Application of antifungal metabolites from Streptomyces philanthi RL-1-178 for maize grain coating formulations and their efficacy as biofungicide during storage.

The major safety risk of maize grain is contamination with mycotoxins. In this study, a maize-coating formulation containing freeze-dried culture filtrate of Streptomyces philanthi RL-1-178 (DCF RL-1-178) was developed and evaluated to prevent the growth of mycotoxins during maize grain storage. In vitro studies using confrontation tests on PDA plates indicated that S. philanthi RL-1-178 inhibited the growth of Aspergillus parasiticus TISTR 3276 (89.0%) and A. flavus PSRDC-4 (95.0%). The maize grain coating formulations containing the DCF RL-1-178 (0, 5, 10, and 15% (v/v)) and the polymer polyvinylpyrrolidone (PVP-K90, 4.0% (w/v)) were tested for their efficacy in In vitro and during 5 months storage. In In vitro assay, maize coating formular containing the optimum concentration (15.0%, v/v) of the DCF RL-1-178 exhibited 54.80% and 54.17% inhibition on the growth of A. parasiticus TISTR 3276 and A. flavus PSRDC-4 respectively. The inhibition was also illustrated by the microstructures of interactions between the coated maize grains with or without the DCF RL-1-178 and the fungal pathogens observed under microscope and SEM. Incorporating the DCF RL-1-178 or fungicidal Metalaxyl® into the polymer PVP-K90 maize grains coating resulted in the complete inhibition of the production of aflatoxin B1 (analysed by HPLC) by the two aflatoxigenic pathogens after 5 months storage at room temperature. However, the shelf-life was shortened to only 3 months during storage at room temperature with 90% relative humidity. Overall, the application of the 10-15% DCF RL-1-178 into the maize grain coating formular provides a new alternative measure to control the mycotoxins during storage for at least 5 months. The In vitro cell cytotoxicity study showed that a concentration of 15% (v/v) or 1000 µg/mL of the DCF RL-1-178 had a strong cytotoxic effect on Vero cells. These findings indicate that DCF RL-1-178 is a potential biofungicide for controlling mycotoxins contamination in maize seed storage for planting, but not maize grain storage for animal feed.

Antifungal and antiaflatoxigenic mechanism activity of freeze-dried culture filtrate of Streptomyces philanthi RL-1-178 on the two aflatoxigenic fungi and identification of its active component.

AIMS: The study reports the antifungal and antiaflatoxigenic mechanism activity of freeze-dried culture filtrate of Streptomyces philanthi RL-1-178 (DCF RL-1-178) against two aflatoxigenic strains (Aspergillus parasiticus and A. flavus) and identification of its active component. METHODS AND RESULTS: Significant inhibition in ergosterol biosynthesis by the DCF RL-1-178 appeared on the plasma membrane. Moreover, the DCF RL-1-178 showed dose-dependent inhibition of methylglyoxal (MG) (an aflatoxin inducer) biosynthesis and exhibited a novel antiaflatoxigenic action mechanism. Significant impairments in enzymatic [superoxide dismutase (SOD) and catalase (CAT)] and nonenzymatic [oxidized and reduced glutathione (GSH) and ratio of oxidized and reduced glutathione (GSSG)] anti-oxidative defense molecules were observed in the two aflatoxigenic cells. The active component of the DCF RL-1-178 was identified as natamycin. The natamycin exhibited against A. parasiticus and A. flavus with the minimum inhibitory concentration (MIC) values of 0.5 and 1.0 µg ml-1, respectively, while the minimum fungicidal concentration values were the same (4.0 µg ml-1). CONCLUSIONS: The DCF RL-1-178 containing natamycin exhibited the following effects: (1) inhibition of cellular ergosterol biosynthesis on plasma membrane, (2) reduction in MG (aflatoxin inducer) confirmed novel antiaflatoxigenic mechanism of action, and (3) caused remarkable debasement in antioxidant defense enzymes (SOD and CAT) and nonenzymatic defense molecules (GSH and GSSG) revealing biochemical mechanism of action.

Exploring the potential of Crotalaria juncea flower extracts as a source of antioxidants, antimicrobials, and cytoprotective agents for biomedical applications.

Plants provide an unlimited source of bioactive compounds, possessing tremendous applications in the pharmaceutical industry. In the search for sources of antioxidants and antimicrobial agents against human pathogens, ethanol extracts of Crotalaria juncea flowers (CJ flower extract) were evaluated. The highest total phenolic (5.65 µg GAE/ml) and flavonoid (0.43 µg QE/ml) contents were observed in the 100 µg/ml CJ flower extract. To assess antioxidant activity, three in vitro antioxidant tests were employed: DPPH radical-scavenging, ABTS+ radical-scavenging, and hydroxyl radical-scavenging assay. The CJ flower extract demonstrated significant (P < 0.05) antioxidant activity, dependent on the percentage of solvent extraction and the specific assays utilized. The highest antioxidant activity was obtained with 100% ethanol extraction and using the hydroxyl radical-scavenging assay (56.63%). Antimicrobial activity was assessed against six human pathogens, including the fungi Microsporum gypseum and five Gram-positive bacteria (Propionibacterium acnes, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pyogenes, and Streptococcus mutans), as well as one Gram-negative bacterium (Escherichia coli ). The CJ flower extract inhibited the growth of both fungal and bacterial pathogens. The cytotoxicity of the CJ flower extract was measured using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, and the highest concentration of the extract (100 µg/ml) did not affect L929 cell viability. Moreover, the CJ flower extract demonstrated the ability to suppress H2O2-induced toxicity in L929 cells. Overall, the CJ flower extract has potential as an alternative source for exploring new antioxidants, antimicrobial agents, and cytoprotectants that could prove valuable for biomedical applications.

Efficacy of the antifungal metabolites of Streptomyces philanthi RL-1-178 on aflatoxin degradation with its application to prevent aflatoxigenic fungi in stored maize grains and identification of the bioactive compound.

Aflatoxin B1 is a potent carcinogen produced by Aspergillus flavus (A. flavus) and Aspergillus. parasiticus (A. parasiticus), mainly during grain storage. The efficacy of the freeze-dried culture filtrate of Streptomyces philanthi (S. philanthi) strain RL-1-178 (DCF) on degradation of aflatoxin B1 (AFB1) were evaluated and its bioactive compounds were identified. The DCF at a concentration of 9.0% (w/v) completely inhibited growth and AFB1 production of A. parasiticus TISTR 3276 and A. flavus PSRDC-4 after 7 days tested in yeast-extract sucrose (YES) medium and on stored maize grains after 28 and 14 days incubation, respectively. This indicated the more tolerance of A. parasiticus over A. flavus. The DCF and bacterial cells of S. philanthi were capable to degrade AFB1 by 85.0% and 100% for 72 h and 8 days, respectively. This confirmed the higher efficacy of the DCF over the cells. After separation of the DCF on thin-layer chromatography (TLC) plate by bioautography bioassay, each active band was identified by liquid chromatography-quadrupole time of flight mass spectrometer (LC-Q-TOF MS/MS). The results revealed two compounds which were identified as azithromycin and an unknown based on mass ions of both ESI+ and ESI- modes. The antifungal metabolites in the culture filtrate of S. philanthi were proved to degrade aflatoxin B1. It could be concluded that the DCF may be applied to prevent the growth of the two aflatoxin-producing fungi as well as the occurrence of aflatoxin in the stored maize grains.

Impact of environmental factors on Streptomyces spp. metabolites against Botrytis cinerea.

Botrytis cinerea is an economically important disease on numerous vegetables including tomato. From our previous studies, a spore suspension of Streptomyces philanthi RL-1-178 and RM-1-138 and Streptomyces mycarofaciens SS-2-243 showed strong inhibition against B. cinerea. In this study, the efficacy of their antifungal metabolites against B. cinerea was investigated after enhancing the production through the optimum culture medium and environmental conditions (temperature, light/dark cycle). In vitro studies indicated that glucose yeast-malt (GYM) agar and incubation at 28°C were optimal for growth and mass spore production of all three Streptomyces strains. Moreover, light/dark conditions had a positive effect on the growth and spore production of S. philanthi RM-1-138 and RL-1-178 but not on S. mycarofaciens SS-2-243. Both strains of S. philanthi possessed an antifungal activity against B. cinerea (100% inhibition) while S. mycarofaciens showed different results on PDA (83% inhibition) and GYM (88% inhibition) at the optimum incubation temperature at 21°C. The antifungal compounds from S. philanthi RM-1-138 exhibited the highest protection efficacy against B. cinerea on tomato leaves (82.89% and 0.33 cm2 lesion areas symptoms). The antifungal compounds RM-1-138, identified by GC-MS, were greatly altered based on components concentration under various temperatures and light/dark conditions. The anti-B. cinerea of S. philanthi RM-1-138 was established at a higher level in several metabolic compounds in the dark condition (11 and 32 antifungal compounds after incubation at 21°C and 28°C, respectively) than in the light condition (11 and 19 antifungal compounds after incubation at 21°C and 28°C, respectively). At 21°C, the dominant component was acetic acid (67.41% and 68.77% in light and dark conditions, respectively) while at 28°C, benzeneacetamide (43.58% in light) and propanamide (20.68% in the dark) were dominant. The results clearly demonstrated the significant influence of environmental factors on the production of antifungal metabolites of Streptomyces spp.

Utilization of palm oil mill effluent as a novel substrate for the production of antifungal compounds by Streptomyces philanthi RM-1-138 and evaluation of its efficacy in suppression of three strains of oil palm pathogen.

AIMS: This study aimed to use palm oil mill effluent (POME) as a renewable resource for the production of antifungal compounds by Streptomyces philanthi RM-1-138 against Ganoderma boninense, Ceratocystis paradoxa and Curvularia oryzae. METHODS AND RESULTS: The efficacy of antifungal compounds RM-1-138 against the three strains of fungal oil palm pathogen was evaluated both in vitro and on oil palm leaf segments. In vitro studies using confrontation tests on glucose yeast-malt extract (GYM) agar plates indicated that the strain RM-1-138 inhibited the growth of all three fungal pathogenic strains. The antifungal compounds produced in the GYM medium exhibited significantly higher inhibition (79%-100%) against the three fungal pathogens than using the diluted POME (50%) medium (80%-83% inhibition). The optimum condition for the production of antifungal compounds from the strain RM-1-138 was as following: POME of 47,966 mg L-1 chemical oxygen demand (COD), the initial pH at 7.0 and supplemented with yeast extract (0.4%). Meanwhile, severe morphological and internal abnormalities in C. oryzae hyphae were observed under a scanning electron microscope and transmission electron microscope. In vivo experiment on oil palm leaf segments indicated that the efficacy of the antifungal compounds RM-1-138 (DSI = 1.3) were not significantly difference in the suppression of Curvularia leaf spot compared with the two commercial chemical fungicides of mancozeb® (DSI = 1.0) and tetraconazole® (DSI = 1.3). CONCLUSIONS: Antifungal compounds produced by S. philanthi RM-1-138 grown in POME have the potential to inhibit fungal pathogens. SIGNIFICANCE AND IMPACT OF THE STUDY: The POME (about 47 mg L-1 COD) with the initial pH of 7.0 and supplementation of 0.4% nitrogen could be used as a culture medium for the growth and production of antifungal compounds of S. philanthi RL-1-138. In addition, the antifungal compound RM-1-138 could suppress the three strains of oil palm fungal pathogen tested on oil palm leaf segment.

Strategies for recovery of imbalanced full-scale biogas reactor feeding with palm oil mill effluent.

BACKGROUND: Full-scale biogas production from palm oil mill effluent (POME) was inhibited by low pH and highly volatile fatty acid (VFA) accumulation. Three strategies were investigated for recovering the anaerobic digestion (AD) imbalance on biogas production, namely the dilution method (tap water vs. biogas effluent), pH adjustment method (NaOH, NaHCO3, Ca(OH)2, oil palm ash), and bioaugmentation (active methane-producing sludge) method. The highly economical and feasible method was selected and validated in a full-scale application. RESULTS: The inhibited sludge from a full-scale biogas reactor could be recovered within 30-36 days by employing various strategies. Dilution of the inhibited sludge with biogas effluent at a ratio of 8:2, pH adjustment with 0.14% w/v NaOH, and 8.0% w/v oil palm ash were considered to be more economically feasible than other strategies tested (dilution with tap water, or pH adjustment with 0.50% w/v Ca(OH)2, or 1.25% NaHCO3 and bioaugmentation) with a recovery time of 30-36 days. The recovered biogas reactor exhibited a 35-83% higher methane yield than self-recovery, with a significantly increased hydrolysis constant (kH) and specific methanogenic activity (SMA). The population of Clostridium sp., Bacillus sp., and Methanosarcina sp. increased in the recovered sludge. The imbalanced full-scale hybrid cover lagoon reactor was recovered within 15 days by dilution with biogas effluent at a ratio of 8:2 and a better result than the lab-scale test (36 days). CONCLUSION: Dilution of the inhibited sludge with biogas effluent could recover the imbalance of the full-scale POME-biogas reactor with economically feasible and high biogas production performance.

Improvement of empty palm fruit bunches biodegradability and biogas production by integrating the straw mushroom cultivation as a pretreatment in the solid-state anaerobic digestion.

Empty fruit bunches (EFB) have low biodegradability and restrict their commercial utilization in biogas plants. Integration of straw mushroom (Volvariella volvacea) cultivation as a function of bio-pretreatment on EFB to improve biodegradability and methane production by solid-state anaerobic digestion (SS-AD) was investigated. The mushroom yield was 47.3 kg·tonne-1 EFB with remaining weight in spent mushroom-EFB (S-mEFB) of 82%. The cellulose, hemicellulose, and lignin of EFB were degraded by 3.3%, 21.3%, and 17.6%, respectively, with an increased surface area of S-mEFB. The biodegradability of S-mEFB (62.7%) was 2 times higher than raw EFB (33.5%) with the highest methane yield and production of 281 mL CH4·g-1 VS and 50.6 m3·tonne-1 S-mEFB, respectively. The co-digestion of S-mEFB with 5% v/w POME had highest methane yield of 405 mL CH4·g-1 VS with biodegradability of 90.8%. Integrating straw mushroom cultivation with SS-AD is a promising strategy for achieving an environmentally friendly and economically feasible process.

Comparative assessment of single-stage and two-stage anaerobic digestion for biogas production from high moisture municipal solid waste.

BACKGROUND: Anaerobic digestion (AD) is a suitable process for treating high moisture MSW with biogas and biofertilizer production. However, the low stability of AD performance and low methane production results from high moisture MSW due to the fast acidify of carbohydrate fermentation. The effects of organic loading and incineration fly ash addition as a pH adjustment on methane production from high moisture MSW in the single-stage AD and two-stage AD processes were investigated. RESULTS: Suitable initial organic loading of the single-stage AD process was 17 gVS L-1 at incineration fly ash (IFA) addition of 0.5% with methane yield of 287 mL CH4 g-1 VS. Suitable initial organic loading of the two-stage AD process was 43 gVS L-1 at IFA addition of 1% with hydrogen and methane yield of 47.4 ml H2 g-1 VS and 363 mL CH4 g-1 VS, respectively. The highest hydrogen and methane production of 8.7 m3 H2 ton-1 of high moisture MSW and 66.6 m3 CH4 ton-1 of high moisture MSW was achieved at organic loading of 43 gVS L-1 at IFA addition of 1% by two-stage AD process. Biogas production by the two-stage AD process enabled 18.5% higher energy recovery than single-stage AD. The 1% addition of IFA into high moisture MSW was useful for controlling pH of the two-stage AD process with enhanced biogas production between 87-92% when compared to without IFA addition. Electricity production and energy recovery from MSW using the coupled incineration with biogas production by two-stage AD process were 9,874 MJ ton-1 MSW and 89%, respectively. CONCLUSIONS: The two-stage AD process with IFA addition for pH adjustment could improve biogas production from high moisture MSW, as well as reduce lag phase and enhance biodegradability efficiency. The coupled incineration process with biogas production using the two-stage AD process was suitable for the management of MSW with low area requirement, low greenhouse gas emissions, and high energy recovery.

Bacillus thermoamylovorans-Related Strain Isolated from High Temperature Sites as Potential Producers of Medium-Chain-Length Polyhydroxyalkanoate (mcl-PHA).

Thermotolerant bacteria producing medium-chain-length polyhydroxyalkanoate (mcl-PHA) were isolated from various thermal sites, including palm oil mill effluent, textile wastewater, and hot spring water, in Thailand. Fifteen strains were isolated at 45 °C using nutrient-rich (NR) medium. However, only six isolates produced mcl-PHA at 0.41 ± 0.01 g/L to 0.80 ± 0.01 g/L, representing a mcl-PHA content of 29.44% to 50.77% of the dry cell weight (DCW). The six strains of bacterial isolates could utilise a variety of substrates; all were identified as Bacillus thermoamylovorans. The highest mcl-PHA content (50.77% of the DCW) was accumulated by the B. thermoamylovorans strain PHA005 isolated from palm oil mill effluent. The mcl-PHA from strain PHA005 was composed of five different monomers, 3-hydroxyoctanoate (3HO), 3-hydroxydecanoate (3HD), 3-hydroxytetradecanoate (3HTD), 3-hydroxyhexadecanoic acid (3HHD), and 3-hydroxyoctadecanoic (3HOD), with a monomer content of 24.12, 15.50, 13.00, 39.25, and 8.13 mol%, respectively. The optimum temperature for B. thermoamylovorans strain PHA005 growth is 45 °C, and it can survive at up to 60 °C. This is a first report of PHA synthesis by a thermotolerant B. thermoamylovorans. Moreover, the high content of 3HHD monomers (39.25 mol%) has never been reported in Bacillus.

The Degradation of Phenanthrene, Pyrene, and Fluoranthene and Its Conversion into Medium-Chain-Length Polyhydroxyalkanoate by Novel Polycyclic Aromatic Hydrocarbon-Degrading Bacteria.

Screening of high-efficient polycyclic aromatic hydrocarbon (PAH)-degrading bacteria is important due to environmental contamination by PAHs. In this study, sediment contaminated with phenanthrene (Phe), pyrene (Pyr), and fluoranthene (Fluo) was used as a source of bacteria. The ability of these isolated bacteria to convert PAHs into valuable products was determined. Based on a primary screening, 20 bacterial isolates were obtained; however, only three strains showed a good PAH-degrading ability, and were identified as Pseudomonas aeruginosa, Pseudomonas sp., and Ralstonia sp. PAH-degrading genes were detected in all isolates. Notably, all selected strains could degrade PAHs using the ortho or meta cleavage pathways due to the presence of catechol dioxygenase genes. The ability of isolated strains to convert PAHs into polyhydroxyalkanoate (PHA) was also evaluated in both single and mixed cultures. Single cultures of P. aeruginosa PAH-P02 showed 100% degradation of PAHs, with the highest biomass (1.27 ± 0.02 g l-1) and PHA content (38.20 ± 1.92% dry cell weight). However, degradative ability and PHA production were decreased when mixtures of PAHs were used. This study showed that P. aeruginosa, Pseudomonas sp., and Ralstonia sp. were able to degrade PAHs and convert them into medium-chain-length (mcl)-PHA. A high content of 3-hydroxydecanoate (3HD, C10) was observed in this study. The formation of mcl-PHA with high 3HD content from Pyr and Fluo, and the assessment of mixed cultures converting PAHs to mcl-PHA, were novel contributions.

Thermotolerant cellulolytic Clostridiaceae and Lachnospiraceae rich consortium enhanced biogas production from oil palm empty fruit bunches by solid-state anaerobic digestion.

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

Immobilized oleaginous microalgae as effective two-phase purify unit for biogas and anaerobic digester effluent coupling with lipid production.

Oleaginous microalga Scenedesmus sp. was immobilized in alginate-gel beads and applied as two-phase purify unit for biogas and anaerobic digester effluent from palm oil mill. Optimal microalgal cell concentration and bead volume ratio were 106 cells mL-1 and 25% v/v, respectively. The use of 20% effluent and light intensity at 128 µmol·proton·m-2 s-1 most promoted CO2 removal by immobilized microalgae and achieved the maximum CO2 removal rate of 4.63 kg-CO2 day-1 m-3. This process upgraded methane content in biogas (>95%) and completely remove nitrogen and phosphorus in the effluent. After process operation, 2.98 g L-1 microalgal biomass with 35.92% lipid content were recovered by simple sieving method. Microalgal lipids are composed of C16-C18 (>98%) with prospect high cetane number and short ignition delay time. This study has shown the promising biorefinery concept which is effective not only in CO2 fixation, biogas upgrading and pollutant removal but also cost-effective production of microalgae-based biofuel.

Inhibitory effects of acetophenone or phenylethyl alcohol as fumigant to protect soybean seeds against two aflatoxin-producing fungi.

The antifungal activity of acetophenone and phenylethyl alcohol prevents seed contamination by Aspergillus flavus TISTR 3041 and A. parasiticus TISTR 3276. Their effects on seed germination were investigated. In vitro results showed that 100 µL L-1 acetophenone completely inhibited (by 100%) growth, conidial germination, and sporulation of the two aflatoxin-producing fungi, while phenylethyl alcohol showed only weak inhibitory activity even at 1000 µL L-1. Exposure to acetophenone at 100 µL L-1 for 6 h could completely kill (100% death) both fungal strains, while phenylethyl alcohol showed much lower efficacy (53.12%). In vivo results revealed that fumigation with 100 µL L-1 acetophenone for 24 h completely controlled (100%) A. flavus TISTR 3041 on soybean seeds during a 14-day test but exhibited weak efficacy on A. parasiticus TISTR 3276 (31.77%). Phenylethyl alcohol (1000 µL L-1) demonstrated weak inhibitory effect against both strains. The two volatile compounds had no adverse effects on seed germination. SEM confirmed that acetophenone could completely inhibit conidia germination, and abnormal growth of both fungal strains was observed. Thus, acetophenone has high potential to protect soybean seeds against aflatoxin-producing fungi.

A rapid method for harvesting and immobilization of oleaginous microalgae using pellet-forming filamentous fungi and the application in phytoremediation of secondary effluent.

A rapid method for harvesting and immobilization of oleaginous microalgae using pellet-forming filamentous fungi was developed. The suitable conditions for pellet formation by filamentous fungi were determined. Among the strains tested, Trichoderma reesei QM 9414 showed superior pellet forming ability. Its pellets were used to harvest oleaginous microalga Scenedesmus sp. With increasing volume ratio of fungal pellets to microalgae culture up to 1:2, >94% of microalgal cells were rapidly harvested within 10 min. The ratio of fungal pellets could manipulate both harvesting time and initial concentration of microalgal cells in the pellets. The microalgae-fungal pellets were successfully used as immobilized cells for effective phytoremediation of secondary effluent from seafood processing plants under nonsterile condition. The chemical oxygen demand, total nitrogen, and total phosphorus removal were >74%, >44%, and >93%, respectively. The scanning electron microscopy showed that the microalgal cells were not only entrapped in the pellets but also got attached to the fungal hyphae with sticky exopolysaccharides, possibly secreted by the fungi. The extracted lipids from the pellets were mainly composed of C16-C18 (>83%) with their suitability as biodiesel feedstocks. This study has shown the promising strategy to rapidly harvest and immobilize microalgal cells and the possible application in phytoremediation of industrial effluent.

Effects of volatile fatty acids in biohydrogen effluent on biohythane production from palm oil mill effluent under thermophilic condition

Background: Biohydrogen effluent contains a high concentration of volatile fatty acid (VFA) mainly as butyric, acetic, lactic and propionic acids. The presence of various VFAs (mixture VFAs) and their cooperative effects on two-stage biohythane production need to be further studied. The effect of VFA concentrations in biohydrogen effluent of palm oil mill effluent (POME) on methane yield in methane stage of biohythane production was investigated. Results: The methane yield obtained in low VFA loading (0.9 and 1.8 g/L) was 15­20% times greater than that of high VFA loading (3.6 and 4.7 g/L). Butyric acid at high concentrations (8 g/L) has the individual significantly negative effect the methane production process (P b 0.05). Lactic, acetic and butyric acid mixed with propionic acid at a concentration higher than 0.5 g/L has an interaction significantly negative effect on the methanogenesis process (P b 0.05). Inhibition condition had a negative effect on both bacteria and archaea with inhibited on Geobacillus sp., Thermoanaerobacterium thermosaccharolyticum, Methanoculleus thermophilus and Methanothermobacter delfuvii resulting in low methane yield. Conclusion: Preventing the high concentration of butyric acid, and propionic acid in the hydrogenic effluent could enhance methane production in two-stage anaerobic digestion for biohythane production.

Immobilized oleaginous microalgae for production of lipid and phytoremediation of secondary effluent from palm oil mill in fluidized bed photobioreactor.

Oleaginous microalga Nannochloropsis sp. was immobilized in alginate gel beads and cultivated under optimal conditions that their growth and lipid production were comparable to those of free cells. The immobilized cells were used in phytoremediation of secondary effluent from palm oil mill and easily recovered by simple sieving method. The immobilized cells contributed to removal of nitrogen and phosphorus >90% and CO2 mitigation >99%. They also gave the biomass and lipid production of 1.300±0.050g/L and 0.356±0.097g/L, respectively. The repeated-batch cultivation improved the biomass and lipid production by 2.66 folds and 1.41 folds, respectively. The scale up in 3L-fluidized bed photobioreactor gave the maximum biomass of 3.280±0.049g/L and lipid production of 0.362±0.010g/L. Fatty acid compositions of Nannochloropsis sp. lipids showed their suitability as biodiesel feedstocks. This system not only contributes as tertiary treatment of industrial effluent and CO2 mitigation but also low-cost production of renewable energy.

Draft genome sequence of Thermoanaerobacterium sp. strain PSU-2 isolated from thermophilic hydrogen producing reactor.

Thermoanaerobacterium sp. strain PSU-2 was isolated from thermophilic hydrogen producing reactor and subjected to draft genome sequencing on 454 pyrosequencing and annotated on RAST. The draft genome sequence of strain PSU-2 contains 2,552,497 bases with an estimated G + C content of 35.2%, 2555 CDS, 8 rRNAs and 57 tRNAs. The strain had a number of genes responsible for carbohydrates metabolic, amino acids and derivatives, and protein metabolism of 17.7%, 14.39% and 9.81%, respectively. Strain PSU-2 also had gene responsible for hydrogen biosynthesis as well as the genes related to Ni-Fe hydrogenase. Comparative genomic analysis indicates strain PSU-2 shares about 94% genome sequence similarity with Thermoanaerobacterium xylanolyticum LX-11. The nucleotide sequence of this draft genome was deposited into DDBJ/ENA/GenBank under the accession MSQD00000000.

Purification and characterization of xylitol dehydrogenase with l-arabitol dehydrogenase activity from the newly isolated pentose-fermenting yeast Meyerozyma caribbica 5XY2.

Meyerozyma caribbica strain 5XY2, which was isolated from an alcohol fermentation starter in Thailand, was found to catabolize l-arabinose as well as d-glucose and d-xylose. The highest production amounts of ethanol from d-glucose, xylitol from d-xylose, and l-arabitol from l-arabinose were 0.45 g/g d-glucose, 0.60 g/g d-xylose, and 0.61 g/g l-arabinose with 21.7 g/L ethanol, 20.2 g/L xylitol, and 30.3 g/l l-arabitol, respectively. The enzyme with l-arabitol dehydrogenase (LAD) activity was purified from the strain and found to exhibit broad specificity to polyols, such as xylitol, d-sorbitol, ribitol, and l-arabitol. Xylitol was the preferred substrate with Km=16.1 mM and kcat/Km=67.0 min-1mM-1, while l-arabitol was also a substrate for the enzyme with Km=31.1 mM and kcat/Km=6.5 min-1 mM-1. Therefore, this enzyme from M. caribbica was named xylitol dehydrogenase (McXDH). McXDH had an optimum temperature and pH at 40°C and 9.5, respectively. The McXDH gene included a coding sequence of 1086 bp encoding a putative 362 amino acid protein of 39 kDa with an apparent homopentamer structure. Native McXDH and recombinant McXDH exhibited relative activities toward l-arabitol of approximately 20% that toward xylitol, suggesting the applicability of this enzyme with the functions of XDH and LAD to the development of pentose-fermenting Saccharomyces cerevisiae.

Cellulose and hemicellulose recovery from oil palm empty fruit bunch (EFB) fibers and production of sugars from the fibers.

A sequential two-step treatment with peracetic acid (PA) and alkaline peroxide (AP) at mild temperatures (20-35°C) removed more than 98% of the lignin from oil palm empty fruit bunch (EFB) fiber. For each kilogram of EFB fiber treated, 200-250g of a solids fraction and 120-170g of a precipitate fraction were recovered after the treatment. Subsequent enzymatic hydrolysis (45°C, 72h) of the recovered solids (excluding the precipitate) resulted in a glucose yield of 629.8±0.5g per kg of the original dry EFB biomass. Enzymatic hydrolysis of untreated EFB yielded only 3.0±0.0g glucose per kg of dry EFB. Therefore, the PA-AP pretreatment enhanced glucose recovery from EFB by nearly 210-fold. The total treatment time was 93h (a 9h PA treatment at 35°C, a 12h treatment with AP (20°C, 4% NaOH), 72h of enzymatic hydrolysis).