., Naresh, K., Venkata Mohan, S., 2012. Predominance of Bacilli and Clostridia in microbial community of biohydrogen producing biofilm sustained under diverse acidogenic operating conditions. Int. J. Hydrogen Energy 37 (5), 4068–4076. Hajji, K.T., Lepine, F., Bisaillon, J.G., Beaudet, R., Hawari, J., Guiot, S.R., 2000. Effects of bioaugmentation strategies in UASB reactors with a methanogenic consortium for removal of phenolic compounds. Biotechnol. Bioeng. 67, 418–423. Karakashev, D., Kotay, S.M., Trably, E., Angelidaki, I., 2009. A strict anaerobic extreme thermophilic hydrogen-producing culture enriched from digested household waste. J Appl Microbiol. 106 (3), 1041–1049.

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Please cite this article in press as: Goud, R.K., et al. Bioaugmentation of potent acidogenic isolates: A strategy for enhancing biohydrogen production at elevated organic load. Bioresour. Technol. (2014), http://dx.doi.org/10.1016/j.biortech.2014.03.049