Potential for using enriched cultures and thermotolerant bacterial isolates for production of biohydrogen from oil palm sap and microbial community analysis

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 H₂/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 H₂/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 H₂/L-OP sap) than the enriched cultures of 81RN1 (1085 mL H₂/L-OP sap).     

Commercial application scenario using patent analysis: Fermentative hydrogen production from biomass

The main purpose of this study is to use patent analysis to investigate scenarios for future commercial applications of dark fermentation or anaerobic fermentation using biomass or organic matter as feedstock materials. The first step in this study includes a patent search procedure and patent content interpretation, in which 29 technology patents were identified from the US patent database and divided into five groups in accordance with the scope of their technical applications. The following five scenarios of commercial applications of biomass fermentation for hydrogen production were established through a combination of group applications: screening and cultivation of hydrogen-producing bacteria, biomass waste sources, biomass energization application, value enhancement of waste or wastewater treatment systems, and the application of a multi-functional hydrogen production system integrated with other technologies.     

Fermentative hydrogen production using a real-time fuzzy controller

This work builds a real-time monitoring and control system for bio-hydrogen production fermentation plants using LabVIEW software. The best fermentation environment factors pH and temperature are successfully estimated with stable control ability to create the best hydrogen production environment. The concentrate molasses fermentation waste is as nutrients to hold biomass hydrogen production by dark fermentation in a continuous stirred anaerobic bioreactor, CSABR. In order to verify the applicability of this system, this study compares the proposed anaerobic bioreactor system which's maximum hydrogen production was 3.12 (L/Day) and the system with the fuzzy controller which's hydrogen production rose to 13.44 (L/Day). The result shows that the proposed fuzzy control can not only control feeding pump and heater operations, but also successfully reduce the energy required for hydrogen production, making sure the growth of micro-organisms is in the best environmental conditions for the best growth rate and raise of the maximum hydrogen production.     

Comparison of UASB and EGSB reactors performance, for treatment of raw and deoiled palm oil mill effluent (POME)

Anaerobic digestion of palm oil mill effluent (POME) and deoiled POME was investigated both in batch assays and continuous reactor experiments using up-flow anaerobic sludge blanket (UASB) and expanded granular sludge bed (EGSB) reactors. The methane potential determined from batch assays of POME and deoiled POME was 503 and 610 mL-CH(4)/gVS-added, respectively. For the treatment of POME in continuously fed reactors, both in UASB and EGSB reactors more than 90% COD removal could be obtained, at HRT of 5 days, corresponding to OLR of 5.8 gVS/(L-reactor.d). Similar methane yields of 436-438 mL-CH(4)/gVS-added were obtained for UASB and EGSB respectively. However, for treatment of deoiled POME, both UASB and EGSB reactors could operate at lower OLR of 2.6 gVS/(L-reactor.d), with the methane yield of 600 and 555 mL-CH(4)/gVS-added for UASB and EGSB, respectively. The higher methane yield achieved from the deoiled POME was attributed to lower portion of biofibers which are more recalcitrant compared the rest of organic matter in POME. The UASB reactor was found to be more stable than EGSB reactor under the same OLR, as could be seen from lower VFA concentration, especially propionic acid, compared to the EGSB reactor.     

Methanogenic digestion using mixed substrate of acetic, propionic and butyric acids

Experiments on methanogenic digestion using high concentrations of mixed substrate were conducted. The major intermediate products of anaerobic digestion such as acetic, propionic and butyric acids were mixed in a ratio of 2:1:1 (COD basis), respectively, and used as a substrate for feeding into continuous-flow chemostat reactors maintained at 35°C. These reactors were operated stably at higher feed substrate concentrations and shorter hydraulic retention times (HRT) than those of using a single component of volatile fatty acids as a substrate. At an HRT of 4.43 days, the methanogenesis occurred normally up to a feed substrate concentration of 70,000 mg COD I⁻¹. At a feed substrate concentration of 20,000 mg COD I⁻¹, the methanogenesis occurred normally up to an HRT of 2.91 days and the minimum SRT for microbial populations was calculated to be 2.42 days. An increase in feed substrate concentration adversely affected the propionate degradation strikingly, while a decrease in HRT significantly adversely affected the acetate and propionate degradation. The methane production was 0.301 g⁻¹ COD utilized, and it was independent of the feed substrate concentration and HRT. Bacilli were predominant in all reactors, but sarcinae appeared in the reactors with high feed substrate concentrations and short HRTs. Phenomena in digester failure due to methanogen washout were also observed.     

Fermentative bioenergy production from distillers grains using mixed microflora

The feasibility of hydrogen production from distillers grains substrate, an industrial cellulosic waste, was investigated. A substrate concentration of 80 g/L gave the maximum production at 50 °C and pH of 6.0 using sewage sludge. Four controllable factors with three levels: seed sludge (two sewage sludges and cow dung), temperature (40, 50, and 60 °C), pH (6, 7 and 8) and seed pretreatment (none, heat, and acid) were selected in Taguchi experimental design to optimize fermentation conditions. The peak hydrogen and ethanol productions were found with heat-treated cow dung seed, substrate concentration 80 g/L, 50 °C and pH 6. The peak hydrogen production rate and hydrogen yield were 7.9 mmol H₂/L/d and 0.40 mmol H₂/g-COD respectively whereas the peak ethanol production was 3050 mg COD/L and rate 0.22 g EtOH/L/d. A total bioenergy yield of 41 J/g substrate was obtained which was 21% and 79% from hydrogen and ethanol respectively.