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.     

Effect of hydraulic retention time on biohydrogen production from palm oil mill effluent in anaerobic sequencing batch reactor

The feasibility of hydrogen generation from palm oil mill effluent (POME), a high strength wastewater with high solid content, was evaluated in an anaerobic sequencing batch reactor (ASBR) using enriched mixed microflora, under mesophilic digestion process at 37 °C. Four different hydraulic retention times (HRT), ranging from 96 h to 36 h at constant cycle length of 24 h and various organic loading rate (OLR) concentrations were tested to evaluate hydrogen productivity and operational stability of ASBR. The results showed higher system efficiency was achieved at HRT of 72 h with maximum hydrogen production rate of 6.7 LH₂/L/d and hydrogen yield of 0.34 LH₂/g COD_feeding, while in longer and shorter HRTs, hydrogen productivity decreased. Organic matter removal efficiency was affected by HRT; accordingly, total and soluble COD removal reached more than 37% and 50%, respectively. Solid retention time (SRT) of 4-19 days was achieved at these wide ranges of HRTs. Butyrate was found to be the dominant metabolite in all HRTs. Low concentration of volatile fatty acid (VFA) confirmed the state of stability and efficiency of sequential batch mode operation was achieved in ASBR. Results also suggest that ASBR has the potential to offer high digestion rate and good stability of operation for POME treatment.     

Enhancement of hydrogen production from palm oil mill effluent via cell immobilisation technique

Clostridium sp. LS2 was immobilised by entrapment in polyethylene glycol (PEG) gel beads to improve the biohydrogen production rate from palm oil mill effluent (POME). We sought to explore and optimise the hydrogen production capability of the immobilised cells by studying the conditions for cell immobilisation, including PEG concentration, cell loading and curing times, as well as the effects of temperature and K₂HPO₄ (500–2000 mg/L), NiCl₂ (0.1–5.0 mg/L), FeCl₂ (100–400 mg/L) MgSO₄ (50–200 mg/L) concentrations on hydrogen production rate. The results showed that by optimising the PEG concentration (10% w/v), initial biomass (2.2 g dry weight), curing time (80 min) and temperature (37 °C), as well as the concentrations of K₂HPO₄ (2000 mg/L), NiCl₂ (1 mg/L), FeCl₂ (300 mg/L) and MgSO₄ (100 mg/L), a maximum hydrogen production rate of 7.3 L/l ‐POME/day and a yield of 0.31 L H₂/g chemical oxygen demand were obtained during continuous operation. We believe that this process may be potentially expanded for sustained and large‐scale hydrogen production. Copyright © 2014 John Wiley & Sons, Ltd.     

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.     

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.     

Thermophilic biohydrogen recovery from palm oil mill effluent

Enhancement of biological H₂ production efficiency with pre-ozonation process of palm oil mill effluent (POME) prior to thermophilic dark fermentation (55 °C) was investigated. H₂ fermentation experiments were conducted using varying concentrations of raw and ozonated POME. Results revealed that H₂ can be produced from both raw and ozonated POME under thermophilic fermentation. Maximum H₂ production yield of 77 mL.g⁻¹COD_removed was obtained from ozonated POME, which was higher than that of 51 mL·g⁻¹ COD_removed obtained from raw POME at the highest concentration of 35,000 mg COD.L⁻¹. Meanwhile, the specific H₂ production rate (R'_max) of 1.9 and 1.5 mL·h⁻¹·g⁻¹ TVS were observed in raw and ozonated POME at the concentration of 25,000 mg COD.L⁻¹, respectively. The main metabolic products during POME fermentation were acetic and butyric acids and trace amount of valeric acid. Propionic acid and ethanol have contributed, which could be reduced H₂ production in all batch experiments for both POME. The highest efficiency of total and soluble COD removal of 24 and 25% was obtained from the raw POME, and those of 19 and 25% was obtained from the ozonated POME. The present study demonstrates that the POME loading was greatly influenced on the H₂ production yields and rates. The comparative results showed that the ozonated POME gave higher H₂ yields than the raw POME. Thus, demonstrating that the ozonation process significantly improved the POME biodegradability, which is able to enhance H₂ production yields. However, the ozone pre-treatment was not improved in the specific H₂ production rates.     

Fermentative hydrogen production from indigenous mesophilic strain Bacillus anthracis PUNAJAN 1 newly isolated from palm oil mill effluent

In the present study, a new mesophilic bacterial strain, identified as Bacillus anthracis strain PUNAJAN 1 was isolated from palm oil mill effluent (POME) sludge, and tested for its hydrogen production ability. Effect of physico-chemical factors such as temperature, initial pH, nitrogen source and carbon sources were investigated in order to determine the optimal conditions for hydrogen production. The maximum hydrogen yield of 2.42 mol H₂/mol mannose was obtained at 35 °C and initial pH of 6.5. Yeast and mannose were used as the main carbon and nitrogen sources respectively in the course of the hydrogen production. Apart from synthetic substrate, specific hydrogen production potentials of the strain using POME was calculated and found to be 236 ml H₂/g chemical oxygen demand (COD). The findings of this study demonstrate that the indigenous strain PUNAJAN 1 could be a potential candidate for hydrogen using POME as substrate.     

Simultaneous thermophilic hydrogen production and phenol removal from palm oil mill effluent by Thermoanaerobacterium-rich sludge

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.     

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.     

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.     

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.     

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.     

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%.     

Optimization of biohydrogen production of palm oil mill effluent by ozone pretreatment

The biohydrogen (H₂) production in batch experiments under varying concentrations of raw and ozonated palm oil mill effluent (POME) of 5000–30,000 mg COD.L⁻¹, at initial pH 6, under mesophilic (37 °C), thermophilic (55 °C) and extreme-thermophilic (70 °C) conditions. Effects of ozone pretreatment, substrate concentration and fermentation temperature on H₂ production using mesophilic seed sludge was undertaken. The results demonstrated that H₂ can be produced from both raw and ozonated POME, and the amounts of H₂ production were directly increased as the POME concentrations were increased. H₂ was successfully produced under the mesophilic fermentation of ozonated POME, with maximum H₂ yield, and specific H₂ production rate of 182 mL.g⁻¹ COD_removed (30,000 mg COD.L⁻¹) and 6.2 mL.h⁻¹.g⁻¹ TVS (25,000 mg COD.L⁻¹), respectively. Thus, indicating that the ozone pretreatment could elevate on the biodegradability of major constituents of the POME, which significantly enhanced yields and rates of the H₂ production. H₂ production was not achieved under the thermophilic and extreme-thermophilic fermentation. In both fermentation temperatures with ozonated POME, the maximum H₂ yield was 62 mL.g⁻¹ COD_removed (30,000 mg COD.L⁻¹) and 63 mL.g⁻¹ COD_removed (30,000 mg COD.L⁻¹), respectively. The highest efficiency of total and soluble COD removal was obtained at 44 and 37%, respectively following the mesophilic fermentation, of 24 and 25%, respectively under the thermophilic fermentation, of 32 and 20%, respectively under the extreme-thermophilic fermentation. The production of volatile fatty acids increased with an increased fermentation time and temperature in both raw and ozonated POME under all three fermentation temperatures. The accumulation of volatile fatty acids in the reactor content were mostly acetic and butyric acids. H₂ fermentation under the mesophilic condition of 37 °C was the better selection than that of the thermophilic and extreme-thermophilic fermentation.     

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.     

Enhancement of biohydrogen production from palm oil mill effluent (POME): A review

Palm oil mill effluent (POME), a wastewater from the most significant agricultural industry in Southeast Asia is produced in tremendous amounts that requires proper management to mitigate its negative environmental effects. The feasibility of treating POME in a closed dark fermentation (DF) system to replace the existing inefficient open ponding treatment has been thoroughly investigated. Theoretically, the maximum H₂ yield obtained by DF process is 4 mol_H2/mol_glucose, however, it is not achievable due to the nature of POME. In this study, several enhancement methods for increasing H₂ yield and DF process stability were discussed. An apprehension into the different pre-treatment methods on POME including physicochemical, chemical and biological and their effects on the characteristics of POME including pH, temperature, sugar content, solid content, viscosity, nutrients and by-product toxicity on the biohydrogen production and effluent quality were reviewed. Various bioreactor designs were used for biohydrogen from POME, the modifications applied on the system design to increase the stability and productivity of POME treatment have been examined. Moreover, higher biohydrogen productivity could be obtained with the addition of nanoparticle nutrients and introducing genetically modified H₂-producing bacteria. Finally, further investigation in the future shall focus on the development of a more inclusive and efficient POME treatment via DF process that favours biohydrogen production, environmental benign and economically viable.     

Identification and cultivation of hydrogenotrophic methanogens from palm oil mill effluent for high methane production

By means of biorefinery, biogas production through anaerobic digestion is one of the most common treatments of wastewater in the palm oil industry. After biogas production, the treated palm oil mill effluent (POME) is generally discharged into the environment. However, certain level of hazardous compounds still exists in the treated wastewater, which can lead to the pollution of water bodies. In this study, we have investigated the dynamics of volatile organic acids dwelling in consecutive POME treatment lagoons as well as identified, and categorized, microbial species responsible for the treatment process. Bacteria and methanogens, both hydrogenotrophic and acetoclastic, related to methane production were identified using mcrA and 16S rRNA genes specific primers. Two hydrogenotrophic methanogens, Methanoculleus marisnigri and Methanoculleus chikugoensis, were found abundant in accordance with high formate concentration throughout the process of anaerobic digestion. This study has also isolated eight consortia of microbes that yielded different methane productions by utilizing formate as the substrate in the synthetic medium. The consortia of a group, containing M. marisnigri, M. chikugoensis, uncultured bacteria, Aminobacterium sp., and Ruminobacillus xylanolyticum, produced the highest methane yield of 259 mL/g COD after 25 days of incubation in the laboratory. The findings from this study are contributing to optimize and increase biogas production in POME, which will allow higher efficiency in palm oil mill wastewater treatment.