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Acceleration of the anaerobic digestion (AD) process is crucial to achieving energy-efficient recycling of organic wastes. Hydrochar is produced by hydrothermal liquefaction of biomass, yet its application in the AD process is rarely reported. The present study showed that sewage sludge-derived hydrochar (SH) enhanced the methane production rate of glucose by 37%. SH increased the methane production rate from acetate but did not affect acidification and the methane production rate from H2/CO2. SH enhanced hydrogenotrophic methanogenesis, which could be due to direct interspecies electron transfer (DIET) by converting H+, e-, and CO2 to methane. Trichococcus and Methanosaeta were dominant in the AD process with SH. Label-free proteomic analysis showed Methanosaeta was involved in DIET as reflected by the up-regulation of proteins involved in hydrogenotrophic methanogenesis. Hydrochars derived from corn straw (CH), Enteromorpha algae (EH), and poplar wood (PH), as well as activated carbon (AC), were also tested in the AD process. SH, CH, and EH obviously increased the methane production rates, which were 39%, 15%, and 20% higher than the control experiment, respectively. It was neither electrical conductivity nor the total redox property of hydrochars and AC but the abundances of surface oxygen-containing functional groups that correlated to the methane production rates.

This study attempted to find potential effective thermotolerant microorganisms producing complex enzymes for use in the hydrolyzing empty fruit bunch (EFB) to reduce cost of enzyme and enhance the efficiency of saccharification. The enrichment process at 45 °C was employed as a strategy to obtain four effective thermotolerant microorganisms. Streptomyces thermocarboxydus ME742, Bacillus subtilis ME751 and Bacillus amyloliquefaciens ASB/TRE produced the highest activity of xylanase (226.2 U/mL), CMCase (3.84 U/mL) and FPase (69.55 U/mL), respectively, while Aspergillus fumigatus A4112 exhibited the highest specific activity of xylanase (637.9 U/mg), CMCase (5.55 U/mg) and FPase (21.58 U/mg). Xylanase of isolated ME742 and A4112, CMCase of isolated ASB/TRE, FPase of isolated ME742, ME751 and ASB/TRE possessed thermostability with 80% remaining activity at 60 °C after 1 h incubation. These four strains were capable to reduce 49–78% (w/w) lignin in raw EFB with simultaneous enzyme production. The EFB residue was reused as substrate for saccharification with the highest amount of reducing sugar using the crude enzymes from S. thermocarboxydus ME742 (9.24 mg/g EFB). The sugar was 3.76 and 3.61 fold higher than that obtained from saccharification of acid- and alkaline-pretreated EFB, respectively. Moreover, the crude enzymes from A. fumigatus A4112 and B. amyloliquefaciens ASB/TRE hydrolyzed palm oil mill effluent (POME) to generate high yield of reducing sugar (61.01–64.63 mg/g TS-POME). Therefore, these selected strains were considered as the potent biological tool applicable in the bioconversion of oil palm biomass to fermentable reducing sugars.

The efficacy of concentrated enzymes from Trichoderma koningiopsis TM3 in hydrolyzing palm oil mill effluent (POME) and oil palm trunk residues (OPTr) at 40 and 50 °C was evaluated prior to methane fermentation. POME hydrolysate containing total sugar concentration of 15.40 g L−1 was obtained from enzymatic hydrolysis using 15 Unit g−1 TVS at 50 °C for 18 h incubation with the hydrolysis yield of 0.35 g total sugars g−1 TVS. The OPTr hydrolysate contained slightly higher total sugar concentration (18.90 g L−1) with the hydrolysis yield of 0.85 g total sugars g−1 TVS under the same condition. Methane production from POME hydrolysate was 6.29% higher than the raw POME. Co-digestion of POME hydrolysate with OPTr gave the maximum methane yield (369 ml CH4 g−1 VS-added) with the increase of 9.28% compared to the raw POME. The methane production rate (Rmax) and the hydrolysis rate constant (kh) of the co-digestion of POME hydrolysate with OPTr were 1.2-fold higher than those of the POME hydrolysate. PCR-DGGE analysis revealed that Clostridium sp. and Petrimonas sp. were dominated bacteria while Methanosarcina sp. and Methanospirillum sp. played an important role in methane production. These results indicated that enzymatic pretreatment and co-digestion of POME hydrolysate with OPTr could improve methane yield from anaerobic fermentation of POME.

In this study, the performance of immobilized cells on granular activated carbon (GAC) for thermophilic biohydrogen production is determined using POME as a fermentation substrate. The immobilized cells are formed at different pH medium using sugar composition characterized in the POME. The pH 6 revealed the optimum pH used for biofilm development with HPR of 2.8 mmol H2/L h. The effect of sugar utilization by the immobilized cells on GAC are determined at different sugar concentration using the Monod model prior validated the performance of the cells in the fluidized bed reactor (FBR). From the model, 0.316 ± 0.013 h−1 of maximum specific growth rate was obtained at 20 g/L sugar used and was keep increasing to the maximum of 30 g/L of sugar used with HPR 2.6–2.8 mmol H2/L h. Lastly, the POME-enriched nutrients are used as the carbon source in the fluidized bed reactor (FBR). The highest HPR obtained was at HRT 12 h, (5.2 mmol H2/L h) and HY of 1.24 mol H2/mol sugar. The screening of the microbial population by DGGE revealed that the Thermoanaerobacterium thermosaccharolyticum sp. was dominant for all the HRTs, thereby indicating that this bacterium is resilient towards environmental disturbances.

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.

The suitability of molasses, Napier grass (Pennisetum purpureum), empty fruit bunches (EFB), palm oil mill effluent (POME), and glycerol waste as a co-substrate with Chlorella sp. TISTR 8411 biomass for biohythane production was investigated. Mono-digestion of Chlorella biomass had hydrogen and methane yield of 23–35 and 164–177 mL gVS−1, respectively. Co-digestion of Chlorella biomass with 2–6% TS of organic wastes was optimized for biohythane production with hydrogen and methane yield of 17–75 and 214–577 mL gVS−1, respectively. The hydrogen and methane yield from co-digestion of Chlorella biomass with molasses, POME, and glycerol waste was increased by 8–100% and 80–264%, respectively. The biohythane production of co-digestion of Chlorella was 6–11 L L-mixed waste−1 with an optimal C/N ratio range of 19–41 and H2/CH4 ratio range of 0.06–0.3. Co-digestion of Chlorella biomass was significantly improved biohythane production in term of yield, production rate, and kinetics.

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.

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.

Background The co-fermentation of syngas (mainly CO, H2 and CO2) and different concentrations of carbohydrate/protein synthetic wastewater to produce volatile fatty acids (VFAs) was conducted in the present study. Results It was found that co-fermentation of syngas with carbohydrate-rich synthetic wastewater could enhance the conversion efficiency of syngas and the most efficient conversion of syngas was obtained by co-fermentation of syngas with 5 g/L glucose, which resulted in 25% and 43% increased conversion efficiencies of CO and H2, compared to syngas alone. The protein-rich synthetic wastewater as co-substrate, however, had inhibition on syngas conversion due to the presence of high concentration of NH4+-N (> 900 mg/L) produced from protein degradation. qPCR analysis found higher concentration of acetogens, which could use CO and H2, was present in syngas and glucose co-fermentation system, compared to glucose solo-fermentation or syngas solo-fermentation. In addition, the known acetogen Clostridium formicoaceticum, which could utilize both carbohydrate and CO/H2 was enriched in syngas solo-fermentation and syngas with glucose co-fermentation. In addition, butyrate was detected in syngas and glucose co-fermentation system, compared to glucose solo-fermentation. The detected n-butyrate could be converted from acetate and lactate/ethanol which produced from glucose in syngas and glucose co-fermentation system supported by label-free quantitative proteomic analysis. Conclusions These results demonstrated that the co-fermentation with syngas and carbohydrate-rich wastewater could be a promising technology to increase the conversion of syngas to VFAs. In addition, the syngas and glucose co-fermentation system could change the degradation pathway of glucose in co-fermentation and produce fatty acids with longer carbon chain supported by microbial community and label-free quantitative proteomic analysis. The above results are innovative and lead to achieve effective conversion of syngas into VFAs/longer chain fatty acids, which would for sure have a great interest for the scientific and engineering community. Furthermore, the present study also used the combination of high-throughput sequencing of 16S rRNA genes, qPCR analysis and label-free quantitative proteomic analysis to provide deep insights of the co-fermentation process from the taxonomic and proteomic aspects, which should be applied for future studies relating with anaerobic fermentation.

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.

Two-stage thermophilic solid state anaerobic digestion (SS-AD) processes for biohythane production from Chlorella sp. biomass was investigated. The hydrogen and methane yield from fresh Chlorella sp. biomass was 124.9 mL H2 gVS−1 and 230.1 mL CH4 gVS−1, respectively, at 16% TS with biodegradation efficiency of 34%. Hydrogen and methane production from hydrothermal pretreated Chlorella sp. biomass was 190.0 mL H2 gVS−1 and 319.8 mL CH4 gVS−1, respectively at 16% TS with biodegradation efficiency of 47%. Pretreatment of Chlorella sp. biomass with hydrothermal follow by two-stage thermophilic SS-AD improved 150% biogas production comparing with fresh biomass. Clostridium sp. was the main bacteria responsible for hydrogen production. Clostridium sp. and Ruminococcus sp. was main bacteria in methane stage, while Methanoculleus sp. and Methanothermobacter sp. was dominated archaea in methane production process. Chlorella sp. biomass with hydrothermal pretreatment has great potential for hydrogen and methane production via two-stage thermophilic SS-AD.

Oil palm trunk (OPT) an abundant agriculture waste was evaluated as an alternative lignocellulosic biomass resource for bioethanol production. Sulfite-based SPORL pretreatment (Sulfite Pretreatment to Overcome Recalcitrance of Lignocellulose) was applied to the OPT to enhance its enzymatic saccharification. The pretreatment conditions were optimized through a designed experiment within the ranges of temperature 170–190oC, time for 30 min, 2–6% sulfite, and initial H2SO4 concentrations 3–7% (w/w). The overall saccharides (hexoses and pentoses) recovery of SPORL pretreated was 62.5%. SPORL pretreatment removed 38.8% lignin from the OPT as lignosulfonate in the liquor with great potential for co-product development, and 93.9% xylose was dissolved, which were the main reasons why the SPORL pretreatment could enhance the enzymatic hydrolysis of the OPT. The results indicated that over 92% of the cellulose in the pretreated OPT with 7% H2SO4 and 6% Na2SO3 at 190oC was enzymatically hydrolyzed to glucose within 48 h with a cellulase loading of 15 FPU/g cellulose, and overall glucose yield was 66%.

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

Hydrothermal liquefaction of sewage sludge to produce bio-oil and hydro-char unavoidably results in the production of high-strength organic wastewater (HTLWW). However, anaerobic digestion (AD) of HTLWW generally has low conversion efficiency due to the presence of complex and refractory organics. The present study showed that granular activated carbon (GAC) promoted the AD of HTLWW in continuous experiments, resulting in the higher methane yield (259 mL/g COD) compared to control experiment (202 mL/g COD). It was found that GAC increased the activities of both aceticlastic and hydrogenotrophic methanogens. The molecular transformation of organics in HTLWW was further analyzed. It was shown GAC promoted the degradation of soluble microbial by-products, fulvic- and humic-like substances as revealed by 3-dimensional fluorescence excitation-emission matrix (3D-EEM) analysis. Gas chromatography mass spectrometry (GC–MS) analysis showed that GAC resulted in the higher degradation of N-heterocyclic compounds, acids and aromatic compounds and less production of new organic species. Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) analysis also showed that GAC promoted the degradation of nitrogenous organics. In addition, it was shown that GAC improved the removal of less oxidized, higher nitrogen content, and higher double bond equivalent (DBE) organic compounds. Microbial analysis showed that GAC not only increased the microbial concentration, but also enriched more syntrophic bacteria (e.g., Syntrophorhabdus and Synergistes), which were capable of degrading a wide range of different organics including nitrogenous and aromatic organics. Furthermore, profound effects on the methanogens and the enrichment of Methanothrix instead of Methanosarcina were observed. Overall, the present study revealed the molecular transformation and microbial mechanism in the AD of HTLWW with the presence of GAC. Keywords: Wastewater treatment, Sludge hydrothermal liquefaction, Granular activated carbon, Anaerobic digestion, Molecular analysis, Microbial analysis

Thermotolerant cellulolytic consortium for improvement biogas production from oil palm empty fruit bunches (EFB) by prehydrolysis and bioaugmentation strategies was investigated via solid-state anaerobic digestion (SS-AD). The prehydrolysis EFB with Clostridiaceae and Lachnospiraceae rich consortium have maximum methane yield of 252 and 349 ml CH4 g−1 VS with total EFB degradation efficiency of 62% and 86%, respectively. Clostridiaceae and Lachnospiraceae rich consortium augmentation in biogas reactor have maximum methane yield of 217 and 85.2 ml CH4 g−1 VS with degradation efficiency of 42% and 16%, respectively. The best improvement of biogas production was achieved by prehydrolysis EFB with Lachnospiraceae rich consortium with maximum methane production of 113 m3 CH4 tonne−1 EFB. While, Clostridiaceae rich consortium was suitable for augmentation in biogas reactor with maximum methane production of 70.6 m3 CH4 tonne−1 EFB. Application of thermotolerant cellulolytic consortium into the SS-AD systems could enhance biogas production of 3–11 times.

The high-solid anaerobic digestion (AD) for anaerobic co-digestion of palm oil mill effluent (POME) and oil palm empty fruit bunches (EFB) has low degradation efficiency. The enhancement of biogas production and EFB degradation efficiency by Bacteroides and Clostridium-rich methanogenic consortium was investigated. A Bacteroides and Clostridium-rich methanogenic consortium have xylanase, FPase, endo-glucanase, and exo-glucanase activity of 9.2, 1.3, 2.1, and 1.5 IU·mL−1, respectively with EFB degradation efficiency of 57.5%. Methane production (79 m3-CH4·tonne−1-EFB) by Bacteroides and Clostridium-rich methanogenic consortium augmentation was increased by 67.2%. The real application of bioaugmentation in the feedstock mixing tank before a feed to the AD reactor significantly increased methane yield (376.7 mL-CH4·g-1-VS−) by 85.5%. Bioaugmentation of Bacteroides and Clostridium-rich methanogenic consortium increased the number of hydrolytic bacteria and strengthened symbiotic relationships with indigenous microorganisms resulting in high biogas production, accelerated hydrolysis, and reduced solids accumulation of the high-solid AD process.

A novel reactor was developed in this research to simultaneously produce biogas from solid- and liquid-state anaerobic digestions in one container for the first time. Excess heat from the outer liquid-state anaerobic digestion (OL-AD) of POME was transferred to the inner solid-state anaerobic digestion (IS-AD) of EFB for promoting thermophilic digestions (55 °C and 70 °C). Continuous operations at various hydraulic retention times were performed. Bacteria and archaea communities were identified. The results showed that the maximum methane yields were 507.07 and 125.84 mL-CH4/g-VS for OL-AD and IS-AD, respectively. In OL-AD, raising temperature increased the methane yield by 6.4%. In IS-AD, the yield was not significantly affected by elevated temperature. However, the pretreatment of EFB with NaOH increased the methane yield by 14.1%. The main bacteria and archaea communities in OL-AD and IS-AD were found similar. The novel reactor could save energy, reduce costs and digester space, and increase the methane yield.

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.

Population genetic structure of Varuna litterata living along the coast of Thailand were examined in this study. The samples were collected from 3 coastal regions: The Andaman sea (Satun, Trang, Phang Nga), the lower Gulf of Thailand (Pattani, Songkhla, Nakhon Si Thammarat) and the upper Gulf of Thailand (Petchburi, Samut Songkram, Rayong, Trat). Intraspecific variation was determined based on partial sequences of the cytochrome oxidase subunits I gene. A total of 182 samples were collected but only 32 haplotypes were obtained from these samples. An excess of rare haplotypes indicated that the female effective population size of V. litterata living along the coast of Thailand is large. Estimated values of haplotype diversity and nucleotide diversity were 0.790 and 0.003, respectively. The AMOVA (analysis of molecular variance) and phylogenetic analysis results showed that based on genetic variation, the population of this organism was found to have 2 genetically different populations: The Andaman sea population and the Gulf of Thailand population. Genetic exchange of V. litterata among populations inhabiting along the coast of Thailand could be described by the stepping stone model. The results of neutrality tests, both Tajima’s D and Fu’s Fs statistics, yielded negative values (-1.992 and -26.877, respectively) and statistically significant deviation from the neutrality, indicating that the V. litterata living along the Thailand coast had experienced population expansion. Mismatch distribution analysis indicated that a possible expansion occurred 211,428 years ago during the Pleistocene glaciations period.

Bacterial Cellulose (BC) was produced by Acetobacterxylinum TISTR 086 use mixing of oil palm shoot juices (OPSJ) and coconut juice (CJ) medium with or without nutrient supplemented or OPSJ was digested with α-amylase. Results showed the highest yield was obtained at OPSJ with CJ at mixing ratios of 50% (4.70±0.11 g/L) and the lowest yield was obtained at OPSJ with CJ at mixing ratios of 0% (3.54±0.11 g/L). OPSJ was digested with 1300 lU/gm at concentration enzyme 650 IU/L at 37 °C for 24 h has highest sugar concentration of 73.93±0.12 g/L. Amylase digested OPSJ mixed with coconut juice’s highest bacterial cellulose production at OPSJ with CJ at mixing ratios of 50% (4.94±0.13 g/L), respectively. Moreover, the BC obtained had molecular weight from 332,122 to 503,233 g/mol. The chemical structure of BC produced was studied using Fourier Transform Infrared Spectroscopy (FT-IR) and X-ray defections. The morphology of BC was investigated by Scanning Electron Microscopy (SEM). Thermal property was further studied using Differential Scanning Calorimetry (DSC) and Thermogravimetic Analysis (TGA). This study obviously showed the ability of OPSJ, a suitable and cheap carbon source, to be used as a substrate in a fermentation medium for production of cellulose by Acetobacterxylinum TISTR 086.

BACKGROUND AND OBJECTIVE Toxic nitrogen compounds are one cause decreasing of shrimp production and water pollution. Indigenous Halomonas spp., isolated from Pacific white shrimp farm are benefitted for saline ammonium waste water treatment. This study aimed to isolate the heterotrophic-halophilic Halomonas spp. and investigate their ammonium removal efficiency. MATERIALS AND METHODS Halomonas spp., were isolated by culturing of samples collected from shrimp farm into modified Pep-Beef-AOM medium. Ammonium converting ability was tested and monitored by nitrite reagent. Ammonium removal efficiency was measured by the standard colorimetric method. Identification and classification of Halomonas spp., were studied by morphological, physiological and biochemical characteristics as well as molecular information. RESULTS There were 5 strains of heterotrophic-halophilic nitrifying bacteria including SKNB2, SKNB4, SKNB17, SKNB20 and SKNB22 were isolated. The identification result based on 16S rRNA sequence analysis indicated that all 5 strains were Halomonas spp., with sequence similarity values of 91-99 %. Ammonium removal efficiency of all strains showed a range of 23-71%. The production of nitrite was low detected of 0.01-0.15 mg-N L-1, while the amount of nitrate was almost undetectable. CONCLUSION This might suggest that the indigenous Halomonas spp., as nitrifying bacteria involved biological nitrification process for decreasing and transforming of ammonia. Due to being heterotrophic, halophilic and ammonium removing bacteria, these Halomonas spp., could be developed for use in treatment of saline ammonium waste water.

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.

Five bacterial strains were screened for polymer production in five synthetic media (GP, PS, GL, PL and PR). The highest polymer yield was obtained from Bacillus subtilis WD161 in PR medium (8.89 g/L). Cultivation at 30, 45 and 60 °C indicated that they were thermotolerant with the maximum biopolymer yield at 45 °C. Using palm oil mill effluent (POME) as medium, the nitrogen had to be supplemented to 1.28 g/L (equal to that in PR medium) with the initial pH adjusted to 7.0 and cultivation at 30 °C. Characterization based on its composition and functional groups revealed that the biopolymer WD161 was glycoprotein with the molecular weight of 1.54 × 10 6 Da. FT-IR analysis revealed the presence of amine (NH 2 ), hydroxyl, carboxyl and carbonyl groups. It was also a bioflocculant and its flocculating activity to precipitate the suspended solid in POME increased by 35% under the optimum condition.

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.

Hydrothermal carbonization aqueous phase (HTC-AP) can be used for methane production by anaerobic digestion (AD). However, it generally had low conversion efficiency due to the formation of complex dissolved organic matters, which depends upon the components of biomass. The present study investigated the characteristics, methane potentials, and recalcitrant chemicals of HTC-AP produced from different combinations of model compounds carbohydrate (α-cellulose, C) and protein (bovine serum albumin, BSA) with mass ratios of 1:0, 0.75:0.25, 0.5:0.5, 0.75:0.25 and 0:1. The methane yields of samples 1:0 (pure C) and 0:1 (pure BSA) were 192 mL/g COD and 187 mL/g COD, respectively, while it was decreased to 105.5 CH4 mL/g COD for sample 0.75:0.25 (C/BSA), indicating more recalcitrant organics were produced with the combination of C and BSA. It was found that the mean MW (209157) of sample 0.75:0.25 was much higher than the other samples (

The highly heterogeneous structure of oil palm empty fruit bunches (EFB) restrict the exploitation of its potential in biogas plants. Fungal pretreatment of EFB with Trichoderma reesei TISTR 3080 and Pleurotus ostreatus DSM 11191 to improve EFB biodegradability and methane production via solid-state anaerobic digestion (SS-AD) was investigated. The pretreatment by T. reesei and P. ostreatus were effective at an EFB moisture content of 80–85 (%w/w) and incubation for 10–15 days. The methane yield was increased by up to 44–52 % compared to raw EFB. A strong linear relationship was observed between the methane yield, cellulose, hemicellulose and lignin degradation for both fungal strains. Maximum methane production from untreated EFB, T. reesei and P. ostreatus pretreated EFB was 36.2, 75.8, and 64.9 m3 CH4 tonne−1 EFB, respectively. The pretreatment of EFB with T. reesei is thus a promising strategy to achieve higher methane yields and a 60–75 % higher biodegradation efficiency without any additional energy input and undesired by-product inhibition.

Hydrogen production from crude glycerol by two-stage processes of dark fermentation using Klebsiella sp. TR17 and photo fermentation using Rhodopseudomonas palustris TN1 (Rps. palustris TN1) was investigated in batch experiments. In dark fermentation, the cumulative hydrogen production and hydrogen yield was 64.24 mmol H2/L and 5.74 mmol H2/g COD consumed, respectively with 80.21% of glycerol conversion rate. The dark fermentation effluent (DFE) was employed for photo fermentation. Effect of DFE concentrations (0–5 times dilution), with and without supplementation of yeast extract (2.3 g/L) + NaHCO3 (0.63 g/L), and glutamate (2–8 mM) were optimized. The optimal conditions for hydrogen production from Rps. palustris TN1 were 5 times dilution of DFE without the supplement of yeast extract + NaHCO3, and 2 mM glutamate. Under the optimum conditions, the cumulative hydrogen production of 3.12 mmol H2/L and hydrogen yield of 0.68 mmol H2/g COD consumed was obtained. The total hydrogen yield of two-stage processes was estimated to be 6.42 mmol H2/g COD consumed which was 10.4% of the theoretical yield.

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.

Anaerobic co-digestion of canned seafood wastewater (CSW) with glycerol waste (GW) and wolffia arrhiza (WA) for methane production was investigated. Methane yields from anaerobic co-digestion of CSW with 1%GW, CSW with 1%GW and 5%WA, CSW with 1%GW and 10%WA and CSW with 1%GW and 15%WA were 577, 789, 545 and 474 mL CH4/g VS-added, respectively. Methane production from CSW with 1%GW and 5%WA increased approximately 4-fold when compared with CSW alone (278 mLCH4/g VS-added). Co-digestion of CSW with 1% GW and 5% WA was the best condition and gave the maximum methane production of 8.8 m3 CH4/m3 mixed wastewater and 96.8% biodegradability. The maximum methane production rate and yield were 3.71 L CH4/L-reactor.day and 858 mL CH4/g VS-added (352 mLCH4/g COD-removed) at OLR of 4 g COD/L. day in UASB reactor. The methane composition in biogas was 62.3%. The Monod, Modified Stover–Kincannon and Grau second-order models were used to explain the performance of UASB reactor. The results showed that the kinetic coefficient of the Modified Stover–Kincannon model could explain the performance of UASB reactor in term of COD removal efficiency and microbial growth by having the regression coefficient (R2) as 0.987.

Hydrogen production from palm oil mill effluent (POME) by Thermoanaerobacterium thermosaccharolyticum PSU-2 was investigated both in batch and continuous reactors using anaerobic sequencing batch reactor (ASBR) and continuous stirred tank reactor (CSTR). The hydrogen production determined from batch experiment of POME at an inoculum size of 0%, 10%, 20% and 30% (v/v) was 161, 201, 246 and 296 mL H2/g-COD with COD removal efficiency of 21%, 23%, 23% and 23%, respectively. Continuous hydrogen production was start-up with 30% (v/v) inoculum in both ASBR and CSTR reactors and more than 30% COD removal could be obtained at HRT of 4 days, corresponding to OLR of 11.3 g COD/ L·day. Similar hydrogen production rates of 2.05 and2.16 LH2/L. day were obtained from ASBR and CSTR, respectively. COD removal efficiency of ASBR was 37.7%, while it was 44.8% for CSTR. However, ASBR was stable in term of alkalinity, while the CSTR was stable in term of hydrogen production, soluble metabolites concentration and alkalinity. Therefore, the CSTR was found to be more stable in hydrogen production than ASBR under the same OLR.

Bio-hydrogen production from glycerol by immobilized Enterobacter aerogenes ATCC 13048 on heat-treated upflow anaerobic sludge blanket (UASB) granules was examined in a UASB reactor. The organic loading rate (OLR) was optimized in order to maximize the hydrogen production rate (HPR). The maximum hydrogen content (37.1% and 24.2%) and HPR (9 and 6.2 mmol H2/L h) were achieved at the optimum OLR of 50 g/L d using pure and waste glycerol as the substrate, respectively. The major soluble metabolite products (SMPs) were ethanol, 1,3-propanediol (1,3-PD), formic acid, and acetic acid. The microbial community and microbial structure, analyzed by fluorescent in situ hybridization (FISH) and scanning electron microscopy (SEM), revealed that the predominant hydrogen producers were E. aerogenes ATCC 13048 and firmicutes bacteria including Clostridium, Bacillus, and Dialister sp.

A limited number of bacteria can convert oil palm ( Elaeis guineensis ) sap to hydrogen with satisfactory yield and productivity. In this study, a total of 18 fermentative enriched cultures and 36 newly isolated thermotolerant bacterial strains were compared for hydrogen production from oil palm (OP) sap. The new isolates were obtained from hot springs, palm oil mill effluent and oil palm sap. The test was conducted in three steps: (i) a test for hydrogen production from mixed substrates (cellulose, starch, xylose, and glucose) and OP sap; (ii) a test for substrate concentration tolerance; and (iii) a test for thermotolerance. Five enriched candidates for each of the hydrogen producers were selected according to the criteria defined for the screening test. The hydrogen production of these selected bacterial strains from hot springs were cultivated in batch fermentation of oil palm sap at room temperature (30 ± 2 °C). Five enriched cultures, namely 81RN1, OPS, 85RN5, 89SR3-2 and 112YL1 were found to give high cumulative hydrogen formation of 1085, 1009, 994, 983 and 778 mL H 2 /L-OP sap, respectively, with the hydrogen content of 29.8, 29.4, 28.7, 27.1 and 27.5%, respectively. PCR–DGGE profiling showed that all these five enriched cultures consisted of species closely related to the genus Clostridium sp. based on the 16S rRNA gene. For pure cultures, the top five hydrogen producers were the isolates encoded as PS-3, PS-4, PS-5, PS-7 and PS-8 exhibiting the hydrogen production of 1973, 1774, 1335, 1170 and 1070 mL H 2 /L-OP sap, respectively, with the hydrogen content of 33.7, 29.6, 32.5, 31.5 and 26.4%, respectively. Identification of these high hydrogen producers using 16S rRNA sequence matching showed that the isolates PS-3 and PS-8 belonged to Clostridium beijerinckii , while the isolate PS-7 belonged to Clostridium acetobutylicum and the isolates PS-4 and PS-5 belonged to Klebsiella sp. and Klebsiella pneumoniae , respectively. Therefore, the pure culture C. beijerinckii PS-3 exhibited 1.8 folds higher hydrogen production (1973 mL H 2 /L-OP sap) than the enriched cultures of 81RN1 (1085 mL H 2 /L-OP sap).

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.

Hydrogen and methane production from desugared molasses by a two-stage thermophilic anaerobic process was investigated in a series of two up-flow anaerobic sludge blanket (UASB) reactors. The first reactor that was dominated with hydrogen-producing bacteria of Thermoanaerobacterium thermosaccharolyticum and Thermoanaerobacterium aciditolerans could generate a high hydrogen production rate of 5600 mL H2/day/L, corresponding to a yield of 132 mL H2/g volatile solid (VS). The effluent from the hydrogen reactor was further converted to methane in the second reactor with the optimal production rate of 3380 mL CH4/day/L, corresponding to a yield of 239 mL CH4/g VS. Aceticlastic Methanosarcina mazei was the dominant methanogen in the methanogenesis stage. This work demonstrates that biohydrogen production can be very efficiently coupled with a subsequent step of methane production using desugared molasses. Furthermore, the mixed gas with a volumetric content of 16.5% H2, 38.7% CO2, and 44.8% CH4, containing approximately 15% energy by hydrogen is viable to be bio-hythane.

A thermotolerant fermentative hydrogen-producing strain was isolated from crude glycerol contaminated soil and identified as Klebsiella pneumoniae on the basis of the 16S rRNA gene analysis as well as physiological and biochemical characteristics. The selected strain, designated as K. pneumoniae TR17, gave good hydrogen production from crude glycerol. Culture conditions influencing the hydrogen production were investigated. The strain produced hydrogen within a wide range of temperature (30–50 °C), initial pH (4.0–9.0) and crude glycerol concentration (20–100 g/L) with yeast extract as a favorable nitrogen source. In batch cultivation, the optimal conditions for hydrogen production were: cultivation temperature at 40 °C, initial pH at 8.0, 20 g/L crude glycerol and 2 g/L yeast extract. This resulted in the maximum cumulative hydrogen production of 27.7 mmol H 2 /L and hydrogen yield of 0.25 mol H 2 /mol glycerol. In addition, the main soluble metabolites were 1,3-propanediol, 2,3-butanediol and ethanol corresponding to the production of 3.52, 2.06 and 3.95 g/L, respectively.

Thermotolerant consortia were obtained by heat-shock treatment on seed sludge from palm oil mill. Effect of the initial pH (4.5–6.5) on fermentative hydrogen production palm oil mill effluent (POME) showed the optimum pH at 6.0, with the maximum hydrogen production potential of 702.52 mL/L-POME, production rate of 74.54 mL/L/h. Nutrients optimization was investigated by response surface methodology with central composite design (CCD). The optimum nutrients contained 0.25 g urea/L, 0.02 g Na 2 HPO 4 /L and 0.36 g FeSO 4 ·7H 2 O/L, giving the predicted value of hydrogen production of 1075 mL/L-POME. Validation experiment revealed the actual hydrogen production of 968 mL/L-POME. Studies on the effect of temperature (25–55 °C) revealed that the maximum hydrogen production potential (985.3 mL/L-POME), hydrogen production rate (75.99 mL/L/h) and hydrogen yield (27.09 mL/g COD) were achieved at 55, 45 and 37 °C, respectively. Corresponding microbial community determined by the DGGE profile demonstrated that Clostridium spp. was the dominant species. Clostridium paraputrificum was the only dominant bacterium presented in all temperatures tested, indicating that the strain was thermotolerant.

Hydrogen production from desugared molasses (DM) was investigated in both batch and continuous reactors using thermophilic mixed cultures enriched from digested manure by load shock (loading with DM concentration of 50.1 g-sugar/L) to suppress methanogens. H 2 gas, free of methane, was produced during batch cultivations, at different (DM) concentrations ranging from 1.5 g-sugars/L to 50.1 g-sugars/L. The highest yield of 237 ml-H 2 /g-sugar was achieved during the DM batch fermentation at concentration of 2.1 g-sugars/L, whereafter the yield decreased with increasing DM concentration. The enriched hydrogen producing mixed culture achieved from the 16.7 g-sugars/L DM batch cultivation was immobilized on heat treated anaerobic sludge granules in an up-flow anaerobic sludge blanket (UASB) reactor. The UASB reactor, operated at a hydraulic retention time (HRT) of 24 h fed with 16.7 g-sugars/L DM showed good performance with a satisfactory hydrogen yield of 269.5 ml-H 2 /g-sugar and rate of 4500 ml H 2 /l⋅d. Fluorescent in situ hybridization (FISH) analysis of the microbial community of sludge from batch fermentation and the UASB-granules after 54 days of operation, was dominated by Thermoanaerobacterium spp., which are key players in fermentative hydrogen production of DM under thermophilic conditions. Furthermore, the granules in the UASB reactor were also significantly containing Thermoanaerobacterium spp. and phylum Firmecutes (most Clotridium , Bacillus and Desulfobacterium ) and Thermoanaerobacterium thermosaccharolyticum with a relative abundance of 36%, 27%, and 10% of total microorganisms, respectively. This study shows that hydrogen production could be efficiently facilitated by using anaerobic granules as a carrier, where microbes from mixed culture enriched in the DM batch cultivation were immobilized on, in an UASB reactor.

We investigated the feasibility of producing biohydrogen from sago starch in wastewater using a thermophilic mixed culture enriched from a hot spring in southern Thailand (PGK). The effects of repeated batch cultivations, the dependence of the initial pH values ranging from 5.0 to 8.0 and the initial sago starch concentrations ranging from 2.5 to 60 g/l as well as the utilization of 4 different starch types (gelatinized dry starch, non-gelatinized dry starch, gelatinized wet starch, and non-gelatinized wet starch) were examined in batch experiments at 60 °C. Microbial community structures were also investigated. The methane-free biogas evolved contained up to 55% hydrogen, with the remainder comprising carbon dioxide. Repeated batch cultivation under identical conditions improved the hydrogen yield. Gelatinized dry starch at an initial pH of 6.5 and an initial starch concentration of 2.5 g/l gave the maximum hydrogen yield of 422 ml-H2/g-starchadded (80% of the theoretical limit). PCR-DGGE profiles of 16S rRNA gene fragments from the cultures showed that the predominant hydrogen producers were closely related to thermophilic anaerobic bacteria, including Thermoanaerobacterium saccharolyticum, Thermoanaerobacterium thermosulfurigenes, and uncultured Thermoanaerobacterium sp., all of which probably produced hydrogen with simultaneous generation of ethanol. The thermophilic bacilli capable of utilizing starch, including Bacillus sp., Anoxybacillus sp., Bacillus lentus, and Geobacillus sp., were also found in the starch cultures, but mainly in the non-gelatinized wet starch culture.

Sediment samples from hot spring were enriched for hydrogen-producing bacteria with xylose, glucose and mixed of both sugars at high temperature (60 °C). Enriched cultures encoded as PGR and YLT showed high cumulative hydrogen production in xylose–glucose mixed and xylose with hydrogen evolution of 1506 and 1487 mL H2/L-medium and hydrogen yields of 301.3 and 297.4 mL H2/g sugarconsumed, respectively. The enriched cultures coded PGR and YLT were produced acetate and butyrate as main soluble metabolites with high hydrogen production. PCR-DGGE profiling showed that the enriched cultures gave best hydrogen production from xylose–glucose mixed comprised of three groups of hydrogen-producing species, (A) relating to genus Bacillus sp. and Anoxybacillus sp., (B) relating to genus Caloramator sp. and Clostridium sp. and (C) relating to efficient hydrogen-producing species (Thermoanaerobacterium sp. and Caldicellulosiruptor sp.). These microbial consortia developed show promising to apply for biohydrogen production from lignocellulosic hydrolysate containing xylose–glucose mixed.

Non-sterile operation of continuous stirred tank reactor (CSTR) augmented with Clostridium butyricum and fed with sugarcane juice was studied at various hydraulic retention time (HRT). The maximum hydrogen production rate and yield of 3.38 mmol H2/L/h and 1.0 mol H2/mol hexose consumed, respectively, were achieved at HRT 4 h. The relationship of the augmented microorganism and normal flora in the fermentation system under non-sterile condition were analyzed by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE). Initially, at HRT 36 h, other species related to Lactobacillus harbinensis and Klebseilla pneumoniae were present as a major group in the reactor. When HRT was decreased to 12, 6 and 4 h, C. butyricum was present with a competition between L. harbinensis and K. pneumoniae. Results indicated that augmented C. butyricum could compete with contaminated microorganisms during non-sterile operation at low HRT (12–4 h) with the support of normal flora (K. pneumoniae).

Xylose and glucose are the major sugar components of lignocellulosic hydrolysate. This study aims to develop thermophilic hydrogen-producing consortia from eight sediments-rich samples of geothermal springs in Southern Thailand by repeated batch cultivation at 60 °C with glucose, xylose and xylose–glucose mixed substrates. Significant hydrogen production potentials were obtained from thermophilic enriched cultures encoded as PGR and YLT with the maximum hydrogen yields of 241.4 and 231.6 mL H2/g sugarconsumed, respectively. After repeated batch cultivation the hydrogen yield from xylose–glucose mixed substrate of PGR increased to 375 mL H2/g sugarconsumed which was 30% higher than that of YLT (287 mL H2/g sugarconsumed). Soluble metabolites from xylose–glucose mixed substrates were composed mostly of butyric acid (20.6–21.8 mM), acetic acid (7.2–13.5 mM), lactic acid (8.2–11.7 mM) and butanol (4.4–13.0 mM). Denaturing gradient gel electrophoresis (DGGE) profiles illustrated small difference in microbial patterns of PGR enriched with glucose, xylose–glucose mixed substrate and xylose. The dominant populations were affiliated with low G + C content Gram-positive bacteria, Thermoanaerobacterium sp., Thermoanaerobacter sp., Caloramater sp. and Anoxybacillus sp. based on the 16S rRNA gene. Cultivation of the enriched culture PGR in oil palm trunk hydrolysate, the maximum hydrogen yield of 301 mL H2/g sugarconsumed was achieved at hydrolysate concentration of 40% (v/v). At higher concentration to 80% (v/v), the hydrogen fermentation process was inhibited. Therefore, the efficient thermophilic hydrogen-producing consortia PGR has successfully developed and has great potential for production of biohydrogen from mixed sugars hydrolysate.

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.

The optimum values of hydraulic retention time (HRT) and organic loading rate (OLR) of an anaerobic sequencing batch reactor (ASBR) for biohydrogen production from palm oil mill effluent (POME) under thermophilic conditions (60 °C) were investigated in order to achieve the maximum process stability. Microbial community structure dynamics in the ASBR was studied by denaturing gradient gel electrophoresis (DGGE) aiming at improved insight into the hydrogen fermentation microorganisms. The optimum values of 2-d HRT with an OLR of 60 gCOD l −1 d −1 gave a maximum hydrogen yield of 0.27 l H 2 g COD −1 with a volumetric hydrogen production rate of 9.1 l H 2 l −1 d −1 (16.9 mmol l −1 h −1 ). The hydrogen content, total carbohydrate consumption, COD (chemical oxygen demand) removal and suspended solids removal were 55 ± 3.5%, 92 ± 3%, 57 ± 2.5% and 78 ± 2%, respectively. Acetic acid and butyric acid were the major soluble end-products. The microbial community structure was strongly dependent on the HRT and OLR. DGGE profiling illustrated that Thermoanaerobacterium spp., such as Thermoanaerobacterium thermosaccharolyticum and Thermoanaerobacterium bryantii , were dominant and probably played an important role in hydrogen production under the optimum conditions. The shift in the microbial community from a dominance of T. thermosaccharolyticum to a community where also Caloramator proteoclasticus constituted a major component occurred at suboptimal HRT (1 d) and OLR (80 gCOD l −1 d −1 ) conditions. The results showed that the hydrogen production performance was closely correlated with the bacterial community structure. This is the first report of a successful ASBR operation achieving a high hydrogen production rate from real wastewater (POME).

Five methods for preparation of hydrogen-producing seeds (base, acid, 2-bromoethanesulfonic acid (BESA), load-shock and heat shock treatments) as well as an untreated anaerobic digested sludge were compared for their hydrogen production performance and responsible microbial community structures under thermophilic condition (60 °C). The results showed that the load-shock treatment method was the best for enriching thermophilic hydrogen-producing seeds from mixed anaerobic cultures as it completely repressed methanogenic activity and gave the a maximum hydrogen production yield of 1.96 mol H 2 mol −1 hexose with an hydrogen production rate of 11.2 mmol H 2 l −1 h −1 . Load-shock and heat-shock treatments resulted in a dominance of Thermoanaerobacterium thermosaccharolyticum with acetic acid and butyric acid type of fermentation while base- and acid-treated seeds were dominated by Clostridium sp. and BESA-treated seeds were dominated by Bacillus sp. The comparative experimental results from hydrogen production performance and microbial community analysis showed that the load-shock treatment method was better than the other four methods for enriching thermophilic hydrogen-producing seeds from anaerobic digested sludge. Load-shock treated sludge was implemented in palm oil mill effluent (POME) fermentation and was found to give maximum hydrogen production rates of 13.34 mmol H 2 l −1 h −1 and resulted in a dominance of Thermoanaerobacterium spp. Load-shock treatment is an easy and practical method for enriching thermophilic hydrogen-producing bacteria from anaerobic digested sludge.

16S rRNA gene targeted oligonucleotide probes for specific detection of genera Thermoanaerobacterium (Tbm1282), Caldicellulosiruptor (Ccs432), and specie Thermoanaerobacterium thermosaccharolyticum (Tbmthsacc184) were designed and used to monitor the spatial distribution of hydrogen producing bacteria in sludge and granules from anaerobic reactors. The designed probes were checked for their specificity and then validated using fluorescence in situ hybridization with target microorganisms and non-target microorganisms. Thermoanaerobacterium spp., T. thermosaccharolyticum and Caldicellulosiruptor spp. were detected with the probes designed with coverage of 75%, 100% and 93%, respectively. Thermophilic (60 °C) hydrogen producing reactors, one fed with sucrose and another, fed with palm oil mill effluent comprised of following major groups of hydrogen producers: Thermoanaerobacterium spp. (49% and 36%), T. thermosaccharolyticum (16% and 10%), phylum Firmicutes (low G+C) gram positive bacteria (15% and 27%). Extreme-thermophilic (70 °C) hydrogen producing reactors, one fed with xylose and another, fed with lignocellulosic hydrolysate comprised of following major groups of hydrogen producers: Caldicellulosiruptor spp. (40.5% and 20.5%), phylum Firmicutes (low G+C) gram positive bacteria (17% and 20%), Archaea (7% and 8.5%), and Thermoanaerobacterium spp. (0% and 5%). Results obtained, showed good applicability of the probes Tbm1282, Tbmthsacc184 and Ccs432 for specific detection and quantification of thermophilic and extreme-thermophilic hydrogen producers in complex environments.

Biohydrogen production from Thermoanaerobacterium thermosaccharolyticum strain PSU-2 was examined in upflow anaerobic sludge blanket (UASB) reactor and carrier-free upflow anaerobic reactor (UA), both fed with sucrose and operating at 60 °C. Heat-pretreated methanogenic granules were used as carrier to immobilize T. thermosaccharolyticum strain PSU-2 in UASB reactor operated at a hydraulic retention time (HRT) ranging from 0.75 to 24 h and corresponding sucrose loading rate from 58.5 to 2.4 mmol sucrose l−1 h−1. In comparison with hydrogen production rate of 12.1 mmol H2 l−1 h−1 obtained by carrier-free reactor upflow anaerobic (UA) system, a greatly improved hydrogen production rate up to 152 mmol H2 l−1 h−1 was demonstrated by the granular cells in UASB system. The biofilm of T. thermosaccharolyticum strain PSU-2 developed on treated methanogenic granules in UASB reactor substantially enhanced biomass retention (3 times), and production of hydrogen (12 times) compared to carrier-free reactor. It appears to be the most preferred process for highly efficient dark fermentative hydrogen production from sugar containing wastewater under thermophilic conditions.

Thermoanaerobacterium-rich sludge acclimated with palm oil mill effluent (POME) in an anaerobic sequencing batch reactor operating at 60 ∘ C was used as a seed in batch experiments to investigate the effects of C/N (carbon/nitrogen) ratio, C/P (carbon/phosphate) ratio and iron concentration in POME on fermentative hydrogen production. A central composite design was performed with the aim of optimizing the hydrogen yield together with POME degradation using response surface methodology (RSM). The RSM results indicated that the presence of 257 mg Fe 2 + / l , a C/N ratio of 74 and a C/P ratio of 559 were optimal for simultaneous hydrogen production and COD (chemical oxygen demand) removal. C/N ratio, C/P ratio and iron concentration all had an individual effect on hydrogen production and COD removal, while iron concentration and C/N ratio had the greatest interactive effect on hydrogen production ( P 0.05 ) while C/N and C/P ratio gave more profound interactive effect on COD removal ( P 0.05 ) . The predicted maximum simultaneous hydrogen production and COD removal were 6.5 l H 2 / l - POME and 58%, respectively. In a confirmation experiment under optimized conditions highly reproducible results were obtained, with a hydrogen production and COD removal efficiency of 6.33 ± 0.142 l H 2 / l - POME and 55 ± 1.5 % , respectively. The total carbohydrate conversion was 92 ± 2.7 % . The hydrogen production rate reached 25.9 mmol H 2 / l / day and increased by 60% as compared with the use of raw POME. Thermoanaerobacterium spp. were found to be dominant and present at a higher population density under optimized conditions than in raw POME fermentation. Optimization of the culture cultivation conditions in POME resulted in a simultaneous increase in biohydrogen production and COD reduction.