Influence of linoleic acid,pH and HRT on anaerobic microbial populations and metabolic shifts in ASBRs during dark hydrogen fermentation of lignocellulosic sugars

In this study, controlling an anaerobic microbial community to increase the hydrogen (H₂) yield during the degradation of lignocelluosic sugars was accomplished by adding linoleic acid (LA) at low pH and reducing the hydraulic retention time (HRT) of an anaerobic sequencing batch reactor (ASBR). At pH 5.5 and a 1.7 d HRT, the maximum H₂ yield for LA treated cultures fed glucose or xylose reached 2.89 ± 0.18 mol mol⁻¹ and 1.94 ± 0.17 mol mol⁻¹, respectively. The major soluble metabolites at pH 5.5 with a 1.7 day HRT differed between the control and LA treated cultures. A metabolic shift toward H₂ production resulted in increased hydrogenase activity in both the xylose (13%) and glucose (34%) fed LA treated cultures relative to the controls. In addition, the Clostridia population and the H₂ yield were elevated in cultures treated with LA. A flux balance analysis for the LA treated cultures showed a reduction in homoacetogenic activity which was associated with reducing the Bacteriodes levels from 12% to 5% in the glucose fed cultures and 16% to 10% in the xylose fed cultures. Strategies for controlling the homoacetogens and optimal hydrogen production from glucose and xylose are proposed.     

Effect of furans and linoleic acid on hydrogen production

The effects of furans (furfural and 5-hydroxymethylfurfural (HMF)) on hydrogen (H₂) production using mixed anaerobic cultures were evaluated by conducting batch experiments. Two mixed anaerobic cultures (culture A and B) fed furans plus glucose and treated with and without linoleic acid (LA) at pH 5.5 were maintained at 37 °C. In the LA inhibited cultures A and B fed 0.75 g L⁻¹ furfural and 0.25 g L⁻¹ HMF, the maximum H₂ yields observed were 1.89 ± 0.27 mol mol⁻¹ glucose and 1.75 ± 0.22 mol mol⁻¹ glucose, respectively. In cultures with maximum H₂ yields, Clostridium sp. and Flavobacterium sp. were dominant. Acetate, butyrate and ethanol were the major soluble metabolites detected in cultures A and B whereas propionate was also dominant in culture B. A canonical correspondence analysis based on the byproducts and the relative abundance of the terminal-restriction fragments revealed less variation between cultures treated with LA and low correlation value between the factors and the species composition.     

Assessing the impact of palmitic,myristic and lauric acids on hydrogen production from glucose fermentation by mixed anaerobic granular cultures

The effects of lauric (LUA), myristic (MA), palmitic (PA), and a mixture of myristic:palmitic (MA:PA) acids on hydrogen (H₂) production from glucose degradation using anaerobic mixed cultures were assessed at 37 °C with an initial pH set at 5.0 and 7.131 mM of each acid. The maximum H₂ yield (2.53 ± 0.18 mol mol⁻¹ glucose) was observed in cultures treated with PA. A principal component analysis (PCA) of the by-products and the microbial population data sets detected similarities between the controls and PA treated cultures; however, differences were observed between the controls and PA treated cultures in comparison to the MA and LUA treated cultures. The flux balance analysis (FBA) showed that PA decreased the quantity of H₂ consumed via homoacetogenesis compared to the other LCFAs. The control culture was dominated by Thermoanaerovibrio acidaminovorans (60%), Geobacillus sp. and Eubacterium sp. (28%), while Clostridium sp. was less than 1%. Treatment with PA, MA, MA:PA, or LUA increased the H₂ producers (Clostridium sp. and Bacillus sp.) population by approximately 48, 67, 86, and 86%, respectively.     

Non-sterile bio-hydrogen fermentation from food waste in a continuous stirred tank reactor (CSTR): Performance and population analysis

Bio-hydrogen production from food waste by anaerobic mixed cultures was conducted in a continuous stirred tank reactor (CSTR). The hydraulic retention time (HRT) was optimized in order to maximize hydrogen yield (HY) and hydrogen production rate (HPR). The maximum hydrogen content (38.6%), HPR (379 mL H₂/L. d) and HY (261 mL H₂/g-VS_added) were achieved at the optimum HRT of 60 h. The major soluble metabolite products were butyric and acetic acids which indicated a butyrate-acetate type fermentation. Operation of CSTR at HRT 60 h could select hydrogen producing bacteria and eliminate lactic acid bacteria and acetogenic bacteria. The microbial community analyzed by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) revealed that the predominant hydrogen producer was Clostridium sp.     

Statistical optimization of conditions for minimum H2 consumption in mixed anaerobic cultures: Effect on homoacetogenesis and methanogenesis

Hydrogen (H₂) production using mixed anaerobic cultures often suffers severe yield reduction due to the syntrophic association between H₂ consumers (methanogens and homoacetogens) and H₂ producers (acidogens). The objective of this study was to uncouple the syntrophic association between H₂ producers and consumers by optimizing conditions for minimum H₂ consumption using a Box–Behnken design approach. The factors investigated in this study include temperature, pH and linoleic acid (LA) concentration. A quadratic response surface model was developed to predict the H₂ consumed by mixed anaerobic cultures and the optimum conditions for minimum H₂ consumption were 38 °C, pH 5.5 and 2 g L⁻¹ LA. Methanogenesis was inhibited in cultures fed 2 g L⁻¹ LA and maintained at pH 6.0 and 53 °C. In comparison, both methanogenesis and homoacetogenesis were inhibited in cultures fed 1–2 g L⁻¹ LA and maintained at a pH of 4.5 (Fig. 2B and 2E and Table 2 Expt. # 1, 2 and 11). Microbial diversity analysis revealed that LA fed cultures was dominated by spore forming Clostridium sp. in addition to Syntrophus aciditrophus. In comparison, control cultures were dominated by Eubacterium sp., Methanocalculus halotolerans and Methanococcoides alaskense. This study described an approach for regulating H₂ consumption in mixed cultures by optimizing process and environmental factors. Understanding the effects of these individual factors and their interaction is important in the full-scale operation of H₂ production facilities.     

Hydrogen production from xylose by moderate thermophilic mixed cultures using granules and biofilm up-flow anaerobic reactors

Continuous H₂ production from xylose by granules and biofilm up-flow anaerobic reactor using moderate thermophilic mixed cultures was investigated. The maximum H₂ yield of 251 mL H₂/g-xylose with H₂production rate of 15.1 L H₂/L⋅d was obtained from granules reactor operating at the organic loading rate (OLR) of 60 g-xylose/L⋅d and hydraulic retention time (HRT) of 4 h. Meanwhile the highest H₂ production rate of 13.3 L H₂/L⋅d with an H₂ yield of 221 mL H₂/g–xylose was achieved from the biofilm reactor. Both reactors were dominated by Thermoanaerobacterium species with acetate and butyrate as main fermentation products. The microbial community of the biofilm reactor was composed of Thermoanaerobacterium species, while granules reactor was composed of Clostridium sp., Thermoanaerobacterium sp. and Caloramator sp. The granular reactor was more microbial diversity and more balance between economic efficiency in term of the hydrogen production rate and technical efficiency in term of hydrogen yield.     

Optimizing hydrogen production from a switchgrass steam exploded liquor using a mixed anaerobic culture in an upflow anaerobic sludge blanket reactor

In this study, the operation of an upflow anaerobic sludge blanket reactor (UASBR) producing hydrogen (H₂) from a steam-exploded switchgrass (SWG) liquor was statistically optimized. The factors consider included pH, hydraulic retention time (HRT) and linoleic acid (LA) concentration. Under optimal operational conditions (pH 5.0, 10 h HRT and 1.75 g L⁻¹ LA), which were close to the predicted conditions using the D-optimality index, the maximum H₂ and methane yield observed were 99.86 ± 5.6 mL g⁻¹ TVS and 0.5 ± 0.1 mL g⁻¹ TVS, respectively. Under maximum H₂-producing conditions, high levels of acetate plus butyrate were observed with low levels of ethanol and lactate. A principal component analysis revealed that clustering of the samples was based on the operating conditions and fermentation metabolites. The microbial profiles revealed that by lowering the HRT from 16 to 8 h or decreasing the pH from 7.0 to 5.0 in the controls caused a 50% reduction in the relative abundance of the terminal restriction fragments belonging to the methanogenic population (Methanobacteria, Methanomicrobia, Methanococci). With LA treatment, H₂ producers (Ruminococcaceae and Clostridiaceae) were dominant and methanogens were inhibited and/or washed-out from the UASBR.     

Effect of inhibitors on hydrogen consumption and microbial population dynamics in mixed anaerobic cultures

The impact of different chemical microbial stressors (2-bromoethanesulfonate (BES), furfural, fish oil, lauric acid (LUA) and linoleic acid (LA)) on the inhibition of mesophilic hydrogen (H₂) consumption was examined in this study. Hydrogen consumption half-life values were used to compare the extent of inhibition by the different microbial stressing agents. A statistical analysis of the percent H₂ consumed using Tukey's analysis revealed the following trend: Control > fish oil = linoleic acid (LA (C18:2)) = furfural > BES > lauric acid (LUA (C12:0). The terminal restriction fragment length polymorphism (T-RLFP) results indicated that aceticlastic methanogens (Methanosaeta sp., Methanosarcina sp.) and hydrogenotrophic methanogens (Methanococcus sp.) were inhibited by the different chemical stressing agents. Cultures fed LUA and LA had a high abundance of Clostridium sp., Clostridium propionicum and Propionibacterium acnes. In comparison, BES and furfural fed cultures contained large fractions of Clostridium sp., Eubacteria sp. and Bacteroides sp. while in the fish oil fed cultures, the dominant organism detected was Eubacteria sp. This study indicated that H₂ consumption was affected by the chemical stressing agent concentration.     

Co-digestion of cassava starch wastewater with buffalo dung for bio-hydrogen production

Batch and continuous modes for bio-hydrogen production by co-digesting cassava starch wastewater with buffalo dung were investigated. Response surface methodology with central composite design was used to optimize the bio-hydrogen production conditions. A hydrogen production potential of 1787 mL H₂/L was achieved under optimal conditions of 2.84 g/L of NaHCO₃, an initial pH of 6.77 and a total chemical oxygen demand (tCOD)/total nitrogen ratio of 42.36. A continuous stirred tank reactor was operated under the optimum conditions from batch mode to investigate the effects of hydraulic retention time (HRT) of 72, 60 and 48 h on hydrogen production. The highest hydrogen content, hydrogen production rate and hydrogen yield of 33%, 839 mL H₂/L.d and 16.90 mL H₂/g-COD_added, respectively, were achieved at a HRT of 60 h. The predominant hydrogen producer under the optimal conditions in batch mode was Clostridium sp. while Clostridium sp., Megasphaera sp. and Chloroflexi sp. were observed in the continuous hydrogen production mode at an optimal HRT.     

Using a food and paper-cardboard waste blend as a novel feedstock for hydrogen production: Influence of key process parameters on microbial diversity

In this study, fermentation of a thermally treated simulated organic solid waste into hydrogen (H₂) was examined using a pretreated anaerobic mixed culture. The culture was fed a steam exploded food waste plus paper-cardboard waste blend liquor with and without linoleic acid (LA). The individual and interaction effects of the initial pH, LA concentration and the initial chemical oxygen demand (COD) concentration on H₂ and methane (CH₄) production was assessed using a Box–Behnken design (BBD). The BBD model predicted a maximum H₂ yield of 87 mL g⁻¹ COD or 98 mL H₂ g⁻¹ VS with 1.6 g L⁻¹ LA, an initial pH of 5.93 and an initial COD of 9.34 g COD L⁻¹. The major microbial populations detected in cultures at pH 5.5 with and without LA included Clostridium sp., Enterococcus asini, Enterococcus faecalis, and Lactobacillus gallinarum. The dendrogram for the 16S rRNA gene T-RFs profiles showed four major groups with a similarity index of 72–75% for Clade III. The major H₂-producing populations were grouped in Clade I with a similarity index range of 55–75%.     

High-yield biohydrogen production from biodiesel manufacturing waste by Thermotoga neapolitana

Efficient conversion of glycerol waste from biodiesel manufacturing processes into biohydrogen by the hyperthermophilic eubacterium Thermotoga neapolitana DSM 4359 was investigated. Biohydrogen production by T. neapolitana was examined using the batch cultivation mode in culture medium containing pure glycerol or glycerol waste as the sole substrate. Pre-treated glycerol waste showed higher hydrogen (H₂) production than untreated waste. Nitrogen (N₂) sparging and pH control were successfully implemented to maintain the culture pH and to reduce H₂ partial pressure in the headspace for optimal growth rate and to enhance hydrogen production from the glycerol waste. It was found that hydrogen production increased from 1.24 ± 0.06 to 1.98 ± 0.1 mol-H₂ mol⁻¹ glycerol_consumed by optimising N₂ sparging and pH control. We observed that in medium containing 0.05 M HEPES, with three cycles of N₂ sparging, the H₂ yield increased to 2.73 ± 0.14 mol-H₂ mol⁻¹ glycerol_consumed, which was 2.22-fold higher than the non-N₂ sparged H₂ yield (1.23 ± 0.06 mol-H₂ mol⁻¹ glycerol_consumed).     

Pretreating mixed anaerobic communities from different sources: Correlating the hydrogen yield with hydrogenase activity and microbial diversity

In this study, granular and flocculated anaerobic mixed cultures were pretreated using heat, shock loading, acid, alkali, linoleic acid (LA) and 2-bromoethane sulphonic acid (BESA). Under mesophilic conditions (37 °C) and an initial pH value of 6.0, higher H₂ yields were observed for the flocculated cultures when compared to the granular cultures. The maximum yield for granular cultures treated acid, BESA or LA were statistically the same. Butyric acid fermentation was dominant in a majority of the treated cultures. The maximum hydrogenase evolution specific activity (ESA) (124 ± 8 U_e mg VSS⁻¹) at 37 °C correlated with the maximum H₂ yield for the LA treated flocculated cultures (1.69 ± 0.18 mol mol⁻¹ glucose). The microbial diversity data clearly showed that the low H₂ yield in the granular cultures was due to the lower proportion of H₂ producers. A principle component analysis (PCA) revealed that the LA treated flocculated and granular cultures were grouped together and showed more diversity in comparison to other pretreatment methods.     

Using a statistical approach to model hydrogen production from a steam exploded corn stalk hydrolysate fed to mixed anaerobic cultures in an ASBR

In this study, hydrogen (H₂) production from the fermentation of steam exploded corn stalk (CS) liquor was statistically optimized using a fractional factorial design approach. The factors under consideration included temperature, pH and hydraulic retention time (HRT). Under optimal conditions at 53 °C, a pH at 4.5 and a 9.5 h HRT, the observed maximum H₂ yield of 98 ± 2 mL g⁻¹ TVS together with negligible CH₄ were similar to the model predicted responses. A flux analysis revealed negligible homoacetogenic activity in cultures at 53 °C and low pH (≤5.5). Both homoacetogenic (R17) and methanogenic (R28 and R29) fluxes accounted for more than 68–90% of H₂ consumption in cultures at low temperatures. The low H₂ yields observed in cultures maintained at 21 °C and 37 °C was associated with high lactate and solvent levels. High H₂ yields in cultures at 53 °C were associated with a higher abundance of Clostrdium sp. and CH₄ production at low temperatures was due to the presence of hydrogenotrophic methanogens (Methanothermobacter marburgensis and Methanobrevibacter ruminatum) and aceticlastic methanogens (Methanosaeta sp. and Methanosarcina sp.). The results obtained from this study (within the factor ranges investigated) indicated that steam exploded CS liquor could be a potential substrate for H₂ production using mixed microbial cultures.     

Impact of culture source and linoleic acid (C18:2) on biohydrogen production from glucose under mesophilic conditions

Genomic and statistical methods were used to demonstrate the effects of linoleic acid (LA) on hydrogen (H₂) production in mixed anaerobic cultures from two sources (designated as A and B). The microbial composition of the control cultures CA and CB were statistically different. Bacteroidaceae (26%) and Clostridiaceae (10%) dominated CA whereas Clostridiaceae (33%) and Bacteroidaceae (10%) dominated CB. Homoacetogens directed 42% of the electron equivalents to acetate production and decreased the H₂ yield by 50% in CA compared to CB. The maximum H₂ yields (3.11 ± 0.02 and 3.11 ± 0.07 mol H₂ mol⁻¹ glucose in LA-treated cultures ALA and BLA, respectively) were statistically the same. Cultures ALA and BLA followed the acetate-butyrate pathway while CA and CB followed propionate and homoacetogenic pathways. LA-treated and control cultures were statistically different based on the type and quantity of metabolites; the differences were also confirmed by principal component analysis (PCA).     

Simultaneous saccharification and fermentation of cellulose for bio-hydrogen production by anaerobic mixed cultures in elephant dung

The objective of this study was to optimize the culture conditions for simultaneous saccharification and fermentation (SSF) of cellulose for bio-hydrogen production by anaerobic mixed cultures in elephant dung under thermophilic temperature. Carboxymethyl cellulose (CMC) was used as the model substrate. The investigated parameters included initial pH, temperature and substrate concentration. The experimental results showed that maximum hydrogen yield (HY) and hydrogen production rate (HPR) of 7.22 ± 0.62 mmol H₂/g CMC_added and 73.4 ± 3.8 mL H₂/L h, respectively, were achieved at an initial pH of 7.0, temperature of 55 °C and CMC concentration of 0.25 g/L. The optimum conditions were then used to produce hydrogen from the cellulose fraction of sugarcane bagasse (SCB) at a concentration of 0.40 g/L (equivalent to 0.25 g/L cellulose) in which an HY of 7.10 ± 3.22 mmol H₂/g cellulose_added. The pre-dominant hydrogen producers analyzed by polymerase chain reaction-denaturing gel gradient electrophoresis (PCR-DGGE) were Thermoanaerobacterium thermosaccharolyticum and Clostridium sp. The lower HY obtained when the cellulose fraction of SCB was used as the substrate might be due to the presence of lignin in the SCB as well as the presence of Lactobacillus parabuchneri and Lactobacillus rhamnosus in the hydrogen fermentation broth.     

Effect of hydraulic retention time on hydrogen production and chemical oxygen demand removal from tapioca wastewater using anaerobic mixed cultures in anaerobic baffled reactor (ABR)

This study evaluated hydrogen production and chemical oxygen demand removal (COD removal) from tapioca wastewater using anaerobic mixed cultures in anaerobic baffled reactor (ABR). The ABR was conducted based on the optimum condition obtained from the batch experiment, i.e. 2.25 g/L of FeSO₄ and initial pH of 9.0. The effects of the varying hydraulic retention times (HRT: 24, 18, 12, 6 and 3 h) on hydrogen production and COD removal in a continuous ABR were operated at room temperature (32.3 ± 1.5 °C). Hydrogen production rate (HPR) increased with a reduction in HRT i.e. from 164.45 ± 4.14 mL H₂/L.d (24 h HRT) to 883.19 ± 7.89 mL H₂/L.d (6 h HRT) then decreased to 748.54 ± 13.84 mL H₂/L.d (3 h HRT). COD removal increased with reduction in HRT i.e. from 14.02 ± 0.58% (24 h HRT) to 29.30 ± 0.84% (6 h HRT) then decreased to 21.97 ± 0.94% (3 h HRT). HRT of 6 h was the optimum condition for ABR operation as indicated.     

Biological hydrogen production from sweet sorghum syrup by mixed cultures using an anaerobic sequencing batch reactor (ASBR)

Continuous biological hydrogen production from sweet sorghum syrup by mixed cultures was investigated by using anaerobic sequencing batch reactor (ASBR). The ASBR was conducted based on the optimum condition obtained from batch experiment i.e. 25 g/L of total sugar concentration, 1.45 g/L of FeSO₄ and pH of 5.0. Feasibility of continuous hydrogen fermentation in ASBR operation at room temperature (30 ± 3 °C) with different hydraulic retention time (HRT) of 96, 48, 24 and 12 hr and cycle periods consisting of filling (20 min), settling (20 min), and decanting (20 min) phases was analyzed. Results showed that hydrogen content decreased with a reduction in HRT i.e. from 42.93% (96 hr HRT) to 21.06% (12 hr HRT). Decrease in HRT resulted in a decrease of solvents produced which was from 10.77 to 2.67 mg/L for acetone and 78.25 mg/L to zero for butanol at HRT of 96 hr-12 hr, respectively. HRT of 24 hr was the optimum condition for ASBR operation indicated by the maximum hydrogen yield of 0.68 mol H₂/mol hexose. The microbial determination in DGGE analysis indicated that the well-known hydrogen producers Clostridia species were dominant in the reacting step. The presence of Sporolactobacillus sp. which could excrete the bacteriocins causing the adverse effect on hydrogen-producing bacteria might responsible for the low hydrogen content obtained.     

Continuous thermophilic hydrogen production and microbial community analysis from anaerobic digestion of diluted sugar cane stillage

The aim of this study was to promote biohydrogen production in an thermophilic anaerobic fluidized bed reactor (AFBR) at 55 °C using a mixture of sugar cane stillage and glucose at approximately 5000–5300 mg COD L⁻¹. During a reduction in the hydraulic retention time (HRT) from 8, 6, 4, 2 and 1 h, H₂ yields of 5.73 mmol g COD_added⁻¹ were achieved (at HRT of 4 h, with organic loading rate of 52.7 kg COD m⁻³ d⁻¹). The maximum volumetric H₂ production of 0.78 L H₂ h⁻¹ L⁻¹ was achieved using stillage as carbon source. In all operational phases, the H₂ average content in the biogas was between 31.4 and 52.0%. Butyric fermentation was the predominant metabolic pathway. The microbial community in accordance with the DGGE bands profile was found similarity coefficient between 91 and 95% without significant changes in bacterial populations after co-substrate removal. Bacteria like Thermoanaerobacterium sp. and Clostridium sp. were identified.     

Effects of linoleic acid and its degradation by-products on mesophilic hydrogen production using flocculated and granular mixed anaerobic cultures

The effects of linoleic acid (LA (C18:2)) and its degradation by-products on hydrogen (H₂) production were examined at 37 °C and an initial pH value of 5.0 using granular and flocculated mixed anaerobic cultures from the same source. In the flocculated cultures, the H₂ consumers were inhibited to a greater extent when compared to the granular cultures. The maximum H₂ yields were 2.52 ± 0.2 and 1.9 ± 0.2 mol mol⁻¹ glucose in the flocculated and granular cultures, respectively. The major long chain fatty acids (LCFAs) detected at which H₂ attained a maximum value were LA (750 mg L⁻¹) and myristic acid (MA) (500 mg L⁻¹).     

Biohydrogen production from cassava starch processing wastewater by thermophilic mixed cultures

Natural microbial consortia from hot spring samples were used to developed thermophilic mixed cultures for biohydrogen production from cassava starch processing wastewater (CSPW). Significant hydrogen production potentials were obtained from three thermophilic mixed cultures namely PK, SW and PR with maximum hydrogen production yields of 249.3, 180 and 124.9 mL H₂/g starch, respectively from raw cassava starch and 252.4, 224.4 and 165.4 mL H₂/g starch, respectively from gelatinized cassava starch. Acetic acid-ethanol and acetic-lactic acid type fermentation were observed in cassava starch fermentation, based on three thermophilic mixed cultures performance. The thermophilic mixed cultures PK, SW and PR exhibited the maximum hydrogen yield of 287, 264 and 232 mL H₂/g starch in CSPW, respectively corresponding to 53%, 48.7% and 42.8% of the theoretical values. Phylogenetic analysis of thermophilic mixed cultures revealed that members involved cassava starch degrading bacteria and hydrogen producers in both raw cassava starch and CSPW were phylogenetically related to the Thermoanaerobacterium saccharolyticum, Thermoanaerobacterium thermosaccharolyticum, Anoxybacillus sp., Geobacillus sp. and Clostridium sp.