Microbial community analysis of mixed anaerobic microflora in suspended sludge of ASBR producing hydrogen from palm oil mill effluent

This study investigated the microbial community of an anaerobic sequencing batch reactor (ASBR) operating at mesophilic temperature under varying hydraulic retention times (HRTs) for evaluating optimal hydrogen production conditions, using palm oil mill effluent (POME) as substrate. POME sludge enriched by heat treatment with hydrogen-producing bacteria was used as inoculum and acclimated with the POME. The microbial community was determined by first isolating cultivable bacteria at each operating HRT and then using polymerase chain reaction (PCR). The PCR products were sequenced and sequence identification was performed using the BLAST algorithm and Genbank database. The findings revealed that about 50% of the isolates present were members of the genus Streptococcus, about 30% were Lactobacillus species and around 20% were identified as species of genus Clostridium. Scanning electron microscopy (SEM) analysis also confirmed the presence of spherical and rod-shaped microbial morphologies in the sludge samples of bioreactor during prolonged cultivation.     

Ozonation aided mesophilic biohydrogen production from palm oil mill effluent

Ozone pretreatment of palm oil mill effluent (POME) was employed to improve sustrate biodegradability prior to biological H₂ production. The H₂ production was conducted at varing pHs from 4.0 to 6.0 to examine the impact of pH on the H₂ mesophilic production (37 °C). The optimal pH for H₂ production was 6.0 for both raw and ozonated POME. The POME concentrations were greatly influenced the yields and rates of H₂ production. At the optimal pH, the maximum H₂ production yield of 182 ± 7.2 mL.g⁻¹ COD (7.96 mmoL.g⁻¹ COD) was achieved at the ozonated POME concentration of 30,000 mg COD.L⁻¹. The maximum H₂ production rate (R_max) of 43.1 ± 2.5 mL.h⁻¹ was obtained at the ozonated POME concentration of 25,000 mg COD.L⁻¹. The highest total COD removal was 44% at of 15,000 mg COD.L⁻¹ ozonated POME. Acetic and butyric acids were dominant products during H₂ fermentation and tended to increase with the increased POME concentrations. Ozonation as a pretreatment process showed significant enhancement of the POME biodegradability , and subsequently improved the H₂ production H₂.     

The use of acidified palm oil mill effluent for thermophilic biomethane production by changing the hydraulic retention time in anaerobic sequencing batch reactor

The feasibility of thermophilic biomethane production from acidified palm oil mill effluent (POME) was assessed in a 5 L anaerobic sequencing batch reactor (ASBR). The effects of various hydraulic retention time (HRT) (10-1 d) on methane production performance and the stability of ASBR in treating acidified POME were evaluated herein. It was found that the highest methane productivity of 5.65 L CH₄/L/d could be attained at HRT of 2 d. However, the removal of chemical oxygen demand (COD) and volatile fatty acid (VFA) at this HRT is rather low (65-62%) hence making it inefficient to operate at HRT 2 d since most of the contaminants remained in the liquid streams. Thus the most recommended HRT was 3 d with maximum methane productivity of 3.96 L CH₄/L/d with corresponding methane yield of 260.3 L CH₄/kgCOD_removed. The COD removal efficiency at 3 d HRT was 71%, and the VFA consumption was more than 80%. The correlation of total VFA: total alkalinity (TVFA: TA) at HRT of 3 d was found to be 0.1. This recommended HRT of 3 is equally shorter than any previously reported application of POME as a substrate for thermophilic biomethane.     

Effects of process,operational and environmental variables on biohydrogen production using palm oil mill effluent (POME)

A batch study for biohydrogen production was conducted using raw palm oil mill effluent (POME) and POME sludge as a feed and inoculum respectively. Response Surface Methodology (RSM) was used to design the experiments. Experiments were conducted at different reaction temperatures (30–50 °C), inoculum size to substrate ratios (I:S) and reaction times (8–24 h). An optimum condition of biohydrogen production was achieved with COD removal efficiency of 21.95% with hydrogen yield of 28.47 ml H₂ g^?1 COD removed. The I:S ratio was 40:60, with reaction temperature of 50 °C at 8 h of reaction time. The study showed that a lower substrate concentration (less than 20 g L^?1) for biohydrogen production using pre-settled POME was achievable, with optimum HRT of 8 h under thermophilic condition (50 °C). This study also found that pre-settled POME is feasible to be used as a substrate for biohydrogen production under thermophilic condition.     

Optimization and microbial community analysis for production of biohydrogen from palm oil mill effluent by thermophilic fermentative process

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 H₂ g COD⁻¹ with a volumetric hydrogen production rate of 9.1 l H₂ 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).     

Start-up study of biohydrogen production from palm oil mill effluent in a lab-scale up-flow anaerobic sludge blanket fixed-film reactor

A start-up study of lab-scale up-flow anaerobic sludge blanket fixed-film reactor (UASFF) was conducted to produce biohydrogen from palm oil mill effluent (POME). The reactor was fed with POME at different hydraulic retention time (HRT) and organic loading rate (OLR) to obtain the optimum fermentation time for maximum hydrogen yield (HY). The results showed the HY, volumetric hydrogen production rate (VHPR), and COD removal of 0.5–1.1 L H₂/g COD_consumed, 1.98–4.1 L H₂ L^?1 day^?1, and 33.4–38.5%, respectively. The characteristic study on POME particles was analyzed by particle size distribution (PSD), Scanning electron microscopy (SEM), and Energy-dispersive X-ray spectroscopy (EDX). The microbial Shannon and Simpson diversity indices and Principal Component Analysis assessed the alpha and beta diversity, respectively. The results indicated the change of bacterial community diversity over the operation, in which Clostridium sensu stricto 1 and Lactobacillus species were contributed to hydrogen fermentation.     

Application of immobilized upflow anaerobic sludge blanket reactor using Clostridium LS2 for enhanced biohydrogen production and treatment efficiency of palm oil mill effluent

Polyethylene glycol (PEG) gel was used to immobilize hydrogen producing Clostridium LS2 bacteria for hydrogen production in an upflow anaerobic sludge blanket (UASB) reactor. The UASB reactor with a PEG-immobilized cell packing ratio of 10% weight to volume ratio (w/v) was optimal for dark hydrogen production. The performance of the UASB reactor fed with palm oil mill effluent (POME) as a carbon source was examined under various hydraulic retention time (HRT) and POME concentration. The best volumetric hydrogen production rate of 365 mL H₂/L/h (or 16.2 mmol/L/h) with a hydrogen yield of 0.38 L H₂/g COD_added was obtained at POME concentration of 30 g COD/L and HRT of 16 h. The average hydrogen content of biogas and COD reduction were 68% and 65%, respectively. The primary soluble metabolites were butyric acid and acetic acid with smaller quantities of other volatile fatty acid and alcohols formed during hydrogen fermentation. More importantly, the feasibility of PEG-immobilized cell UASB reactor for the enhancement of the dark-hydrogen production and treatment of wastewater is demonstrated.     

Production of methane from ozonated palm oil mill effluent

Methane (CH₄) production from palm oil mill effluent (POME) pre-treated by ozonation was conducted under mesophilic (37 °C) condition. The results demonstrated that methane can be produced from both non-ozonated and ozonated POME at a concentration range of 3,000 to 15,000 mg COD L⁻¹. Methane yield rised 54% when POME was pre-treated by ozonation at POME concentration of 15,000 mg COD L⁻¹. The methane yield increased the POME concentration was increased. At POME above 15,000 mg COD L⁻¹, the methane yield was dropped dramatically. The methane production rates (R_max) and yields exerted similar trend regarding the POME concentration. Accumulation of volatile fatty acids in the reactor posed the drop of methane production. Ozonation pretreatment process of POME can improve the biodegradability of the complex organic matter in POME and enhanced methane yield and rate at POME concentration range of 3,000–15,000 mg COD L⁻¹.     

Analysis of biohydrogen production from palm oil mill effluent using a pilot-scale up-flow anaerobic sludge blanket fixed-film reactor in life cycle perspective

This study aims to analyse the life-cycle assessment of biohydrogen production from palm oil mill effluent (POME) in a pilot-scale up-flow anaerobic sludge blanket fixed-film reactor. The SimaPro LCA software and ReCiPe 2016 impact assessment method were used. Electricity usage was found to be a significant source of environmental impacts, with 50–98% of the total impacts. Furthermore, an improvement analysis was conducted, resulted in a reduction in all impacts, especially global warming impact with 77% reduction from 818 to 189 kg CO₂-eq per kg biohydrogen. While shifting the pilot reactor to Sarawak may further lessen the impact to 142 kg CO₂-eq due to cleaner grid in that region. Besides, if the environmental burden avoided due to usage of POME is considered, the global warming impact can be further reduced to 54.9 kg CO₂-eq. Thus, the pilot reactor has huge potential, especially in utilizing waste to produce bioenergy.     

Biohydrogen production by Clostridium butyricum EB6 from palm oil mill effluent

A hydrogen producer was successfully isolated from anaerobic digested palm oil mill effluent (POME) sludge. The strain, designated as Clostridium butyricum EB6, efficiently produced hydrogen concurrently with cell growth. A controlled study was done on a synthetic medium at an initial pH value of 6.0 with 10 g/L glucose with the maximum hydrogen production at 948 mL H₂/L-medium and the volumetric hydrogen production rate at 172 mL H₂/L-medium/h. The supplementation of yeast extract was shown to have a significant effect with a maximum hydrogen production of 992 mL H₂/L-medium at 4 g/L of yeast extract added. The effect of pH on hydrogen production from POME was investigated. Experimental results showed that the optimum hydrogen production ability occurred at pH 5.5. The maximum hydrogen production and maximum volumetric hydrogen production rate were at 3195 mL H₂/L-medium and 1034 mL H₂/L-medium/h, respectively. The hydrogen content in the biogas produced was in the range of 60–70%.     

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

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

Effect of pH on continuous biohydrogen production from liquid swine manure with glucose supplement using an anaerobic sequencing batch reactor

pH is considered as one of the most important factors governing the hydrogen fermentation process. In this project, five pH levels, ranging from 4.4 to 5.6 at 0.3 increments, were tested to evaluate the pH effect on hydrogen production from swine manure supplemented with glucose in an anaerobic sequencing batch reactor system with 16 h of hydraulic retention time (HRT). The optimal hydrogen yield (1.50 mol H₂/mol glucose) was achieved at pH 5.0 when the maximum production rate of 2.25 L/d/L was obtained. Continuous hydrogen production was achieved for over 3 weeks for pH 5.0, 4.7, and 4.4, with no significant methane produced. However, as pH increased to 5.3 and 5.6, methane production was observed in the biogas with concurrent reductions in hydrogen production, indicating that methanogens could become increasingly activated for pH 5.3 or higher. Acetate, propionate, butyrate, valerate, and ethanol were the main aqueous products whose distribution was significantly affected by pH as well.     

Microbial community analysis of thermophilic mixed culture sludge for biohydrogen production from palm oil mill effluent

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.     

Effect of pre-treatment and hydraulic retention time on biohydrogen production from organic wastes

This study investigated the effect of pre-treatment and hydraulic retention time (HRT) on biohydrogen production from organic wastes. Various pre-treatments including thermal, base, acid, ultrasonication, and hydrogen peroxide were applied alone or in combination to enhance biohydrogen production from potato and bean wastewater in batch tests. All the pre-treated samples showed higher hydrogen production than the control tests. Hydrogen peroxide pre-treatment achieved the best results of 939.7 and 470 mL for potato and bean wastewater, respectively. Continuous biohydrogen production from sucrose, potato and bean wastewater was significantly influenced by reducing the HRT as 24, 18 and 12 h. Sucrose and potato showed similar behavior, where the hydrogen production rate (HPR) increased with decreasing the HRT. Optimum hydrogen yield results of 320 mL-H₂/g-VS (sucrose) and 150 mL-H₂/g-VS (potato) were achieved at HRT of 18 h. Bean wastewater showed optimum HPR of 0.65 L/L.d with hydrogen yield of 80 mL-H₂/g-VS at 24 h HRT.     

Pilot-scale of biohythane production from palm oil mill effluent by two-stage thermophilic anaerobic fermentation

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

Enhancing biohydrogen production from chemical wastewater treatment in anaerobic sequencing batch biofilm reactor (AnSBBR) by bioaugmenting with selectively enriched kanamycin resistant anaerobic mixed consortia

The basic aim of this study was to investigate the feasibility of bioaugmentation strategy in the process of enhancing biohydrogen (H₂) production from chemical wastewater treatment (organic loading rate (OLR)—6.3 kg COD/m³-day) in anaerobic sequencing batch biofilm reactor (AnSBBR) operated at room temperature (28±2$28\pm 2$ °C) under acidophilic microenvironment (pH 6) with a total cycle period of 24 h. Parent augmented inoculum (kanamycin resistant) was acquired from an operating upflow anaerobic sludge blanket (UASB) reactor treating chemical wastewater and subjected to selective enrichment by applying repetitive/cyclic pre-treatment methods [altering between heat-shock treatment (100 °C; 2 h) and acid treatment (pH 3; 24 h)] to eliminate non-spore forming bacteria and to inhibit the growth of methanogenic bacteria (MB). Experimental data revealed the positive influence of bioaugmentation strategy on the overall H₂ production. Specific H₂ production almost doubled after augmentation from 0.297 to 0.483 mol H₂/kg COD_R-day. Chemical wastewater acted as primary carbon source in the metabolic reactions involving molecular H₂ generation leading to substrate degradation. The augmented culture persisted in the system till the termination of the experiments. The survival and retention of the augmented inoculum and its positive effect on process enhancement may be attributed to the adopted reactor configuration and operating conditions. Scanning electron microscope (SEM) images documented the selective enrichment of morphologically similar group of bacteria capable of producing H₂ under acidophilic conditions in anaerobic microenvironment. This depicted work corroborated successful application of bioaugmentation strategy to improve H₂ production rate from anaerobic chemical wastewater treatment.     

High efficient biohydrogen production from palm oil mill effluent by two-stage dark fermentation and microbial electrolysis under thermophilic condition

Biohydrogen production from palm oil mill effluent by two-stage dark fermentation and microbial electrolysis was investigated under thermophilic condition. The optimum chemical oxygen demand (COD) concentration and pH for dark fermentation were 66 g·L⁻¹ and 6.5 with a hydrogen yield of 73 mL-H₂·gCOD⁻¹. The dark fermentation effluent consisted of mainly acetate and butyrate. The optimum voltage for microbial electrolysis was 0.7 V with a hydrogen yield of 163 mL-H₂·gCOD⁻¹. The hydrogen yield of continuous two-stage dark fermentation and microbial electrolysis was 236 mL-H₂·gCOD⁻¹ with a hydrogen production rate of 7.81 L·L⁻¹·d⁻¹. The hydrogen yield was 3 times increased when compared with dark fermentation alone. Thermoanaerobacterium sp. was dominated in the dark fermentation stage while Geobacter sp. and Desulfovibrio sp. dominated in the microbial electrolysis cell stage. Two-stage dark fermentation and microbial electrolysis under thermophilic condition is a highly promising option to maximize the conversion of palm oil mill effluent into biohydrogen.     

Biohydrogen production from organic solid waste in a sequencing batch reactor: An optimization of the hydraulic and solids retention time

Hydrogen production from organic solids waste was evaluated using a sequencing batch reactor (SBR) under mesophilic conditions, to investigate the effect of the hydraulic retention time (HRT) and solids retention time (SRT) on hydrogen production. The examined HRT and SRT values were from 4.6 to 27 h and 17–102 h, respectively. The results showed high hydrogen production rates (1.86 LH₂/L·d) and a yield of 127.26 mLH₂/gCOD_removed for an SRT of 60 h and an HRT of 16 h. The highest chemical oxygen demand (COD) removal (38.6 ± 6.9%) was also obtained under those conditions. The highest substrate hydrolysis percentage (73.0 ± 11.4%) was obtained at an HRT of 16 h and an SRT of 102 h. A short SRT of 20 h affected hydrogen production, which decreased up to 90%. With an SRT of 20 h and an HRT of 16 h, acetic acid-like fatty acids were mainly obtained. In experiments with a long SRT (60 h), the obtained fatty acid was butyrate. The conversion efficiencies for converting particulate material into fatty acids were 51–47% using a long SRT; a short HRT resulted in percentages of 37–40%. A 3D surface analysis was performed using the maximum hydrogen yield conditions as the central point, showing that the optimal hydrogen production can be obtained with an HRT of 16 h and an SRT of 55 h. Microbial analysis showed the predominance of the Olsenella genus at an HRT< 8 h and the presence of Clostridium at an HRT of 16 h. The HRT is the main parameter leading the community composition in the process.     

Effect of initial pH,nutrients and temperature on hydrogen production from palm oil mill effluent using thermotolerant consortia and corresponding microbial communities

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 Na₂HPO₄/L and 0.36 g FeSO₄·7H₂O/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.