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This research aimed to study the diversity and antimicrobial activity of culturable haloarchaea in soil samples of solar saltern in Pattani Province, Thailand and Southern Thai salt-fermented food. Seventy-seven extremely haloarchaea were isolated on Halophilic medium agar containing 25% NaCl at 37°C for 7-21 days. They were grouped by Amplified Ribosomal DNA Restriction Analysis (ARDRA) with two restriction enzymes, RsaI and HindIII. The ARDRA patterns illustrated 6 different Operation Taxonomic Units (OTUs). Partial 16S rRNA gene sequences (938 bp) of the representative of each OTUs were aligned with the GenBank database. The results showed that the representatives of OTU1, OTU2, OTU3, OTU4, OTU5, and OTU6 had 99-100% similarity to Halobacterium salinarum, 99-100% similarity to Halostagnicola larsenii, 99% similarity to Natronococcus sp., 99-100% similarity to Haloferax alexandrinus, 99-100% similarity to Natrialba sp. and 97% similarity to Halococcus sp., respectively. The antimicrobial activity testing of the 17 isolated haloarchaeal strains from solar saltern was performed against 5 tested strains of Hbt. salinarum and 1 strain of Natrialba sp. were isolated from fermented food. Seven (41.2%) were candidate halocin-producing strains. In addition, no phage activity was observed. The 14-day fermentation broth of Natronococcus sp. SS13 showed the highest antimicrobial activity against Hbt. salinarum PK08, BD07 and BD09 (>5,120 AU/ml).

Temperature plays a significant role in anaerobic digestion (AD) as it affects the microbial communities and ultimately controls the efficiency of the process. Few studies have looked at temperature-adjusted AD, but it is unclear how the temperature shifts affect biogas production and the dynamics of microorganisms involved in methanogenesis. This study tested two temperature shift scenarios in fed-batch mode using anaerobically digested sewage sludge and glucose-based substrate. The first scenario was acclimatized to upshifting temperatures from 42 °C to 48 °C while the second was acclimatized to downshifting temperatures from 55 °C to 45 °C. Both temperature shift scenarios resulted in a decrease in biogas production, especially at 45 °C. The upshifted scenario experienced a maximum decrease of 83%, and the downshifted scenario experienced a 16–33% decrease in methane production. Next-generation 16S rRNA sequencing revealed the domination of Methanoculleus in the upshifted scenario. However, a low correlation between the number of Methanoculleus and the other hydrogenotrophic methanogens to biogas production indicates inhibition in the hydrogenotrophic pathway. The downshifted scenario showed better biogas production due to the substantial domination of acetoclastic Methanosaeta and the low abundance of sulfate-reducing bacteria. Hence, the temperature shift affects the microbial communities, significantly affecting biogas production performance.

Food waste is categorized as organic solid waste, which has a negative impact on environmental sustainability. Food waste was simultaneously used for the feasible generation of mixed volatile fatty acids (VFAs) and bio-hydrogen by deploying dark fermentation. Original anaerobic digested sludge was prepared via the shock technique with 50 g/L glucose under thermophilic temperature (55 °C). The pretreated inoculum was found capable of converting 10 g VS/L food waste to hydrogen with a rather high yield of 135.2 ± 7 mL H2/VSadded. The effect of various concentrations of food waste, including 10.2, 16.3, 20.3, and 26.4 g VS/L, on mixed VFAs production was subsequently carried out in batch dark fermentation. The highest butyric acid concentration (5.26 ± 0.22 g/L) in soluble metabolites was obtained from batch dark fermentation with 26.4 g VS/L of food waste. The dominant Clostridium thermobutyricum,Clostridium sporogenes, and Octadecobacter sp. found in the batch of dark fermentation of food waste could confirm the effectiveness of the load shock pretreatment method for inoculum preparation. The continuous stirred tank reactor (CSTR) inoculated with mixed cultures, also prepared via the load shock pretreatment method and without the addition of external nutrients, was operated by feeding 26.4 g VS/L food waste at the kinetically designed HRT for 4 days, corresponding to an organic loading rate (OLR) of 7.6 g VS/L·d. Under steady state conditions, promising butyric acid (5.65 ± 0.51 g/L)-rich mixed VFAs were achieved along with the hydrogen yield of 104.9 ± 11.0 mL-H2/g VSadded, which is similar to the upper side of the previously reported yields (8.8 ± 0.6–103.6 ± 0.6 mL-H2/g VS).

Some species of the genus Clostridium are efficient acetate producers and have been deemed useful for upgrading industrial biogas. An acetogenic, strictly anaerobic, Gram-stain-positive, subterminal endospore-forming bacterium designated strain PL3T was isolated from peatland soil enrichments with H2 and CO2. Cells of strain PL3T were 0.8–1.0×4.0–10.0 µm in size and rod-shaped. Growth of strain PL3T occurred at pH 6.0–7.5 (optimum, pH 7.0), at 20–40 °C (optimum, 30 °C) and with 0–1.5 % (w/v) NaCl (optimum, 0.5%). Biochemical analyses revealed that strain PL3T metabolized lactose, maltose, raffinose, rhamnose, lactic acid, sorbitol, arabinose and glycerol. Acetic acid was the predominant metabolite under anaerobic respiration with H2/CO2. The major cellular fatty acids were C16 : 0, C16 : 1 cis 9 and C17 : 0 cyc. The main polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, aminolipid and aminophospholipid. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain PL3T belongs to the genus Clostridium with the highest sequence similarity to Clostridium aciditolerans DSM 17425T (98.6 %) followed by Clostridium nitrophenolicum (97.8 %). The genomic DNA G+C content of strain PL3T was 31.1 mol%.The genomic in silico DNA–DNA hybridization value between strain PL3T and C. aciditolerans DSM 17425T was 25.1 %, with an average nucleotide identity of 80.2 %. Based on phenotypic, chemotaxonomic and phylogenetic differences, strain PL3T was suggested to represent a novel species of the genus Clostridium , for which the name Clostridium thailandense sp. nov. is proposed. The type strain is PL3T (=DSM 111812T=TISTR 2984T).

Methane production from solid-state anaerobic digestion (SS-AD) of empty fruit bunches (EFB) using recycling solid-anaerobic digested (S-AD) sludge and liquid-anaerobic digested (L-AD) sludge as inoculum was investigated under thermophilic (55 °C) and mesophilic conditions (40 °C). The methane production rate of L-AD sludge (2.98–3.27 L-CH4 L−1 reactor d −1) was 1–2 times higher than recycling S-AD sludge (1.36 L-CH4 L−1 reactor d −1) as inoculum. Methane production of L-AD and S-AD sludge inoculum was 53.5–54.1 and 31.2–39.0 m3 CH4 tonne-1 EFB, respectively. The SS-AD of EFB with L-AD and S-AD sludge under thermophilic condition had specific methane activity of 34–96 times higher than the mesophilic condition. Clostridium sp., Methanosaeta sp., and Methanoculleus sp. were responsible for the SS-AD of EFB with L-AD sludge inoculum. The L-AD sludge was suitable inoculum for SS-AD of EFB in terms of methane production rate, cellulose degradation efficiency, biogas production, and microbial activity.

Improvement of biohythane production from oil palm industry solid waste residues by co-digestion with palm oil mill effluent (POME) in two-stage thermophilic fermentation was investigated. A two-stage co-digestion of solid waste with POME has biohythane production of 26.5–34 m3/ton waste. The co-digestion of solid waste with POME increased biohythane production of 67–114% compared to digestion POME alone. Co-digestion of solid waste with POME enhanced hydrolysis constant (kh) from 0.07 to 0.113 to 0.120–0.223 d−1. The hydrolysis constant (kh) of co-digestion was 10 times higher than the single digestion of solid waste. Clostridium sp. was predominated in the hydrogen stage, while Methanosphaera sp. was predominant in methane stage. The co-digestion of solid waste with readily biodegradable organic matter (POME) could significantly increase biohythane production with achieving the significant cost reduction for pretreatment of solid wastes.

The impact of the heavy metal contamination and acidity on the bacterial community was studied in samples collected from the Akhtala copper mine tailing using molecular approaches. The bacterial community structure analysis by PCR-DGGE fingerprinting revealed an abundance of Firmicutes, Acidobacteria, and Proteobacteria in different layers of the Akhtala tailing. 454 pyrotag sequence analyses revealed that a significant part of the sequences originated from Proteobacteria (49%) and Bacteroidetes (43%). Bacterial taxa are distributed also in phyla Saccharibacteria (2%), Verrucomicrobia (1.5%), Gammatimonadetes (1%), and some minor additional bacterial groups. The main primary producers in the Akhtala tailing appear to be obligate autotrophic Thiobacillus and Sulfuritalea species. Representatives of Lutibacter and Lysobacter genera are the most abundant acid-tolerant heterotrophs in the studied tailing. The presence of a large number of yet-uncultivated species emphasizes the importance of the future exploration of the tailing as an important source of novel bacteria.

The pilot-scale of two-stage thermophilic (55 °C) for biohythane production from palm oil mill effluent (POME) was operated at hydraulic retention time (HRT) of 2 days and organic loading rate (OLR) of 27.5 gCOD/L⋅d) for first stage and HRT of 10 days and OLR of 5.5 gCOD/L⋅d for second stage. Biohythane production rate was 1.93 L-gas/L⋅d with biogas containing 11% H2, 37% CO2, and 52% CH4. Recirculation of methane effluent mixed with POME at a ratio of 1:1 can control pH in the first stage at an optimal range of 5.0–6.5. Microbial community in hydrogen stage dominated by Thermoanaerobacterium sp., while methane stage dominated by Methanosarcina sp. The H2/CH4 ratio of biohythane was 0.13–0.18 which suitable for vehicle fuel. Biohythane production from POME could be promising cleaner biofuel with flexible and controllable H2/CH4 ratio.

The effect of trace metals supplementation into palm oil mill effluent on biohythane production and responsible microbial communities in thermophilic two-stage anaerobic fermentation was investigated. High biohythane yields were linked to Ni/Co/Fe supplementation (10, 6 and 20 mg L−1, respectively) with maximum H2 and CH4 yields of 139 mL H2 gVS−1 and 454 mL CH4 gVS−1, respectively. The Ni/Co/Fe supplementation resulted in higher numbers of Bacillus sp., Clostridium sp. and Thermoanaerobacterium sp. together with increasing hydrogenase expression level leading to increasing hydrogen yields of 90.4%. The numbers of Methanosarcina, Methanomassiliicoccus, and Methanoculleus were enhanced by Ni/Co/Fe addition, accompanied by 21.7% higher methane yields. No correlation between methyl coenzyme-M reductase expression level and methane yields was observed. The Ni/Co/Fe supplementation improved gas production in the two-stage biohythane process via enhancing a number of viable hydrogen-producing bacteria together with hydrogenase activity in H2 stage and enhancing number methanogens in the CH4 stage.

A sudden pH drops always inhibits the anaerobic digestion (AD) reactor for biogas production from palm oil mill effluent (POME). The pH adjustment of POME by oil palm ash addition and the biogas effluent recycling effect on the preventing of pH drop and change of the archaea community was investigated. The pH adjustment of POME to 7.5 increased the methane yield two times more than raw POME (pH 4.3). The optimal dose for pH adjustment by oil palm ash addition was 5% w/v with a methane yield of 440 mL-CH4/gVS. The optimal dose for pH adjustment by biogas effluent recycling was 20% v/v with a methane yield of 351 mL-CH4/gVS. Methane production from POME in a continuous reactor with pH adjustment by 5% w/v oil palm ash and 20% v/v biogas effluent recycling was 19.1 ± 0.25 and 13.8 ± 0.3 m3 CH4/m3-POME, respectively. The pH adjustment by oil palm ash enhanced methane production for the long-term operation with the stability of pH, alkalinity, and archaea community. Oil palm ash increased the number of Methanosarcina mazei and Methanothermobacter defluvii. Oil palm ash is a cost-effective alkali material as a source of buffer and trace metals for preventing the pH drop and the increased methanogen population in the AD process.

Third generation biomass, i.e. microalgae, has emerged as a promising alternative to first and second generation biomass for biohydrogen production. However, its utilization is still low at present, due to several reasons including the strong and rigidity of the microalgal cell wall that limit the hydrolysis efficiency during dark fermentation (DF) and photofermentation (PF) processes. To improve the utilization efficiency of microalgal biomass, it is crucial that important aspects related to the production of the biomass and the following processes are elaborated. Thus, this article provides detailed overview of algal strains, cultivation, and harvesting. It also presents recent research and detailed information on microalgal biomass pretreatment, and biohydrogen production through DF, PF, and co-digestion of microalgal biomass with organic materials. Furthermore, factors affecting fermentation processes performance and the use of molecular techniques in biohydrogen production are presented. This review also discusses challenges and future prospects towards biohydrogen production from microalgal biomass.

Background Anaerobic digestion (AD) is a suitable process for treating high moisture MSW with biogas and biofertilizer production. However, the low stability of AD performance and low methane production results from high moisture MSW due to the fast acidify of carbohydrate fermentation. The effects of organic loading and incineration fly ash addition as a pH adjustment on methane production from high moisture MSW in the single-stage AD and two-stage AD processes were investigated. Results Suitable initial organic loading of the single-stage AD process was 17 gVS L−1 at incineration fly ash (IFA) addition of 0.5% with methane yield of 287 mL CH4 g−1 VS. Suitable initial organic loading of the two-stage AD process was 43 gVS L−1 at IFA addition of 1% with hydrogen and methane yield of 47.4 ml H2 g−1 VS and 363 mL CH4 g−1 VS, respectively. The highest hydrogen and methane production of 8.7 m3 H2 ton−1 of high moisture MSW and 66.6 m3 CH4 ton−1 of high moisture MSW was achieved at organic loading of 43 gVS L−1 at IFA addition of 1% by two-stage AD process. Biogas production by the two-stage AD process enabled 18.5% higher energy recovery than single-stage AD. The 1% addition of IFA into high moisture MSW was useful for controlling pH of the two-stage AD process with enhanced biogas production between 87–92% when compared to without IFA addition. Electricity production and energy recovery from MSW using the coupled incineration with biogas production by two-stage AD process were 9,874 MJ ton−1 MSW and 89%, respectively. Conclusions The two-stage AD process with IFA addition for pH adjustment could improve biogas production from high moisture MSW, as well as reduce lag phase and enhance biodegradability efficiency. The coupled incineration process with biogas production using the two-stage AD process was suitable for the management of MSW with low area requirement, low greenhouse gas emissions, and high energy recovery.

Empty fruit bunches (EFB) have low biodegradability and restrict their commercial utilization in biogas plants. Integration of straw mushroom (Volvariella volvacea) cultivation as a function of bio-pretreatment on EFB to improve biodegradability and methane production by solid-state anaerobic digestion (SS-AD) was investigated. The mushroom yield was 47.3 kg·tonne−1 EFB with remaining weight in spent mushroom-EFB (S-mEFB) of 82%. The cellulose, hemicellulose, and lignin of EFB were degraded by 3.3%, 21.3%, and 17.6%, respectively, with an increased surface area of S-mEFB. The biodegradability of S-mEFB (62.7%) was 2 times higher than raw EFB (33.5%) with the highest methane yield and production of 281 mL CH4·g−1 VS and 50.6 m3·tonne−1 S-mEFB, respectively. The co-digestion of S-mEFB with 5% v/w POME had highest methane yield of 405 mL CH4·g−1 VS with biodegradability of 90.8%. Integrating straw mushroom cultivation with SS-AD is a promising strategy for achieving an environmentally friendly and economically feasible process.

This study aimed to evaluate the possibility of acidogenic phase as the pretreatment method of filter cake (FC) before being co-digested with biogas effluent (BE), and anaerobic sludge (AS) for methane production. The effects of separation of acidogenic and methanogenic phases on the improvement of the methane production process from a co-digestion of FC with BE, and AS were also investigated. FC was pretreated through acidogenic phase anaerobic digestion (AD) using a different substrate to inoculum ratios (SIRs) and substrate concentrations. The optimum conditions for acidogenic phase AD of FC were SIRs of 4:1 and substrate concentration of 15.0% total solid (TS). Resultantly, the maximum volatile fatty acids (VFA) yield of 0.17 g-VFAs/g-VS (volatile solids) was obtained. The major VFA components were butyric and acetic acid. The acidogenic phase of FC was semi-continuously conducted in a continuous stirred tank reactor (CSTR) at various hydraulic retention time (HRT). The highest concentration and yield of VFAs of 7.29 g/L and 0.39 g-VFAs/g-VS, respectively, were observed at HRT of 5 d. The major VFAs observed were butyric acid (48.8%), and acetic acid (36.8%). Subsequently, the proportions of pretreated FC, BE, and AS to produce methane were optimized in batch experiments. The highest methane yield (MY) of 195.9 mL-CH4/g-VS was achieved at the proportions of pretreated FC and BE of 37.75 and 22.25 g-VS/L, respectively, without the addition of AS. The optimum substrate proportion was further used to determine the MY in a 10 L CSTR with the various HRT. The maximum MY of 130.5 mL-CH4/g-VS was achieved at an HRT of 20 d which produced an EY of 4.7 kJ/g-VS. Analysis of microbial community by 16S rRNA amplicon sequencing on an illumination platform indicated that genera Rummeliibacillus, Lactobacillus, and Bacillus were the dominant bacteria found in the acidogenic phase. Methanoculleus and Methanosarcina were found to be the most dominant genera in methane fermentation. Results suggested that acidogenic phase digestion can be used as an alternative method for FC pretreatment to improve methane production. Furthermore, the separation of acidogenic and methanogenic phases improves the process stability.

Homoacetogens consortium was enriched from peatland soil, digested sludge, and rumen fluid for simultaneous carbon dioxide (CO2) consumption and acetic acid production in the biogas upgrading process. The homoacetogens consortium enriched from peatland soil (PL3) has a maximum CO2 consumption and acetic acid production of 95% and 120 mg/L, respectively. The methane content in upgraded biogas and acetic acid production of 98% and 543 mg/L was obtained at optimum hydrogen (H2) to CO2 at a ratio of 2:1, pH 8, and temperature 30 °C. The upgraded biogas (CH4 > 95%) can be used as vehicle fuel or injected into the natural gas grid. The homoacetogens consortium PL3 was dominated by Clostridium sp., Proteiniclasticum sp., and Petrimonas sp. Clostridium species are the main homoacetogens responsible for CO2 reduction using H2 as an energy source. The homoacetogens consortium PL3 could provide an opportunity for simultaneous biogas upgrading to biomethane and acetic acid production.

Background: Biohydrogen effluent contains a high concentration of volatile fatty acid (VFA) mainly as butyric, acetic, lactic and propionic acids. The presence of various VFAs (mixture VFAs) and their cooperative effects on two-stage biohythane production need to be further studied. The effect of VFA concentrations in biohydrogen effluent of palm oil mill effluent (POME) on methane yield in methane stage of biohythane production was investigated. Results: The methane yield obtained in low VFA loading (0.9 and 1.8 g/L) was 15–20% times greater than that of high VFA loading (3.6 and 4.7 g/L). Butyric acid at high concentrations (8 g/L) has the individual significantly negative effect the methane production process ( P P Geobacillus sp., Thermoanaerobacterium thermosaccharolyticum , Methanoculleus thermophilus and Methanothermobacter delfuvii resulting in low methane yield. Conclusion: Preventing the high concentration of butyric acid, and propionic acid in the hydrogenic effluent could enhance methane production in two-stage anaerobic digestion for biohythane production. Normal 0 false false false ES-CL X-NONE X-NONE /* Style Definitions */ table.MsoNormalTable {mso-style-name:"Tabla normal"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-parent:""; mso-padding-alt:0cm 5.4pt 0cm 5.4pt; mso-para-margin-top:0cm; mso-para-margin-right:0cm; mso-para-margin-bottom:8.0pt; mso-para-margin-left:0cm; line-height:107%; mso-pagination:widow-orphan; font-size:11.0pt; font-family:"Calibri",sans-serif; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin; mso-bidi-font-family:"Times New Roman"; mso-bidi-theme-font:minor-bidi; mso-ansi-language:ES-CL;} Normal 0 false false false EN-US X-NONE TH

Recovery of bioenergy and biochemicals from industrial and agricultural wastewater has attracted growing and widespread interests. The two-stage fermentation process offers an appealing avenue to achieve stepwise and directional substrate conversion in separated stages. The two-stage fermentation process could enhance the degradation of the substrate with high product yield and quality. The two-stage fermentation process has been developed for the production of biomethane, biohydrogen, biohythane, bioelectricity, and medium-chain carboxylic acids (MCCAs) with advantages over the conventional one-stage process. Two-stage fermentation process promising for future applications in industrial and agricultural wastewater treatment with simultaneous value-added production and pollution control. This chapter provides an overview of the various two-stage fermentation process for bioenergy and biochemicals production, introduces the recent advances, biochemical pathways, microorganisms, process parameters, technology assessment, and discusses the significant challenges with future perspectives for practical application.

Valorization of biodiesel-derived crude glycerol and its hydrolysis product (ethanol) as a co-substrate supplemented to palm oil mill effluent (POME) to enhance biogas production efficiency under mesophilic anaerobic digestion was investigated. Based on our previous studies, the concentrations of each co-substrate were varied in the range of 1–5% v⋅v−1. The optimum concentrations of crude glycerol and ethanol were 1% and 5% v/v, respectively. They could increase both methane production (nearly 2 fold) and methane yield (1.98–2.09 fold based on volatile solids (VS) and 1.6–1.87 fold based on COD). However, they exhibited oppositely in the methane production rate; the 1% crude glycerol was 38% lower while 5% ethanol exhibited a 67% higher methane production rate than the control (POME). The results clearly indicated that the methane production rate from 5% ethanol was 2.7 times higher than that from the 1% crude glycerol. Microbial community analysis revealed that the dominant bacteria in both co-substrates biogas production process were Sandarakinotalea sp., Lachnobacterium sp., Blautia sp., Marinobacter sp., Anaerotruncus sp., Clostridium sp., Enterobacter sp., and Acinetobacter sp. The Blautia sp. was dominant in the presence of 1% crude glycerol than 5% ethanol. The archaea communities were dominated by hydrogenotrophic methanogen (Methanoregula sp. and Methanoculleus sp.) and acetoclastic methanogen (Methanosarcina sp. and Methanospirillum sp.). Therefore, ethanol was a better co-substrate than crude glycerol. Nevertheless, biogas production from POME with crude glycerol as co-substrate was anticipated to strongly increase by enhancing the growth (or bioaugmentation) of Blautia sp. and Methanosarcina sp.

Two-stage thermophilic fermentation and mesophilic methanogenic process with methanogenic effluent recirculation to hydrogen reactor for biohythane production from palm oil mill effluent (POME) was investigated. Maximum hydrogen (4.1 L H2/L-POME) and methane production (16.6 L CH4/L-POME) from POME in batch tests were achieved at 30% recirculation rate of methanogenic effluent. Continuous hydrogen production of without and with recirculation phase was 2.2 and 3.8 L H2/L-POME, respectively. Continuous methane production of without and with recirculation phase was 12.2 and 14 L CH4/L-POME, respectively. Both reactor effluents were effective to keep at optimal pH with 2 times increasing in hydrogen production. The hydrogen and methane yield were 135 mL H2/gVS and 414 mL CH4/gVS, respectively. Biohythane gas composition was composed 13.3% of hydrogen, 54.4% of methane and 32.2% carbon dioxide. Two-stage process with methanogenic effluent recirculation flavored Thermoanaerobacterium sp. in the hydrogen reactor and efficiently for energy recovery from POME.

Co-fermentation of palm oil mill effluent (POME) for hydrogen production by Thermoanaerobacterium thermosaccharolyticum PSU-2 under thermophilic condition was investigated. Hydrogen production from co-fermentation POME with 5 g/L decanter cake and without was 2.6 and 1.7 L-H 2 /L-POME. Co-fermentation of POME with decanter cake enhanced hydrogen production. Tbm. thermosaccharolyticum PSU-2 augmentation at 20% v/v was found to be an efficient start-up strategy in continuous hydrogen production from co-fermentation of POME with decanter cake. Tbm. thermosaccharolyticum PSU-2 augmentation could suppress normal flora bacteria in POME and decanter cake. Hydrogen production of 7.2 L-H 2 /L-POME and hydrogen production rate of 18.1 mmol-H 2 /L/h was achieved from continuous hydrogen in long term operation. The soluble metabolites were dominated by acetic acid and butyric acid. Tbm. thermosaccharolyticum PSU-2 augmentation could use for control undesired bacteria in the hydrogen production system with flavored Thermoanaerobacterium sp. as dominant species.

Thermoanaerobacterium sp. strain PSU-2 was isolated from thermophilic hydrogen producing reactor and subjected to draft genome sequencing on 454 pyrosequencing and annotated on RAST. The draft genome sequence of strain PSU-2 contains 2,552,497 bases with an estimated G + C content of 35.2%, 2555 CDS, 8 rRNAs and 57 tRNAs. The strain had a number of genes responsible for carbohydrates metabolic, amino acids and derivatives, and protein metabolism of 17.7%, 14.39% and 9.81%, respectively. Strain PSU-2 also had gene responsible for hydrogen biosynthesis as well as the genes related to Ni-Fe hydrogenase. Comparative genomic analysis indicates strain PSU-2 shares about 94% genome sequence similarity with Thermoanaerobacterium xylanolyticum LX-11. The nucleotide sequence of this draft genome was deposited into DDBJ/ENA/GenBank under the accession MSQD00000000.

Moderate thermophilic fermentation appears to be superior on hydrogen production rate (98–152 mmol/L/h) with satisfactory hydrogen yield of 2–2.4 mol/mol C6, while extreme thermophilic fermentation has a lower hydrogen production rate (15–30 mmol/L/h) with rather high hydrogen yield of 2.8–3.5 mol/mol C6 when using pure sugar as substrate. Both moderate thermophilic and extreme thermophilic fermentation has similar hydrogen yield for the real-world substrate (2.4–2.8 mol H2/mol C6), such as wastewater and organic wastes. Extreme thermophilic fermentation has less variety of side-products than moderate thermophilic fermentation. Significant improvement in H2 production yield could be achieved to near maximum of 4 mol H2/mol C6 by in situ gas separation, microbial population optimization, and metabolic engineering in the microorganisms. Bioreactor design by integrating dark fermentation with volatile fatty acids oxidation by microbial electrolysis could have the potential to increase the hydrogen yield gone beyond the current maximum of 4 mol H2/mol C6 and address technical barriers that currently limit the technoeconomic feasibility of fermentative hydrogen production.

A two-stage thermophilic fermentation and mesophilic methanogenic process for biohythane production from palm oil mill effluent (POME) was investigated. The hydrogen and methane potential from POME were 170–200 L H 2 kgCOD −1 and 210–292 L CH 4 kgCOD −1 , respectively. A continuous two-stage process with hydraulic retention time (HRT) of 2 d for hydrogen reactor and 15 d for methane reactor, obtained 34% higher energy yield than single stage methane production. The hydrogen and methane yields from two-stage were 210 L H 2 kgCOD −1 and 315 L CH 4 kgCOD −1 , respectively with total energy yield of 15.34 MJ kgCOD −1 . Mixed hydrogen and methane (biohythane) production rate was 4.4 L biogas L −1 d −1 with containing of 51% CH 4 , 14% H 2 and 35% CO 2 . Hydrogen reactor was dominated with hydrogen producing bacteria of Thermoanaerobacterium thermosaccharolyticum , while acetoclastic Methanoculleus sp. was the dominant methanogen in methane reactor. Two-stage process for biohythane production could efficiently for energy recovery from POME.

The microbial community structure of thermophilic mixed culture sludge used for biohydrogen production from palm oil mill effluent was analyzed by fluorescence in situ hybridization (FISH) and 16S rRNA gene clone library techniques. The hydrogen-producing bacteria were isolated and their ability to produce hydrogen was confirmed. The microbial community was dominated by Thermoanaerobacterium species (∼66%). The remaining microorganisms belonged to Clostridium and Desulfotomaculum spp. (∼28% and ∼6%, respectively). Three hydrogen-producing strains, namely HPB-1, HPB-2, and HPB-3, were isolated. 16S rRNA gene sequence analysis of HPB-1 and HPB-2 revealed a high similarity to Thermoanaerobacterium thermosaccharolyticum (98.6% and 99.0%, respectively). The Thermoanaerobacterium HPB-2 strain was a promising candidate for thermophilic fermentative hydrogen production with a hydrogen yield of 2.53 mol H2 mol−1hexose from organic waste and wastewater containing a mixture of hexose and pentose sugars. Thermoanaerobacterium species play a major role in thermophilic hydrogen production as confirmed both by molecular and cultivation-based analyses.

Thermoanaerobacterium-rich sludge was used for hydrogen production and phenol removal from palm oil mill effluent (POME) in the presence of phenol concentration of 100–1000 mg/L. Thermoanaerobacterium-rich sludge yielded the most hydrogen of 4.2 L H2/L-POME with 65% phenol removal efficiency at 400 mg/L phenol. Butyric acid and acetic acid were the main metabolites. The effects of oil palm ash, NH4NO3 and iron concentration (Fe2+) on hydrogen production and phenol removal efficiency from POME by Thermoanaerobacterium-rich sludge was investigated using response surface methodology (RSM). The RSM results indicated that the presence of 0.2 g Fe2+/L, 0.3 g/L NH4NO3 and 20 g/L oil palm ash in POME could improved phenol removal efficiency, with predicted hydrogen production and phenol removal efficiency of 3.45 L H2/L-POME and 93%, respectively. In a confirmation experiment under optimized conditions highly reproducible results were obtained, with hydrogen production and phenol removal efficiency of 3.43 ± 0.12 L H2/L-POME and 92 ± 1.5%, respectively. Simultaneous hydrogen production and phenol removal efficiency in continuous stirred tank reactor at hydraulic retention time (HRT) of 1 and 2 days were 4.0 L H2/L-POME with 85% and 4.2 L H2/L-POME with 92%, respectively. Phenol degrading Thermoanaerobacterium-rich sludge comprised of Thermoanaerobacterium thermosaccharolyticum, Thermoanaerobacterium aciditolerans, Desulfotomaculum sp., Bacillus coagulans and Clostridium uzonii. Phenol degrading Thermoanaerobacterium-rich sludge has great potential to harvest hydrogen from phenol-containing wastewater.

Effect of organic loading rate (OLR), hydraulic retention time (HRT) and temperature variation on process stability of fermentative hydrogen production from palm oil mill effluent (POME) under thermophilic condition was investigated. High OLR (>70 gCOD/l/d) and low HRT (4 d) has significant affected on hydrogen productivity, process stability and microbial community. Hydrogen production at OLR of 55, 60, 65 and 70 gCOD/l/d was 2.4, 3.4, 4.9 and 3.7 L H 2 /L-POME, respectively. The decreasing in pH and alkalinity and increasing in total VFA could cause the instability of process. The microbial community was changed at OLR >70 gCOD/l/d, HRT 4 d and stop feeding. The dominant microbial community changed from Thermoanaerobacterim sp. to Clostridium sp., Thermoanaerobacterim sp. and Bacillus sp. Hydrogen production at HRT of 2, 4, stop feeding and 2 d was 2.7, 0.8, 0, and 2.6 L H 2 /L-POME, respectively. Hydrogen production, process stability and microbial community was not affected by variation of temperature. Hydrogen production before and after temperature variation was 2.5 and 2.45 L H 2 /L-POME.