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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.

The effect of pretreatment methods for improved biodegradability and biogas production of oil palm empty fruit bunches (EFB) and its co-digestion with palm oil mill effluent (POME) was investigated. The maximum methane potential of POME was 502 mL CH4/g VS-added corresponding to 33.2 m3 CH4/ton POME and 98% biodegradability. Meanwhile, the maximum methane potential of EFB was 202 mL CH4/g VS-added corresponding to 79.1 m3 CH4/ton EFB with 38% biodegradability. Co-digestion of EFB with POME enhanced microbial biodegradability and resulted in 25–32% higher methane production at mixing ratios of 0.4:1, 0.8:1 and 2.3:1 on VS basis than digesting EFB alone. The methane yield was 276–340 mL CH4/g VS-added for co-digestion of EFB with POME at mixing ratios of 0.4:1–2.3:1, while minor improvement was observed at mixing ratios of 6.8:1 and 11:1 (175–197 mL CH4/g VS-added). The best improved was achieved from co-digestion of treated EFB by NaOH presoaking and hydrothermal treatment with POME, which resulted in 98% improvement in methane yield comparing with co-digesting untreated EFB. The maximum methane production of co-digestion treated EFB with POME was 82.7 m3 CH4/ton of mixed treated EFB and POME (6.8:1), corresponding to methane yield of 392 mL CH4/g VS-added. The electricity production of 1 ton mixture of treated EFB and POME would be 1190 MJ or 330 kW h of electricity. The study shows that there is a great potential to co-digestion treated EFB with POME for bioenergy production.

A biodiesel wastewater treatment technology was investigated for neutral alkalinity and COD removal by microbial fuel cell. An upflow bio-filter circuit (UBFC), a kind of biocatalyst MFC was renovated and reinvented. The developed system was combined with a pre-fermented (PF) and an influent adjusted (IA) procedure. The optimal conditions were operated with an organic loading rate (OLR) of 30.0 g COD/L-day, hydraulic retention time (HRT) of 1.04 day, maintained at pH level 6.5-7.5 and aerated at 2.0 L/min. An external resistance of circuit was set at 10 kΩ. The purposed process could improve the quality of the raw wastewater and obtained high efficiency of COD removal of 15.0 g COD/L-day. Moreover, the cost of UBFC system was only US$1775.7/m3 and the total power consumption was 0.152 kW/kg treated COD. The overall advantages of this invention are suitable for biodiesel wastewater treatment.

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.

Poultry slaughterhouse sludge from chicken processing wastewater treatment plant was tested for their suitability as a substrate and inoculum source for fermentation hydrogen production. Dual digestion of poultry slaughterhouse sludge was employed to produce hydrogen by batch anaerobic self-fermentation without any extra-seeds. The sludge (5% TS) was dual digested by aerobic thermophilic digestion at 55 °C with the varying retention time before using as substrate in anaerobic self-fermentation. The best digestion time for enriching hydrogen-producing seeds was 48 h as it completely repressed methanogenic activity and gave the maximum hydrogen yield of 136.9 mL H 2 /g TS with a hydrogen production rate of 2.56 mL H 2 /L/h. The hydrogen production of treated sludge at 48 h (136.9 mL H 2 /g TS) was 15 times higher than that of the raw sludge (8.83 mL H 2 /g TS). With this fermentation process, tCOD value in the activated sludge could be reduced up to 30%.

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).

The effect of pH and medium composition on extreme-thermophilic (70 °C) dark fermentative simultaneous hydrogen and ethanol production (process performance and microbial ecology) was investigated. Hydrogen and ethanol yields were optimized with respect to glucose, peptone, FeSO4, NaHCO3, yeast extract, trace mineral salts, vitamins, and phosphate buffer concentrations as well as initial pH as independent variables. A combination of low levels of both glucose (≤2 g/L) and vitamin solutions (≤1 mL/L) and high levels of initial pH (≥7), mineral salts solution (≥5 mL/L) and FeSO4 (≥100 mg/L) stimulated the hydrogen production, while high level of glucose (≥5 g/L) and low levels of both initial pH (≤5.5) and mineral salts solution (≤1 mL/L) enhanced the ethanol production. High yield of simultaneous hydrogen and ethanol production (1.58 mol H2/mol glucose combined with an ethanol yield of 0.90 mol ethanol/mol glucose) was achieved under extreme-thermophilic mixed culture environment. Results obtained showed that the shift of the metabolic pathways favouring either hydrogen or ethanol production was affected by the change in cultivation conditions (pH and medium composition). The mixed culture in this study demonstrated flexible ability for simultaneous hydrogen and ethanol production, depending on pH and nutrients formulation. The microorganisms involved could be regarded as simultaneous hydrogen/ethanol producers, as hydrogen and ethanol fermentation under all conditions was carried out by a group of extreme-thermophilic bacterial species related to Thermoanaerobacter, Thermoanaerobacterium and Caldanaerobacter.

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.

A thermophilic H 2 -producing bacterial strain was isolated from a biohydrogen reactor fed with palm oil mill effluent (POME) and identified as Thermoanaerobacterium thermosaccharolyticum using 16S rRNA gene analysis. The isolated bacterium, designated as T. thermosaccharolyticum PSU-2, showed a high yield and production rate of H 2 . Temperature optimum, pH optimum and substrate utilization for H 2 production were investigated in batch conditions. All of tested substrate was utilized for H 2 production, while sucrose, xylose and starch were the preferred substrates. The strain produced H 2 within a wide range of pH (4.5–8) and temperature (45– 70 ∘ C ), with the optimal temperature 60 ∘ C and optimal initial pH about 6.25. Maximum of H 2 production rate was registered from hour 8 to hour 16 in late exponential phase. The H 2 production was drastically reduced in a prolonged fermentation (24 h) and stopped at pH 4.5 due to the accumulation of organic acids. The maximum H 2 production yield and rate at sucrose concentration of 20 g l - 1 , pH 6.25 and temperature 60 ∘ C were 2.53 mol H 2 mol - 1 hexose and 12.12 mmol H 2 l - 1 h - 1 , respectively. Organic nitrogen amended medium improved the H 2 production with 68% compared to inorganic nitrogen amended medium. The strain performed ethanol–acetate type fermentation in inorganic nitrogen amended medium, while it performed butyrate–acetate type fermentation in organic nitrogen amended medium.

This study aimed to evaluate the Biochemical Methane Potential (BMP) of different types of wastewaters and sizes of granules. The granules (CS: from a cassava, SS: a seafood, and PS: a palm oil factory) and wastewaters initial Chemical Oxygen Demand (COD) were 18,800, 4,200 and 100,000 mg/l respectively). Modified Gompertz equation was used to compare the data from the experiments. Wastewater from a cassava factory gave the highest BMP when used with only granules from its own source (CS). Wastewater from seafood factory had the highest nitrogen content thus, represented the most imbalance nutrient source. In this case, mix- granules (SS+CS) gave highest BMP. Palm oil mill effluent did not match COD: N ratio criterion and had too high COD level which caused substrate inhibition. Here the mix-granules (PS+CS) gave highest BMP. In general, the larger granule size and the nutrient balance could improve the efficiency and hence increase the biogas production rate. The initial COD or different substrate has a strong effect on BMP and the maximum specific methane rates whereas the different sizes of granule have an effect on the length of lag phase period. In most cases, it was sufficient to represent the experimental data with traditional modified Gompertz equation and Monod models.

Thermophilic microflora was seeded into an anaerobic sequencing batch reactor for hydrogen production from palm oil mill effluent and supplemented with nitrogen, phosphorus and iron sources for biostimulants. The nutrient supplementation increased the hydrogen production yield from 1.6 ± 0.1 to 2.24 ± 0.03 mol H 2 mol −1 hexose and hydrogen production rate from 4.4 ± 0.38 to 6.1 ± 0.03 l H 2 l −1 POME d −1 . Pollution reduction also increased as follows: COD removal from 35.5 ± 9.8 to 62.2 ± 2.8%, suspended solids removal from 91 ± 3.8 to 93.6 ± 1.1% and oil recovery from 75 ± 3 to 80 ± 1.5%, respectively. The nutrient supplementation strategy increased the bacterial diversity in the reactor and promoted in particular the growth of hydrogen-producing bacteria, e.g. Thermoanaerobacterium thermosaccharolyticum , as assessed by denaturing gradient gel electrophoresis.

Anaerobic digestion by two-stage process, containing hydrogen-producing (acidogenic) first stage and methanogenic second stage, has been proposed to degrade substrates which are difficult to be treated by single stage anaerobic digestion process. This research was aimed to evaluate the bio-hydrogen and the bio-methane potentials (BHP and BMP) of skim latex serum (SLS) by using sequential batch hydrogen and methane cultivations at thermophilic conditions (55°C) and with initial SLS concentrations of 37.5-75.0% (v/v). The maximal 1.57 L H2/L SLS for BHP and 12.2L CH4/L SLS for BMP were both achieved with 60% (v/v) SLS. The dominant hydrogen-producing bacteria in the H2 batch reactor were Thermoanaerobacterium sp. and Clostrdium sp. Meanwhile, the CH4 batch reactor was dominated by the methanogens Methanosarcina mazei and Methanothermobacter defluvii. The results demonstrate that SLS can be degraded by conversion to form hydrogen and methane, waste treatment and bioenergy production are thus combined.

A complete conversion of the hexose and pentose sugars in the food wastes hydrolysates (FWH) to ethanol is a prerequisite for maximizing the profitability of an industrial process for bioethanol production. Response surface methodology (RSM) was employed to optimize the effects of nitrogen source [(NH4)2SO4], phosphorus source (KH2PO4), yeast extract and inoculum size on ethanol production from FWH by co-culture of Zymomonas mobilis and Candida shehatae under non-sterile condition. The optimal conditions for ethanol production were 1.15 g/L of (NH4)2SO4, 0.95 g/L of KH2PO4, 1.38 g/L of yeast extract and 14.75%v/v of inoculum. The results indicate that the most significant parameters affecting ethanol production from FWH by co-culture under non-sterile condition was yeast extract. Ethanol production of 77.6 g/L obtained under optimized condition was 56% increased as compared with the use of raw FWH (34 g/L) and was in good agreement with the value predicted by quadratic model (79.98 g/L), thereby confirming its validity. Ethanol yield of FWH in batch fermentation by co-culture was 0.15 g-ethanol/g-food waste (77.6 g/L), which was 94.6% of the theoretical yield while Z. mobilis alone yielded 0.11 g-ethanol/g-food waste (54.2 g/L) and C. shehatae alone yielded 0.09 g-ethanol/g-food waste (48 g/L). Ethanol production from FWH in 1-L fermentor by co-culture also gave ethanol yield of 0.16 g-ethanol/g-food waste (78.8 g/L) which was 96% of the theoretical yield. Despite of being a waste, an ethanol yield of 0.16 g-ethanol/g-food waste demonstrated the potential of food waste as a promising biomass resource for ethanol production. Key words: Co-culture, food waste hydrolysates, non-sterile fermentation, response surface methodology, optimization.

Anaerobic digestion foaming is a common operation problem in biogas plants with negative impacts on the biogas plants economy and environment. A survey of 16 Danish full-scale biogas plants on foaming problems revealed that most of them had experienced foaming in their processes up to three times per year. Foaming incidents often lasted from one day to three weeks, causing 20–50% biogas production loss. One foaming case at Lemvig biogas plant has been investigated and the results indicated that the combination of feedstock composition and mixing pattern of the reactor was the main cause of foaming in this case. Moreover, no difference in bacterial communities between the foaming and non-foaming reactors was observed, showing that filamentous bacteria were not the main reason for foaming in this case.

The effect of alkaline and acid pretreatment on solubilization of solid organic matter in palm oil mill effluent (POME) and hydrogen production by Thermoanaerobacterium-rich sludge was studied. Organic matter solubilization of alkaline and acid pretreatments were increased up to 28 and 15.7% of initial soluble COD in POME, respectively. That corresponds to carbohydrate solubilization of 41 and 32% of initial soluble carbohydrate in POME, respectively. A maximum hydrogen production yield of 4.6 l H /l-POME was achieved 2 from POME pretreated with 1.5% w/v NaOH, which was 3-fold greater than raw POME and 1-fold greater than POME pretreated with 1.5% w/v HCl. Optimum conditions for biohydrogen production from alkaline pretreated POME using Thermoanaerobacterium-rich sludge was found at initial pH 5.5 and temperature of 60°C, which gives a maximum hydrogen production yield of 5.2 l H /l-POME and increased 51% as compared to raw POME. 2 The biogas was mainly composed of hydrogen and carbon dioxide with the percentage of hydrogen ranging from 55-60% of biogas and free of methane. During the conversion of alkaline pretreated POME into hydrogen, the acetic and butyric acids were main by-products in the metabolism. The results showed that alkaline pretreatment is an effective pretreatment methods for enhancing the hydrogen production yield from POME. The use of Thermoanaerobacterium-rich sludge is promising for future engineering practice of biohydrogen production from alkaline pretreated POME.

The newly isolated extreme thermophilic ethanologen Thermoanaerobacter pentosaceus was immobilized in different support materials in order to improve its ethanol production ability. In batch fermentation, a maximum ethanol yield of 1.36 mol mol−1 consumed sugars was obtained by T. pentosaceus immobilized on rapeseed straw. Additionally, immobilized T. pentosaceus’ ethanol production was improved by 11% in comparison to free cells. In continuous mode, it was shown that hydraulic retention time (HRT) affected ethanol yield, and a dramatic shift from ethanol to acetate and lactate production occurred at an HRT of 6 h. The maximum ethanol yield and concentration, 1.50 mol mol−1 consumed sugars and 12.4 g l−1, were obtained with an HRT of 12 h. The latter represented an improvement of 60% in relation to previously obtained results. This indicates that immobilization of T. pentosaceus is an effective strategy to improve its ethanol production ability.

The purpose of this research was to explore the diversity and utilization potential of indigenous vegetables and to investigate the factors that influence the situation of the indigenous vegetables at Kong Ra district, Phatthalung province. This research, performed in conjunction with Participatory Action Research (PAR), during a 6 months study (between June – November 2009), found that there are 50 families, 85 genera and 123 species of local edible plants. The parts most often eaten are buds, leaves, fruits, flowers, sprouts, stems, tubers and vines. Observation of the distribution and diversity of indigenous vegetables demonstrated that the several species can be found in all seasons. The most abundant species found in 6 months of exploration were Leucaena leucocphala followed by Alocarsia indica, Barringtonia acutangula Gaertn. subsp. spicata, Garcinia cowa, Litsea petiolata and Polygonum odoratum. Utilization study of the edible plants revealed that indigenous vegetables can be processed several ways, including eating raw or by cooking. Diversity and distribution of the indigenous vegetables may be associated with physical characteristics of the area in Kong Ra district. Our observations indicated that several species of indigenous vegetables were found in different areas. In addition, this work revealed several factors that have affected local interest in indigenous vegetables such as changes in cultivation behavior of the farmer, changes in consumption behavior and ignorance from the new generation was the most important factor affecting indigenous vegetables.

The two-stage process for extreme thermophilic hydrogen and thermophilic methane production from wheat straw hydrolysate was investigated in up-flow anaerobic sludge bed (UASB) reactors. Specific hydrogen and methane yields of 89 ml-H(2)/g-VS (190 ml-H(2)/g-sugars) and 307 ml-CH(4)/g-VS, respectively were achieved simultaneously with the overall VS removal efficiency of 81% by operating with total hydraulic retention time (HRT) of 4 days . The energy conversion efficiency was dramatically increased from only 7.5% in the hydrogen stage to 87.5% of the potential energy from hydrolysate, corresponding to total energy of 13.4 kJ/g-VS. Dominant hydrogen-producing bacteria in the H(2)-UASB reactor were Thermoanaerobacter wiegelii, Caldanaerobacter subteraneus, and Caloramator fervidus. Meanwhile, the CH(4)-UASB reactor was dominated with methanogens of Methanosarcina mazei and Methanothermobacter defluvii. The results from this study suggest the two stage anaerobic process can be effectively used for energy recovery and for stabilization of hydrolysate at anaerobic conditions.

Hydrolysate was tested as substrate for hydrogen production by extreme thermophilic mixed culture (70 degrees C) in both batch and continuously fed reactors. Hydrogen was produced at hydrolysate concentrations up to 25% (v/v), while no hydrogen was produced at hydrolysate concentration of 30% (v/v), indicating that hydrolysate at high concentrations was inhibiting the hydrogen fermentation process. In addition, the lag phase for hydrogen production was strongly influenced by the hydrolysate concentration, and was prolonged from approximately 11 h at the hydrolysate concentrations below 20% (v/v) to 38 h at the hydrolysate concentration of 25% (v/v). The maximum hydrogen yield as determined in batch assays was 318.4 +/- 5.2 mL-H(2)/g-sugars (14.2 +/- 0.2 mmol-H(2)/g-sugars) at the hydrolysate concentration of 5% (v/v). Continuously fed, and the continuously stirred tank reactor (CSTR), operating at 3 day hydraulic retention time (HRT) and fed with 20% (v/v) hydrolysate could successfully produce hydrogen. The hydrogen yield and production rate were 178.0 +/- 10.1 mL-H(2)/g-sugars (7.9 +/- 0.4 mmol H(2)/g-sugars) and 184.0 +/- 10.7 mL-H(2)/day L(reactor) (8.2 +/- 0.5 mmol-H(2)/day L(reactor)), respectively, corresponding to 12% of the chemical oxygen demand (COD) from sugars. Additionally, it was found that toxic compounds, furfural and hydroxymethylfurfural (HMF), contained in the hydrolysate were effectively degraded in the CSTR, and their concentrations were reduced from 50 and 28 mg/L, respectively, to undetectable concentrations in the effluent. Phylogenetic analysis of the mixed culture revealed that members involved hydrogen producers in both batch and CSTR reactors were phylogenetically related to the Caldanaerobacter subteraneus, Thermoanaerobacter subteraneus, and Thermoanaerobacterium thermosaccharolyticum.

We investigated the feasibility of using Microcystis sp; blue green algae (MB) as a co-substrate to improve the mesophilic anaerobic digestion of cannery seafood wastewater (CSW) supplemented with 1% (v/v) glycerol waste (GW), to maximize bio-methane production. The MB content was set at 5, 10 and 15% (v/v) to find a near optimal methane yield. The maximum 291 mL CH 4 /g VS-added methane yield, corresponding to 4.4 m 3 -CH 4 /m 3 - mixed wastewater, was achieved with a CSW: GW: MB mixture at the volumetric 94:1:5 ratio. The methane yield of CSW digested alone was 278 mL CH 4 /g VS-added (2.2 m 3 CH 4 / m 3 of wastewater). The yields from our other experiments ranged within 81– 150 mLCH 4 /g VS-added. Ratios of MB: CSW exceeding 5% (v/v) gave lower than optimal methane yields. The energy content of methane from 1 m 3 of mixed wastewater, with the near optimal mixture ratio 94:1:5, was 157 MJ or equivalently 44 kWh.

Ethanol production from oil palm frond (OPF) by simultaneous saccharification and Saccharomyces cerevisiae TISTR5048 fermentation was investigated. Solid fraction of OPF (20% TS) was pretreated by 2% H 2 SO 4 , 2% NaOH and 2% NaOH in H 2 O 2 presoaking at room temperature for 24 hours. Pretreated OPF by presoaking in 2% H 2 SO 4 , 2% NaOH and 2% NaOH in H 2 O 2 contained 37%, 42% and 49% of cellulose, respectively. Pretreated OPF was simultaneous saccharification by cellulase enzyme (Cellic CTec2, Novozymes) and sequentially fermentation. Sugar concentration in OPF cellulose hydrolysis of 2% H 2 SO 4 , 2% NaOH and 2% NaOH in H 2 O 2 presoaking was 45.72, 55.73 and 56.94 g/l, respectively. Ethanol concentration of 2% H 2 SO 4 , 2% NaOH and 2% NaOH in H 2 O 2 presoaking was 14.5, 15.0 and 17.2 g/L, respectively. 2% NaOH in H 2 O 2 presoaking was the best pretreatment with 82.11% of total solids recovery and containing 49.9% of cellulose with enzyme digestion ability of 37.6%.

The laboratory-scale UASB reactors were operated at five different hydraulic retention times (HRTs). The various sizes of granules from three different sources: a cassava factory (CS), a seafood factory (SS), and a palm oil mill (PS), having the size range of 1.5-1.7 mm, 0.7-1.0 mm and 0.1-0.2 mm. respectively, were used as inocula for anaerobic digestion of cassava wastewater. For comparison, the first reactor with only granules from its own source (R1, CS) was treated as control. The other two reactors were inoculated with mixed granules from different sources (R2, CS+SS and R3, CS+PS). As HRT decreased from 5 days to 1 day, the organic removal efficiencies decreased from 91.49 to 43.23%, 89.36 to 45.13% and 87.23 to 32.69% for R1, R2 and R3 respectively (or inversely with increasing OLR). In this study selected mathematical models including Monod, Contois, Grau second-order and Modified Stover-Kicannon kinetic models were applied to determine the substrate removal kinetics of UASB reactors. Kinetic parameters were determined through linear regression using experimental data obtained from the steady-state experiments and subsequently used to predict effluent COD. The results showed that Grau second-order and Modified Stover-Kicannon kinetic models were more suitable than the others for predicting the substrate removal for all different sizes of granules. In addition the Upflow Anaerobic Sludge Blanket (UASB) reactor with only granules from a cassava factory gave the best performance.

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.

Manure contains several compounds that can potentially cause foaming during anaerobic digestion. Understanding the effect of substrates and intermediate compounds on foaming tendency and stability could facilitate strategies for foaming prevention and recovery of the process. In this study, the effect of physicochemical properties of substrates and intermediate compounds on liquid properties such as surface tension, surfactant property, and hydrophobicity were investigated and compared with the effect on foaming tendency and foam stability. The results showed that there was no consistent correlation between foaming potential and hydrophobicity, oil displacement area (ODA) or surface tension of the tested solutions, and the best way to determine the foaming property of the solution was to directly measure foaming tendency and foam stability. Na-oleate and acetic acid showed the highest potential to create foam in a manure digester. Moreover, high organic loading of lipids and protein, and high concentrations of acetic and butyric acids also showed a strong tendency to create foaming during anaerobic digestion. Due to their great ability to stabilize foam, high organic loadings of Na-oleate or gelatine were considered to be the main potential foaming problem.

Foaming is one of the major problems that occasionally occur in the biogas plants, affecting negatively the overall digestion process and results in adverse operational, economical and environmental impacts. The most dominant factors contributing to foaming are organic overloading, feedstock composition and the presence of specific microorganisms. The filamentous microorganisms are known to be the major cause of foaming in sludge digester as they are attached to the gas bubbles and accumulated on the surface of the reactor.The present case study investigated the microbial composition of one manure-based digester of Lemvig biogas plant that was facing foaming problem, comparing with three non-foaming digesters. The research was focused on the quantitative and qualitative analysis of Bacteria and Archaea population and on the identification of Gordonia sp. The reactor samples were analysed for foaming properties and microbial analysis. The dynamic population of Bacteria and Archaea were studied by PCR-DGGE method.The results obtained from this study showed that the composition of Bacteria in all reactors was not significantly different indicating that foaming was not caused by Bacteria. In contrary, the quantitative and qualitative analysis of Archaea revealed significant differences in their population and composition.