Microbial diversity of hydrogen producers and their metabolic characteristics

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2 Microbial diversity of hydrogen producers and their metabolic characteristics

3 Bacteria used in this study were characterized for their ability to grow at different physiological conditions such as salt and metals, antibiotics and different ph values. Subsequently these microbes were checked for their abilities to produce H 2 from a wide range of sugars. The process of H 2 production was optimized and the enhancement of the process was achieved through immobilization of microbes under continuous culture conditions. The mixed hydrolytic cultures were used to hydrolyze biowaste and produce H 2 from the hydrolysate with the help of well defined mixed microbial culture (MMC) of H 2 producers. 4.1 Biodiversity of microbial isolates Exploration and exploitation of the microbial biodiversity for the preparation of microbial consortia for hydrolysis of complex substrates such as biowastes and H 2 production from them are likely to lead to the development of robust systems, which may ensure process economy. Microbial isolates to represent high biodiversity were collected from a wide range of natural and synthetic ecosystems such as Contaminated food sample (Pickle waste), Marine coastal Water (Goa), MRL sludge sample (Madras Refinery Limited), IIT ETP sludge sample (Pesticide ETP, Mumbai), Pesticide ETP sludge sample (Gardha ETP, Chennai). These microbial isolates were segregated on the basis of their various physiological parameters such as ability to grow on media at different ph from 2-12, salt concentrations (NaCl, % - nil, 0.5, 2.5, 5), sensitivity to metals i.e. nickel, and cobalt (1 mm) and their sensitivity to 12 different antibiotics (Ampicillin, Carbenicillin, Chloramphenicol, Gentamycin, Kanamycine, Nalidixic acid, Penicillin, PolymyxinB, Rifampicin, Streptomycin, Tetracycline and Vancomycin). The microbial biodiversity was validated by the magnitude of variation in their 16S rrna gene (~1.5 kb) sequences. The best match of the 16S rdna sequences were found among genera belonging to CFB Group of bacteria; firmicutes- Aneuribacillus, Anoxybacillus, Planomicrobium and Bacillus; α- proteobacteria - Rhodobacter; β-proteobacteria - Bordetella and γ-proteobacteria - Alcanivorax, Enterobacter, Marinobacter, Microbacterium, Proteus, Pseudomonas and a marine bacterium. 58

4 4.2 Metabolic characteristics of strains Out of 47 strains, 10 strains: 7 belonging to Bacillus, 2 belonging to Proteus and 1 belonging to Enterobacter had good metabolic characteristics such as the ability to metabolize a wide range of sugars, etc. (Table 4.1). Table 4.1: Biochemical characterization of microbes tested for hydrogen producing abilities S. Substrate Bacterial strain No. EGU HPC Monosaccharides 1 Glucose + a + - b Dextrose Fructose Glucosamine Ribose Xylose L-arabinose - w c D-arabinose Disaccharides 9 Sucrose - + w Maltose + - w Trehalose Lactose w Galactose w Saccharose Raffinose Melibiose w 17 Cellobiose Polysaccharides 18 Inulin w Sugar alcohol 19 Glycerol w Sorbitol w Mannitol Amino acid metabolism 22 Lysine decarboxylase w + w w - 23 Ornithine decarboxylase w 24 Phenylalanine deaminase Others 25 Citrate utilization Malonate Alkaline phosphatase Sodium gluconate Esculin ONPG β-galactosiadse + w Arginine utilization Urease Nitrate reductase a: Positive response; b: Negative response; c: Weak response. 59

5 These strains have shown positive response towards 33 different substrates out of 47 tested. Proteus strains EGU32 and EGU34 showed positive response (change in colour) on arabinose, ribose, xylose, dextrose, trehalose and glycerol, whereas P. mirabilis EGU32 could also utilize fructose, glucose and saccharose. Out of the 7 Bacillus strains, EGU41 could metabolize a large number of sugars but could not grow on citrate and malonate. The other 6 Bacillus strains EGU385, EGU399, EGU542, HPC459, HPC464 and HPC686 showed a behavior which was complementary to that of EGU41. The 10 strains were able to utilize different nitrogen sources. These strains were not able to metabolize certain substrates such as mannose, rhamnose, melezitose, sorbose, α-methyl-d-mannose, dulcitol, inositol, adonitol, xylitol, indole, salicin and methyl red. In view of wide variability in their metabolic activities, a consortium of these strains may result in efficient solubilization of a wide range of complex organic matter components. Since these strains did not produce H 2 S, this may prove beneficial for improving H 2 production process. The ability of Proteus strains to hydrolyze esculin (a glycoside) into glucose and esculetin, which possess multiple pharmacological activities, can also be exploited further. 4.3 Growth curve of microbial cultures Hydrolytic bacteria Different hydrolytic bacteria (used for preparing different mixtures) revealed very good growth pattern on NB medium (Figure 4.1). Almost all the strains showed very good growth from an initial OD 660 value of 0.05 to around after 20 h of incubation. Figure 4.1: Growth curves of hydrolytic mixed culture strains on NB medium 60

6 4.3.2 Hydrogen producers H 2 producing bacteria (used for preparing mixed microbial cultures) were found to grow well on NB medium (Figure 4.2a) and M-9 medium (0.05x) (Figure 4.2b). Growth was found to get stabilized after 18 h on NM and within 9 h of incubation on M-9 medium. The final OD 660 at these stages was in the range Thus it can be concluded that these media are suitable for bacteria used in this study. Figure 4.2a: Growth curves of mixed microbial culture strains in NB medium Figure 4.2b: Growth curves of mixed microbial culture strains in M-9 medium (0.05x) 4.4 Hydrolytic abilities of microbial isolates In order to exploit the H 2 producing abilities of different microbes, it is desirable to produce it from cheap raw materials such as biological wastes. It is economical to use those microbes which also solubilize the various constituents of the biowastes. So, strains were checked for amylase, lipase and protease activities over a wide range of ph 5-12 (Table 4.2). The proteolytic activities of certain strains of Bacillus sp., Bordetella avium and P. mirabilis were quite high, with abilities to produce a zone of hydrolysis in 61

7 the range of mm. Bacillus strains EGU444, EGU445 and EGU447 were among the highest protease producers. On the other hand P. mirabilis strains EGU21, EGU30, EGU32 and EGU34 had a zone of hydrolysis in the range of mm and also had a high relative protease activity of Similarly, B. cereus EGU48, B. thuringiensis strains EGU47 and EGU378, B. subtilis EGU475, B. sphaericus strains EGU385, EGU399 and EGU542, Pseudomonas stutzeri EGU396 and marine bacterium EGU409 had a zone of hydrolysis in the range of mm with a relative lipase activity up to 2.7. The abilities of these microbial isolates to metabolize starch were quite variable i.e mm and the relative amylase activities varied from 1-5. Maximum amylase activities of 23 mm were recorded with B. thuringiensis EGU378, Pseudomonas sp. EGU448 and marine bacterium strain EGU409. Table 4.2: Hydrolytic activities of selected bacterial isolates from diverse environmental samples Strain Enzyme activities a Lipase Amylase Protease ph for activity ZOH b RLA c ZOH RAA d ZOH RPA e Range Optimum EGU EGU EGU EGU EGU EGU nz f na g EGU EGU EGU EGU EGU a: Lipase activities of the strains observed data of 7 day, for amylase and protease were 2 day; b: Zone of hydrolysis; c: Relative lipase activity; d: Relative amylase activity; e: Relative protease activity; f: No zone; g: Not applicable. 4.5 Hydrogen production abilities of microbial isolates H 2 producing abilities of bacteria belonging to CFB Group of bacteria- Myroides odoratus; firmicutes - Aneuribacillus aneurinilyticus, Anoxybacillus flavithermus, Anoxybacillus sp., Bacillus sp., B. cereus, B. thuringiensis, B. subtilis, B. licheniformis, 62

8 B. pumilus, B. sphaericus, B. megaterium, Planomicrobium sp.; α-proteobacteria- R. sphaeroides; β-proteobacteria - B. avium and γ-proteobacteria - Alcanivorax sp., E. aerogenes, Marinobacter aqueolei, Microbacterium paraoxydans, P. mirabilis, Pseudomonas sp., P. stutzeri and marine bacterium (Table 4.3) varied considerably from as low as 100 ml by B. licheniformis EGU14 to as high as 420 ml by E. aerogenes EGU16 i.e., a 4.2 fold higher yield from the same amount of feed. This amounted to a H 2 yield of mol/mol of glucose fed. Of the total biogas (a mixture of H 2 and CO 2 ), H 2 comprised 31-70% and CO %. No CH 4 was observed in any of the reactions. Most of the isolates have shown that a large proportion (>80%) of the total biogas evolution takes place within 2-3 days of incubation. The process becomes slower thereafter. The evolution of H 2 was seen to follow a pattern in the drop of ph, which were 4.94 on an average at the end of every 24 h of incubation. On the subsequent days the ph was observed to fall to 5.65 and The microbial growths were in the range OD for B. megaterium HPC686 and B. licheniformis EGU14, respectively. At high partial pressure of H 2 the biochemical reactors are endergonic and inhibited by the product - H 2. Accumulation of organic acids and a possible drop of ph contribute to biochemical inhibition of the H 2 production (Valdez-Vazquez et al., 2005). Table 4.3: Hydrogen producing abilities of bacterial isolates S. No. CFB Group of bacteria Organism * Biogas a (ml) Vol (ml) Hydrogen (H 2 ) Yield b % Final c OD Myroides odoratus EGU882 (EF633227) Firmicutes 2 Anoxybacillus hidirlerensis EGU Anoxybacillus sp. EGU145 (EF633214) Anoxybacillus sp. EGU146 (EF633214) Bacillus sp. EGU15 (DQ487038) Bacillus sp. EGU85 (DQ768239) Bacillus sp. EGU91 (DQ915850) Bacillus sp. EGU367 (DQ768236) Bacillus sp. EGU444 (DQ768240) Bacillus sp. EGU445 (DQ915849) Bacillus sp. EGU447 (DQ508976) Bacillus sp. HPC459 (DQ460033) Bacillus cereus EGU3 (DQ487039)

9 S. No. Organism * Biogas a (ml) Vol (ml) Hydrogen (H 2 ) Yield b % Final c OD B. cereus EGU41 (DQ508967) B. cereus EGU43 (DQ508969) B. cereus EGU44 (DQ508970) B. cereus EGU46 (DQ508972) B. cereus EGU48 (DQ508974) B. licheniformis EGU14 (DQ768246) B. licheniformis EGU90 (DQ768243) B. thuringiensis EGU45 (DQ508971) B. thuringiensis EGU47 (DQ508973) B. thuringiensis EGU378 (DQ487033) B. subtilis EGU17 (DQ915853) B. subtilis EGU163 (DQ508966) B. pumilus EGU49 (DQ508975) B. pumilus HPC464 (DQ460035) B. sphaericus EGU385 (DQ487032) B. sphaericus EGU399 (DQ487036) B. sphaericus EGU542 (DQ508975) B. megaterium HPC686 (AY818016) Aneuribacillus aneurinilyticus EGU Planomicrobium sp. EGU782 (EF633306) α- proteobacteria 34 Rhodobacter sphaeroides EGU50 (DQ915852) β- proteobacteria 35 Bordetella avium EGU31 (DQ915851) γ- proteobacteria 36 Enterobacter aerogenes EGU16 (DQ768244) Proteus mirabilis EGU21 (DQ768232) P. mirabilis EGU30 (DQ487041) P. mirabilis EGU32 (DQ508964) P. mirabilis EGU34 (DQ508965) Alcanivorax sp. EGU619 (EF633237) Pseudomonas stutzeri EGU394 (DQ487034) P. stutzeri EGU396 (DQ487035) Pseudomonas sp. EGU448 (DQ768241) Marinobacter aqueolei EGU893 (EF633261) Microbacterium paraoxydans EGU Unclassified 47 Marine bacterium EGU409 (DQ487037) Values based on two sets of experiments and two repetitions. Standard deviation was less than 10%. a: Biogas is a mixture of H 2 + CO 2 ; b: mol H 2 /mol of glucose fed; c: Initial OD 660 was 0.4. Medium used: M-9 (1x) supplemented with 2% glucose. Total volume of feed used: 250 ml. Inoculum added at the rate of 40 µg cell protein/ml feed. * View accession no. at 64

10 CFB Group of bacteria- M. odoratus has ability to produce H 2 yield of 0.23 mol/mol glucose fed, which has constituted 32% H 2 of total biogas. Among the various Bacillus species, B. cereus and B. thuringiensis were equally competent to produce 390 ml H 2 (observed volume) equivalent to 0.63 mol H 2 /mol of glucose fed. However, there was a large variability in H 2 producing abilities among the strains belonging to the two Bacillus spp.: from mol/mol of glucose added for B. cereus strains and from mol/mol of glucose added for B. thuringiensis strains. Bacillus sp. strains have abilities to produce H 2 in the range of mol/mol glucose fed. The other 5 species of Bacillus had a relatively low H 2 yielding range: mol/mol of glucose fed. R. sphaeroides EGU50 produced 210 ml of H 2 equivalent to a yield of mol/mol glucose fed. Anoxybacillus spp. show quite large variation in the H 2 production yields, which were in the range of mol/mol glucose fed, where H 2 constitutes 39-60% of total biogas. On the other hand, A. aneurinilyticus EGU345 and Planomicrobium sp. EGU782 produced mol H 2 /mol glucose fed. Proteus and Pseudomonas strains produced moderate to low amounts of H 2 : mol/mol of glucose fed, equivalent to 39-65% of the total biogas. High H 2 yielding strains also had higher H 2 content, invariably in the range of 60-70%. It may be remarked here that these H 2 producing capacities of the various strains were found to be negatively influenced by the growth of the cultures. Alcanivorax sp., M. aqueolei and M. paraoxydans produced H 2 yield of 0.27, 0.33 and 0.26 mol/mol glucose fed, respectively. Marine bacterium produces 240 ml of H 2 equivalent to yield of 0.39 mol/mol glucose fed. It may be reasonable to conclude at this stage that the H 2 yields observed here can be improved by optimization of various process parameters of H 2 producers and by preparing different consortia. 4.6 Batch culture hydrogen production abilities of mixed microbial consortia Mixed microbial consortia of H 2 producers were prepared based on Plackett-Burman design. Eleven different consortia were developed on the basis of 11 best H 2 producers: B. cereus EGU41 and EGU43; B. thuringiensis EGU45; Bacillus sp. EGU91 and HPC459; B. pumilus HPC464; B. megaterium HPC686; E. aerogenes EGU16; P. mirabilis EGU21 and EGU30; and B. avium EGU31. Each consortium had 6 strains each. On individual basis, these 11 strains have been found to evolve ml of biogas from 2% glucose 65

11 solution (250 ml). Here, H 2 constituted 61-70% of the total biogas (Table 4.4). The H 2 producing abilities of 11 bacterial strains in various combinations designated as MMC1- MMC11 have been presented in Table 4.4. On mixing these strains in equal proportion as in MMC1-MMC11, we expected to generate ml H 2 from 2% glucose solution (250 ml). However, the observed H 2 productions by different MMCs were in the range of ml. Thus H 2 yield on mixing different cultures were much lower than the average of their individual capacities. Out of the 11 MMCs, only two - MMC4 (B. cereus EGU43, B. thuringiensis EGU45, Bacillus sp. HPC459, B. pumilus HPC464, E. aerogenes EGU16 and P. mirabilis EGU21) and MMC6 (B. cereus EGU41, B. megaterium HPC686, B. pumilus HPC464, E. aerogenes EGU16, P. mirabilis EGU21 and EGU30) evolved H 2 in the range of ml equal to mol/mol glucose, which were 32.5 and 20.5% lower than the expected values of 385 and 380 ml, respectively. We expected these two consortia, MMC4 and MMC6 to perform well on optimization of different physiological conditions. Table 4.4: Hydrogen production by mixed microbial cultures constituted on the basis of Plackett-Burman design MMC a Bacterial isolates Hydrogen (H 2 ) EGU strain No. HPC strain No. Vol (ml) Exp b Obs c Y d % e MMC1 + f + - g MMC MMC MMC MMC MMC MMC MMC MMC MMC MMC Values based on three set of experiments and three repetitions. Standard deviation was less than 10%. a: Mixed microbial culture; b: Expected volume of H 2, based on the average of H 2 producing capacities of individual strains in the mixed microbial culture under similar experimental conditions; c: Observed volume of H 2 ; d: Yield (mol H 2 /mol glucose utilized); e: % of H 2 in the biogas (H 2 + CO 2 ); f: Present; g: Absent. Medium used: M-9 (1x). Total volume of feed: 250 ml. Inoculum: 40 µg cell protein/ml feed. 66

12 Optimization of hydrogen production process from sugars and enhancement in its hydrogen yield by immobilization of microbes

13 4.7 Optimization of process parameters for hydrogen production by mixed microbial cultures under batch culture Out of 11 MMCs tested for H 2 production from glucose, MMC4 and MMC6 were selected for the optimization and enhancing H 2 yields Effect of sugars MMCs were checked for their ability to metabolize different sugars to H 2. This was primarily done in anticipation of the fact that we may be employing different biowastes as feed materials. Here we can expect a wide variation in their carbohydrate composition. It was found that MMC4 and MMC6 vary in their abilities to produce H 2 from glucose, fructose, maltose, sucrose and lactose at different feed concentrations (Table 4.5). Table 4.5: Effect of sugars on hydrogen producing abilities of mixed microbial cultures Sugar Conc. (%) Glucose Fructose Maltose Sucrose Lactose Hydrogen (H 2 ) Vol a Y b % a Vol Y c % Vol Y c % Vol Y c % Vol Y c % Mixed microbial culture: MMC4 d Mixed microbial culture: MMC6 d Values based on three set of experiments and two repetitions. Standard deviation was less than 10%. a: Volume (ml) and % of H 2 in the biogas (H 2 + CO 2 ); b: mol H 2 /mol glucose utilized; c: mol H 2 /mol hexose fed; d: See Table 4.4 for the composition of the mixed microbial culture. Medium used: M-9 (1x). Total volume of feed: 250 ml. Inoculum: 40 µg cell protein/ml feed. 67

14 H 2 production from lactose as feed was quite low with either of the MMCs. Here H 2 production abilities of MMC4 and MMC6 were in the range of mol/mol hexose utilized. On glucose as feed material, the highest H 2 yields of 0.75 and 1.1 mol/mol hexose utilized were observed with MMC4 and MMC6, respectively. The maximum H 2 yields were observed at 0.5% w/v concentration of glucose, with both the MMCs. There was a steady decline in H 2 yield with increase in glucose concentration, which was as low as mol/mol hexose at 5% w/v glucose concentration. The substrate utilization with both the mixed microbial cultures was in the range of 77-80%. Although H 2 component in the biogas declined by 27% (from 63% at 5% w/v glucose to 46% at 0.5% w/v glucose) with MMC4 and by 23% (from 65 to 52%) with MMC6, it was proportionately less dramatic than that recorded for H 2 yield, which declined by 50% with either of the MMCs. It reflects up on a negative co-relation between feed concentration and H 2 yield. The H 2 yields were in general in the range of mol/mol hexose with fructose and sucrose as feed material. In contrast, with maltose as feed material, the H 2 yields were quite high at 0.5 and 1% concentration (w/v), which were in the range of mol/mol hexose fed. In all these fermentations, a negative correlation between sugar concentration in the feed and H 2 yield was observed. In our experiments, in spite of a large variation in overall H 2 yields, the composition of biogas varied little with respect to H 2 component. It constituted between 40-65% of the total biogas evolved by MMC4 and MMC6. H 2 generation by MMC4 and MMC6 with other sugars as feed indicates the potential of these bacteria to metabolize a wide range of substrates rich in these sugars. A review of all the cases reveals that with both the mixed cultures - MMC4 and MMC6, H 2 yields on glucose were quite high and stable. As glucose has been used by many researchers because it allows better comparability and reproducibility, we proceeded to optimize the H 2 production process with M-9 medium (1x) supplemented with glucose (0.5% w/v). It was also noted here that under these conditions, the overall growth of bacteria were quite high with a final OD 660 of around It was thus desirable to check for any un-necessary microbial growth, which might be affecting H 2 yield negatively. 68

15 4.7.2 Effect of inoculum size A wide range of initial inoculum sizes ( µg cell protein/ml medium) were found to have a profound effect on H 2 yield with both the MMCs (Table 4.6). It was realized that at higher inoculum size the H 2 yield was adversely affected such that it got reduced from 0.76 mol/mol glucose utilized at 10 µg cell protein/ml medium to 0.25 mol/mol glucose utilized at 120 µg cell protein/ml medium with MMC4 and from 1.05 to 0.6 mol/mol glucose utilized under similar increase in inoculum size with MMC6. Table 4.6: Effect of inoculum size on hydrogen producing abilities of mixed microbial cultures Inoculum a Mixed microbial culture b MMC4 MMC6 Hydrogen (H 2 ) Vol (ml) Yield c % d Vol (ml) Yield % Values based on three set of experiments and two repetitions. Standard deviation was less than 10%. a: µg cell protein/ml; b: See Table 4.4 for the composition of the mixed microbial culture; c: mol H 2 /mol glucose utilized; d: % of H 2 in the biogas (H 2 + CO 2 ). Medium used: M-9 (1x). Total volume of feed: 250 ml Effect of medium concentration It was observed that medium concentration (0.1-10x of M-9) had a dramatic effect on H 2 yield and on bacterial growth as well (Table 4.7). Here, a 10 fold reduction in medium (M-9) concentration from 1 to 0.1x resulted in an increase in H 2 yield from or mol/mol glucose utilized with MMC4 and MMC6, respectively. Such an increase in H 2 yield was also accompanied by a 30-35% reduction in bacterial growth. 69

16 Table 4.7: Effect of medium concentration on hydrogen producing abilities of mixed microbial cultures Medium concentration (x) a MMC4 Mixed microbial culture b MMC6 Hydrogen (H 2 ) Vol (ml) Yield c % d Vol Yield % Values based on three set of experiments and two repetitions. Standard deviation was less than 10%. a: Medium M-9; b: See Table 4.4 for the composition of the mixed microbial culture; c: mol H 2 /mol glucose utilized; d: % of H 2 in the biogas (H 2 + CO 2 ). Total volume of feed: 250 ml. Inoculum: 10 µg cell protein/ml feed. 4.8 Continuous culture hydrogen production and enhancement in yields by immobilization of mixed microbial cultures Continuous culture process is employed for sustainable H 2 production with MMCs and its enhancement in yield with immobilization on ligno-cellulosic materials from biological origins as compared to synthetic Optimization of process parameters for hydrogen production by mixed microbial cultures under continuous culture Immobilized support material utilization Since certain Bacillus spp. have an ability to utilize some cellulosic material, we also checked the H 2 producing abilities of MMC4 and MMC6 on M9 minimal medium without any glucose supplementation under batch culture conditions (Table 4.8). No H 2 evolution was observed in reactors with BL and CC as support materials. However, a total of ml of H 2 was observed in reactors where GS and PS were 70

17 used as support material. This quantity of H 2 is produced only once and that too within the first 2-3 days (out of a total of 7 days of incubation). It is unlikely to make any significant impact on the final H 2 production rates, particularly in our case, where the best results have been recorded with BL and CC and the total volume of H 2 evolved is quite high. Table 4.8: Hydrogen production abilities of mixed microbial cultures MMC4 and MMC6 from ligno-cellulosic material used as support material Support material Mixed microbial culture a MMC4 MMC6 Biogas b (ml) H 2 c Vol % Biogas (ml) H 2 Vol % Pea-shells (PS) Banana leave (BL) nd d na e na Nd na na Coconut coir (CC) nd na na Nd na na Groundnut shells (GS) Values based on two set of experiments and two repetitions. Standard deviation was less than 10%. a: See Table 4.4 for the composition of the mixed microbial culture; b: Biogas is a mixture of H 2 +CO 2 ; c: Volume in ml and % of H 2 in biogas; d: Not detected; e: Not applicable. Medium used: M-9 (0.05x). Total volume of feed: 250 ml. Inoculum: 10 µg cell protein/ml feed Effect of substrate concentration The observations made on the effect of glucose concentration on daily fed culture H 2 production by MMCs immobilized on different ligno-cellulosic biological wastes materials - BL, CC, GS or PS packed in to PVC tubes have been presented in Table 4.9. The H 2 production abilities of immobilized MMC4 on ligno-cellulosic materials were in the range of ml of total biogas evolved at an HRT of 4 days. These values of H 2 production were higher than those recorded with FF MMC4. Almost similar H 2 production values were recorded with MMC6. It may be remarked here that H 2 production in a continuous manner was observed even after 20 days of regular feeding only in the case of 2% glucose as feed. At 0.5 and 1.0 % glucose as feed, there was a regular decline in H 2 production. However, as the H 2 production efficiency was higher at 0.5% glucose level, we preferred to optimize the process by checking the influence of medium concentration at 0.5% glucose as feed. 71

18 Table 4.9: Effect of glucose concentration on immobilized hydrogen producing mixed microbial cultures under daily fed culture conditions Support material a H 2 b Glucose concentration (%) % c H 2 % H 2 % H 2 % H 2 % H 2 % MMC4 Mixed microbial culture d MMC6 PVC+PS 40 e e e e PVC+ BL 30 e e e e PVC+CC 20 e e e e PVC+GS 35 e e e e PVC 80 e e FF e Values based on two set of experiments and two repetitions. Standard deviation was less than 10%. a: PVC: Polyvinylchloride ring, PS: Pea-shells, BL: Banana leaves, CC: Coconut coir, GS: Groundnut shells, FF: Fee floating; b: H 2 (ml/day) values represent 20 days of steady state equal to 5 cycles at an HRT of 4 days; c: % of H 2 in biogas; d: See Table 4.4 for the composition of the mixed microbial culture; e: Gas production declined within 5-10 days of starting the experiment. Medium used: M-9 (1x) supplemented with 0.5-2% glucose. Total volume of feed: 680 ml with 120 ml (15%) additional volume occupied by support material in a 1 L reactor. Inoculum: 10 µg cell protein/ ml feed Effect of medium concentration The effect of medium concentration on 0.5% glucose as feed on H 2 production by MMCs immobilized on different ligno-cellulosic biological wastes has been presented in Table FF bacterial cultures evolved ml H 2 with MMC4 and ml H 2 with MMC6 at a medium concentration of x. In both the cases, maximum H 2 generation was recorded at 0.1x medium concentration. With PVC alone as support material, H 2 generation was slightly better than FF MMCs. Here MMC4 performed better at lower medium concentration of 0.05x and produced 140 ml of H 2 compared to MMC6, which gave better performance than FF, 190 ml H 2 at 0.1x. These results indicated that M-9 medium at a concentration of x are more desirable than 0.5x. In contrast to FF and PVC, MMCs immobilized on PS, BL, CC, GS had enhanced H 2 production of ml at 0.05x and ml at 0.1x with MMC4 and ml at 0.05x with MMC6 (Table 4.10). H 2 production was lower at M-9 concentration of 0.5x. Best performance of 250 ml H 2 with MMC4 immobilized on PS and BL were quite comparable to 265 ml H 2 with MMC6 immobilized on PS under similar experimental conditions. Higher biomass retention in the reactor was recorded by observing the OD 660 of the effluent of the fermentation being carried out with 0.05x M-9 medium concentration. It was higher in the range of with FF compared to with PVC and with CC, GS, PS and BL. 72

19 Table 4.10: Effect of medium concentration on immobilized hydrogen producing mixed microbial cultures under daily fed culture conditions Support material a H 2 d MMC4 Medium conc. (x) c Mixed microbial cultures b MMC6 Medium conc. (x) % d H 2 % H 2 % H 2 % H 2 % H 2 % PVC+PS PVC+ BL PVC+CC PVC+GS PVC FF Values based on two set of experiments and two repetitions. Standard deviation was less than 10%. a: PVC: Polyvinylchloride tubes, PS: Pea-shells, BL: Banana leaves, CC: Coconut coir, GS: Groundnut shell, FF: Free floating; b: See Table 4.4 for the composition of the mixed microbial culture; c: Medium used: M-9 supplemented with 0.5% glucose; d: Volume (ml) and % of H 2 in the biogas (H 2 + CO 2 ). Total volume of feed: 680 ml with 120 ml (15%) additional volume occupied by support material in a 1 L reactor. Inoculum: 10 µg cell protein/ml feed Effect of hydraulic retention time A: Hydrogen production at HRT 4 days At a HRT of 4 days, with 0.5% glucose supplemented with 0.05x medium, MMC4 was found to evolve ml of H 2 i.e mol H 2 /mol glucose utilized with different support materials compared to 95 ml with FF MMC4 (Table 4.11). On the other hand, MMC6 was found to perform at a slightly lower levels ( ml H 2 i.e mol H 2 /mol glucose) in comparison to MMC4. The substrate utilization with both the MMCs was in the range of 85-90%. Here PS performed as the best support material for daily fed culture H 2 production (1.40 mol/mol glucose utilized) whereas BL, CC, and GS were almost equal in the range of mol/mol glucose utilized. With MMC6, BL was found to be a better support for immobilizing MMCs for H 2 production compared to other support materials. In these MMCs, H 2 component of the biogas was found to be in the range of 50-65% and no CH 4 was observed in any of them. It may be remarked here that this combination of 0.5% glucose along with 0.05x medium supplement was found to evolve H 2 in a stable manner (Figure 4.3a, b) over a period of 41 days which is equivalent to 10 cycles at an HRT of 4 days. 73

20 Table 4.11: Effect of hydraulic retention time on daily fed culture conditions hydrogen production by immobilized mixed microbial cultures Support material a MMC4 Mixed microbial culture b MMC6 Hydrogen (H 2 ) Hydrogen (H 2 ) Vol c Yield d % c Vol Yield % Hydraulic Retention Time: 4 Days PVC+PS PVC+ BL PVC+CC PVC+GS PVC FF Hydraulic Retention Time: 2 Days PVC+PS PVC+ BL PVC+CC PVC+GS PVC FF Values based on two set of experiments and two repetitions. Standard deviation was less than 10%. a: PVC: Polyvinylchloride tubes, PS: Pea-shells, BL: Banana leaves, CC: Coconut coir, GS: Groundnut shell, FF: Free floating; b: See Table 4.4 for the composition of the mixed microbial culture; c: Volume (ml) and % of H 2 in the biogas (H 2 + CO 2 ); d: mol H 2 /mol glucose utilized. Total volume of feed: 680 ml with additional volume of 120 ml (15%) occupied by support material in a 1 L reactor. Medium used: M-9 (0.05x) supplemented with 0.5% glucose. Inoculum: 10 µg cell protein/ml feed. Values represent 40 days of fermentation at an HRT of 4 days and 60 days of fermentation at an HRT of 2 days. Figure 4.3a: Hydrogen production profiles of mixed microbial culture MMC4 under continuous culture at HRT of 4 days 74

21 Figure 4.3b: Hydrogen production profiles of mixed microbial culture MMC6 under continuous culture at HRT of 4 days ph profile at HRT 4 days The daily profiles of ph within the reactor during 41 days of daily fed culture at HRT of 4 days with MMC4 and MMC6 have been presented in Figures 4.4a, b. At a HRT of 4 days, ph was lower during the initial 3-4 cycles and becomes nearly stable there after in the range of were immobilized cultures as compared to PVC and FF, where it was observed to be in the ranges of Figure 4.4a: ph profiles under continuous culture hydrogen production at HRT 4 days with mixed microbial culture MMC4 Figure 4.4b: ph profiles under continuous culture hydrogen production at HRT 4 days with mixed microbial culture MMC6 75

22 Growth profile at HRT 4 days The bacterial biomass in the effluents from the reactors was in the range of OD 660 of with MMC4 (Figure 4.5a) and with MMC6 (Figure 4.5b). Figure 4.5a: Growth profiles under continuous culture hydrogen production at HRT 4 days with mixed microbial culture MMC4 Figure 4.5b: Growth profiles under continuous culture hydrogen production at HRT 4 days with mixed microbial culture MMC6 With MMC4, under immobilized conditions the OD 660 in the effluent was lower in most cases except BL in comparison to that observed in controls: PVC and FF (Table 4.5a). This was in contrast to the values observed with MMC6, where the effluent from reactor has an OD 660 of 0.3 with FF and 0.4 with immobilized cultures (Table 4.5b). B: Hydrogen production at HRT 2 days At a HRT of 2 days, with 0.5% glucose supplemented with 0.05x medium, MMC4 was found to evolve ml of H 2 i.e mol H 2 /mol glucose utilized with different support materials compared to 40 ml observed with FF MMC4 (Table 4.11). On the other hand, MMC6 was found to perform at a slightly lower levels (60-76

23 300 ml) H 2 i.e mol H 2 /mol glucose) in comparison to MMC4. The substrate utilization with both the MMCs was in the range of 80-86%. Here CC performed as the best support material for daily fed culture H 2 production (1.65 mol/mol glucose utilized) whereas BL, PS, and GS were almost equal in the range of mol/mol glucose utilized. With MMC6, BL was found to be a better support for immobilizing MMCs for H 2 production compared to other support materials. In these MMCs, H 2 component of the biogas was found to be in the range of 20-65% and no CH 4 was observed in any of them. At an HRT of 2 days, a steady state H 2 production was observed to be achieved within cycles of daily fed culture condition with MMC4 (Figures 4.6a) and MMC6 (Figures 4.6b). It may be remarked here that this combination of 0.5% glucose along with 0.05x medium supplement was found to evolve H 2 in a stable manner (Figure 4.6a, b) over a period of 60 days which is equivalent to 30 cycles at an HRT of 2 days. Figure 4.6a: Hydrogen production profiles of mixed microbial culture MMC4 under continuous culture at HRT of 2 days Figure 4.6b: Hydrogen production profiles of mixed microbial culture MMC6 under continuous culture at HRT of 2 days 77

24 ph profile at HRT 2 days The daily profiles of ph within the reactor during 60 days of daily fed culture at HRT of 2 days with MMC4 and MMC6 have been presented in Figures 4.7a,b. At a HRT of 2 days, ph was lower during the initial 4-6 cycles and becomes nearly stable there after in the range of were immobilized cultures as compared to PVC and FF, where it was observed to be in the ranges of with MMC4. Almost similar trends in ph profiles were recorded with MMC6: with immobilized cultures and with FF and PVC. Figure 4.7a: ph profiles under continuous culture hydrogen production at HRT 2 days with mixed microbial culture MMC4 Figure 4.7b: ph profiles under continuous culture hydrogen production at HRT 2 days with mixed microbial culture MMC6 Growth profile at HRT 2 days The bacterial biomass in the effluents from the reactors was in the range of OD 660 of with MMC4 (Figure 4.8a) and with MMC6 (Figure 4.8b). 78

25 With MMC4, under immobilized conditions the OD 660 in the effluent was lower in all cases in comparison to that observed in FF (Figure 4.8a). This was in contrast to the values observed with MMC6, where the effluent from reactor has an OD 660 of 0.3 with FF and 0.4 with immobilized cultures (Figure 4.8b). Figure 4.8a: Growth profiles under continuous culture hydrogen production at HRT 2 days with mixed microbial culture MMC4 Figure 4.8b: Growth profiles under continuous culture hydrogen production at HRT 2 days with mixed microbial culture MMC6 In summary, it can be concluded that optimization of fermentation conditions has lead to improvement in H 2 production from ml under batch culture to ml (H 2 yield of mol H 2 /mol glucose utilized) in daily fed culture conditions. An additional feature is the reduction in incubation period of 4 days in batch culture to 2 days under daily fed culture conditions with MMC4 and MMC6. In order to exploit the H 2 producing abilities of different microbes, it is desirable to produce it from cheap raw materials such as biological wastes. Due to complex nature of biowaste(s) it is necessary to hydrolyze them. Biological pretreatments are more 79

26 suitable and eco-friendly in comparison to physical and chemical methods. In order to solubilize the various constituents of the biowastes, it is necessary to use microbes with high hydrolytic activities. So, here we have checked our strains for their hydrolytic enzyme activities such as amylase, lipase and protease to metabolize carbohydrate, fat and protein, respectively, which are the major components of biowaste materials. 80

27 Optimization of microbial hydrogen production process from biowaste and improvement in its yields

28 4.9 Bioconversion of biowaste to hydrogen With mixed hydrolytic bacterial cultures PSS was subjected to a hydrolysis with 11 HCs (HC1-HC11) composition presented in Table 4.12, prepared from 11 bacterial strains, belonging to firmicutes: Bacillus sp. strains EGU85, EGU367, EGU444 and EGU447; B. thuringiensis EGU378; B. subtilis EGU475; B. sphericus strains EGU385 and EGU542; γ-proteobacteria: P. mirabilis strains EGU30 and EGU32; and marine bacterium strain EGU409. These bacteria were observed to have high amylase, protease and lipase activities. Since these hydrolytic bacteria had been shown to have low H 2 producing abilities on individual basis (Table 4.3), here we have checked their potential to evolve H 2 from biowaste as mixed cultures. Table 4.12: The Plackett-Burman design for mixed hydrolytic bacterial consortia preparation for hydrolysis of pea-shells Mixed hydrolytic microbial consortia Bacterial EGU strain HC1 + a + - b HC HC HC HC HC HC HC HC HC HC a: Present; b: Absent Effect of medium H 2 evolution from PSS (2% TS) without any medium as supplement by HCs (HC1- HC11) was found to vary from ml with yields equivalent to L/kg TS fed. Here H 2 constituted % of the total biogas (Table 4.13). Media (M-9 and GM-2) supplementation resulted in lower H 2 evolution in the range of ml with M-9 and ml in GM-2 media (0.05x). Here, H 2 yields varied in the range 81

29 of and L/kg TS, respectively. Lowering of H 2 yields may due to the utilization of metabolites for microbial growth. The two consortia HC2 and HC5 were observed to be efficient for H 2 production yield in the range of L/kg TS fed. It is reflects that these consortia are quite compatible as compared to others for H 2 production from PSS. Table 4.13: Hydrogen producing potential of hydrolytic consortia from pre-hydrolysed peashells slurry a Mixed hydrolytic bacterial culture b Without medium Vol c Medium M-9 GM-2 Hydrogen (H 2 ) H 2 H 2 Yield d % Vol Yield % Vol Yield % HC HC HC HC HC HC HC HC HC HC HC Values based on two set of experiments and two repetitions. Standard deviation was less than 10%. a: Total volume of feed: 250 ml (2% TS); b: Bacterial composition used as inoculum at the rate of 10 µg cell protein/ml feed; c: Observed volume (ml) of H 2 in the biogas (H 2 + CO 2 ); d: L/kg TS fed. Media M-9 and GM-2 compositions of 0.05x With mixed microbial cultures Here, we initially checked all the MMCs for their potential to evolve H 2 from PSS (2% TS) in the absence of any added mixed hydrolytic bacterial cultures. It thus served as control to find out the base level of H 2 which can be expected to be evolved from PSS (Table 4.14). H 2 evolution was found to vary with MMCs, from ml/250 ml of PSS, which constituted % of the total biogas evolved. It was equivalent to a yield of L H 2 /kg TS fed of the feed. The background H 2 production from PSS (with inherent microflora alone) but in the absence of either 82

30 HCs or MMCs, was observed to be only 10 ml/250 ml feed. The best performers on PSS were MMC4 and MMC6, which evolved H 2 in the range of ml, equivalent to a yield of L/kg TS fed. Hence, we performed the subsequent experiments with these two MMCs. Table 4.14: Hydrogen producing potential of mixed microbial cultures from pea-shells slurry a Mixed microbial Biogas c (ml) Hydrogen (H 2 ) culture b Vol d (ml) Yield e % MMC MMC MMC MMC MMC MMC MMC MMC MMC MMC MMC Control Values based on two set of experiments and two repetitions. Standard deviation was less than 10%. a: Total volume of feed: 250 ml (2% TS); b: Bacterial composition used as inoculum at the rate of 10 µg cell protein/ml feed; c: Biogas is a mixture of H 2 + CO 2 ; d: Observed volume of H 2 in the biogas; e: L/kg TS fed With a combination of mixed cultures of hydrolytic and H 2 producing bacteria The addition of MMC4 and MMC6 to PSS pre-hydrolyzed with HCs proved beneficial for enhancing H 2 production. With MMC4 as H 2 producers, PSS was found to evolve ml of H 2 ( % of the total biogas). On the other hand, MMC6 proved to be slightly lower than MMC4 in its H 2 producing ability: ml H 2 ( % of the total biogas) (Table 4.15). The most interesting feature of these HC and MMC combinations is the performance of HC2, HC5 and HC6 along with MMC4 and HC2, HC5 and HC8 along with MMC6. The overall H 2 evolution was 230 ml with HC6 + MMC4 and 275 ml with HC2 + MMC4, equivalent to a yield of 46 L and 55 L/kg TS fed, respectively (Table 4.15). It can be concluded that the 83

31 performances of HC or MMC individually can be improved by combining the two treatments, such that the relative enhancement with HC2 and MMC4 is 1.45 and 1.89 fold respectively, over their individual performances. Table 4.15: Hydrogen producing potential of mixed microbial cultures from pre-hydrolysed pea-shells slurry a Mixed Mixed microbial culture c hydrolytic bacterial Control MMC4 MMC6 culture b Hydrogen (H 2 ) H 2 H 2 Vol d Yield e % Vol Yield % Vol Yield % HC HC HC HC HC HC HC HC HC HC HC Values based on two set of experiments and two repetitions. Standard deviation was less than 10%. a: Total volume of feed: 250 ml (2% TS) pretreated for 2 days with mixed hydrolytic bacterial culture; b: Bacterial composition used as inoculum at the rate of 10 µg cell protein/ml feed; c: Mixed microbial culture used as inoculum at the rate of 10 µg cell protein/ml feed; d: Observed volume (ml) of H 2 in the biogas (H 2 + CO 2 ); e: L/kg TS fed Optimization of process parameters for hydrogen production by mixed microbial cultures under batch culture from prehydrolyzed pea-shells slurry Effect of inoculum size and fibrous sheath An increase in the inoculum size of HCs has adversely effected the H 2 production with the MMCs. The observed H 2 evolution decreased from ml in the case of HC2 + MMC4, from ml in the case of HC6 + MMC4, from ml in the case of HC5 + MMC6 and from ml in the case of HC8 + MMC6 combinations (Table 4.16). Here H 2 constituted 45-61% of the total biogas evolved. 84

32 Incidentally, incubation of pre-hydrolyzed PSS without fibrous matter also, led to very poor H 2 yields at both inoculums size. The observed H 2 evolution was quite low in the range of ml/250 ml PSS at inoculum size of 1x and ml/250 ml PSS at inoculum size of 2x. The effective H 2 yields were in the range of 4-11 L/kg TS fed. Table 4.16: Hydrogen producing potential of mixed microbial culture from pre-hydrolysed pea-shells slurry a : Effect of inoculum size Inoculum (x) With fibrous sheath MMC4 Mixed microbial culture b Without fibrous sheath With fibrous sheath MMC6 Hydrogen (H 2 ) Hydrogen (H 2 ) Without fibrous sheath Vol c Yield d % Vol Yield % Vol Yield % Vol Yield % HC2 e HC5 e HC6 e HC8 e Values based on two set of experiments and two repetitions. Standard deviation was less than 10%. a: Total volume of feed: 250 ml pretreated for 2 days with mixed hydrolytic bacterial culture; b: Mixed microbial culture used as inoculum at the rate of 10 µg cell protein/ml feed; c: Observed volume (ml) of H 2 in the biogas (H 2 + CO 2 ); d: L/kg TS fed; f: See Table 4.12 for details of mixed hydrolytic bacterial culture composition used Effect of incubation period and total solids The pre-hydrolysis period of PSS was increased from 2-4 days, the yields were found to be adversely affected. The observed H 2 evolution decreased from ml in the case of HC2 + MMC4 and from ml in the case of HC6 + MMC4 combination, which constituted H 2 in then range of 55-61% of the total biogas evolved. Incidentally, incubation of pre-hydrolyzed PSS without fibrous matter, led to very poor H 2 yields. The observed H 2 evolution was quite low in the range of ml/250 ml PSS at 2 days of pretreatment period and 20 ml/250 ml PSS at 4 days pretreatment period. The effective H 2 yields were 4-11 L/kg TS fed. H 2 production with MMCs from pre-hydrolysed PSS (TS in the range of 1-5%) has been presented in Table H 2 evolution varied in different combinations of HCs 85

33 and MMCs as follows: ml with HC2 + MMM4, ml with HC6 + MMC4, ml with HC5 + MMC6 and in the range of ml with HC8 + MMC6. H 2 constituted 45-69% of total biogas evolved. It was observed that 2% TS have high H 2 yield of 55 L/kg of TS fed. An increase or decrease in TS%, resulted in reduction in H 2 yields. Table 4.17: Hydrogen producing potential of mixed microbial culture from pre-hydrolysed pea-shells slurry a : Effect total solid Total solid (%) With fibrous sheath MMC4 Mixed microbial culture b Without fibrous sheath With fibrous sheath MMC6 Hydrogen (H 2 ) Hydrogen (H 2 ) Without fibrous sheath Vol c Yield d % Vol Yield % Vol Yield % Vol Yield % HC2 e HC5 e HC6 e HC8 e Values based on two set of experiments and two repetitions. Standard deviation was less than 10%. a: Total volume of feed: 250 ml (2% TS) pretreated for 2 days with mixed hydrolytic bacterial culture; b: Mixed microbial culture used as inoculum at the rate of 10 µg cell protein/ml feed; c: Observed volume (ml) of H 2 in the biogas (H 2 + CO 2 ); d: L/kg TS fed; f: See Table 4.12 for details of mixed hydrolytic bacterial culture composition used as inoculum at the rate of 10 µg cell protein/ml feed Optimization of various parameters for hydrolysis of biowaste (250 ml PSS, 2% TS) with HCs in combination with H 2 -producers (MMCs) lead to the production of 55 L H 2 /kg TS fed under batch culture conditions. 86

34 Up-scaling of hydrogen production from biowaste

35 4.11 Up-scaling of hydrogen production under batch culture conditions Up-scaling of batch culture observation was conducted with HC2 and MMC4 at different feed quantity levels (Table 4.18 and Figure 4.9)). It was observed that at 0.75 and 1.5 L of feed, the evolved H 2 volumes were 0.9 L and 1.8 L equivalent to L/kg TS fed. However, at 4 L of feed, the H 2 evolution was found to be slightly higher in the range of 5.2 L equivalent to 65 L/kg TS fed. In all these cases H 2 was found to vary from 54-56% of the total biogas evolved. Table 4.18: Batch culture up-scaling of hydrogen production by mixed microbial culture (MMC4) a from pre-hydrolysed (HC2) b pea-shells slurry c Feed (L) Biogas d (L) Hydrogen (H 2) Vol e (L) Yield f % Data based on three replicates and two repetitions. Standard deviation was less than 10%. a: See Table 4.4 for details of bacterial composition used as inoculum at the rate of 10 µg cell protein/ml feed; b: See Table 4.12 for details of bacterial composition used as inoculum at the rate of 10 µg cell protein/ml feed; c: TS (2%); d: Biogas is a mixture of H 2 + CO 2 ; e: Observed volume (ml) of H 2 in the biogas; f: L/kg TS fed. Figure 4.9: Up-scaling of hydrogen production from pea-shells In our analysis, intermediate metabolites produced in the reactors were observed to be acetic acid ( mg/l) and butyric acid ( mg/l) (Figure 4.10). These VFAs are indicative of efficient H 2 production. In fact, this was further supported by low concentrations of propionic acid, which varied in the range of mg/l. In 87

36 fact, efficient H 2 production from sewage sludge was observed when butyric acid was the major VFA and propionic acid was produced in very low concentrations. Since we did observe ethanol production in the range of mg/l, the efficiency of our system in terms of H 2 production can be further enhanced by manipulating physiological parameters leading to reduction in ethanol production. Figure 4.10: Profile of volatile fatty acids during the up-scaling hydrogen production from pea-shells It is thus very interesting to record that this H 2 yield is 1.51 fold higher than that obtained previously from PS digested with undefined mixed cultures of hydrolytic and H 2 producing bacteria (Kalia and Joshi, 1995). Total reduced solid after hydrolysis and H 2 production in the range of 46-52% and COD reduction in the range of 40-45%. In summary, we have observed that the strains isolated from different environmental habitats have abilities to grow on different salt concentrations, wide range of ph values and can tolerate metals to certain extent. The strains with the high H 2 producing and hydrolytic abilities of lipase, amylase and protease can be employed for the preparation of defined mixed cultures (HC1-HC11 and MMC1-MMC11) of bacteria belonging to - Bacillus spp., P. mirabilis, B. avium, E. aerogenes, and a marine bacterium. Exploring their abilities of diverse metabolic characteristics led to the realization that these bacteria can utilize different sugars to evolve H 2, with best yields of 1.4 mol/mol glucose utilized under optimized batch culture conditions with MMCs (MMC4 and MMC6). Under optimized continuous culture conditions (HRT 2 days), MMC6 and MMC4, immobilized on ligno-cellulosic wastes - BL and CC evolved ml H 2 /day. Here, H 2 constituted 58-62% of the total biogas evolved. 88