Poonsuk Prasertsan

Evaluation of methods for preparing hydrogen-producing seed inocula under thermophilic condition by process performance and microbial community analysis.

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 degrees 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.

Two-stage conversion of crude glycerol to energy using dark fermentation linked with microbial fuel cell or microbial electrolysis cell.

Crude glycerol is a main byproduct of the biodiesel industry, and the beneficial use of waste glycerol has been a major challenge. This study characterises the conversion of crude glycerol into bioenergy such as H2 and electricity using a two-stage process linking dark fermentation with a microbial fuel cell (MFC) or microbial electrolysis cell (MEC). The results showed that fermentation achieved a maximum H2 rate of 332 mL/L and a yield of 0.55 mol H2/mol glycerol, accompanied by 20% of organic removal. Fed with the raw fermentation products with an initial COD of 7610 mg/L, a two-chamber MFC produced 92 mW/m(2) in power density and removed 50% of COD. The Columbic efficiency was 14%. When fed with 50% diluted fermentation product, a similar power output (90m W/m(2)) and COD removal (49%) were obtained, but the CE doubled to 27%. Similar substrates were used to produce H2 in two-chamber MECs, and the diluted influent had a higher performance, with the highest yield at 106 mL H2/g COD and a CE of 24%. These results demonstrate that dark fermentation linked with MFC/MEC can be a feasible option for conversion of waste glycerol into bioenergy.

Optimization of furfural production from hemicellulose extracted from delignified palm pressed fiber using a two-stage process.

This study aims to optimize the conditions for furfural production from hemicellulose extracted from delignified palm pressed fiber (dPPF) via two-stage process: acid hydrolysis followed by dehydration, using response surface methodology (RSM). The extracted hemicellulose contained 80.8% xylose. In order to convert hemicellulose to xylose in the acid hydrolysis step, there were four important parameters consisting of reaction temperature (100-150°C), sulfuric acid concentration (1-10% v/v), ratio of sulfuric acid to hemicellulose (L/S ratio) (10, 9, and 8 v/w), and reaction time (30-120min). The maximum xylose production (12.58g/L) was achieved at 125°C, 5.5% sulfuric acid, L/S ratio of 9mL/g for 30min with the determination coefficient (R(2)) value of 0.90. For the dehydration process, two parameters; reaction temperature (120-160°C) and reaction time (30-150min), were optimized. The maximum furfural production (8.67g/L) was achieved at a reaction temperature of 140°C for 90min with the determination coefficient (R(2)) value of 0.93.

Nutrient optimization for production of polyhydroxybutyrate from halotolerant photosynthetic bacteria cultivated under aerobic-dark condition

Three halotolerant bacterial strains; Rhodobacter sphaeroides ES16 (the wild type) and the two mutant strains of R. sphaeroides ES16, namely N20 and U7, were cultivated in glutamate-malate (GM) medium and screened for production of polyhydroxybutyrate (PHB). The mutant strains N20 and U7 were found to accumulate PHB (53.9 and 42.0% of DCW, respectively) 3.6 and 2.8 times higher than the wild type strain (19.5% of DCW), respectively. R. sphaeroides N20 were selected for studies on the effects of nutrient and environmental conditions on PHB accumulation. The optimal condition was 4 g/l acetate, 0.02 g/l (NH 4 ) 2 SO 4 , C/N ratio of 6:1, 1.0 g/l K 2 HPO 4 , 1.0 g/l KH 2 PO 4 and 3% NaCl with initial pH at 7.0. Under this optimal condition, the maximum PHB accumulation increased from 53.9% to 88% of DCW and 9.11 ± 0.08 g/l biomass, 8.02 ± 0.10 g/l PHB concentration were achieved after 60 hrs cultivation at 37oC. These results are the highest values ever obtained from photosynthetic bacteria reported so far.

Optimization for xylanase and cellulase production from Aspergillus niger ATTC 6275 in palm oil mill wastes and its application

Optimization of enzyme production from Aspergillus niger ATCC 6275 under both submerged and solid-substrate cultivation was investigated. Results from submerged cultivation using palm oil mill effluent revealed that pretreatment of ground palm cake did not improve enzyme production. Addition of 0.60g NH4NO3/l generated maximum activity of xylanase and cellulase (CMCase). The optimum aeration rate was 1.2 v/v min. Under solid-substrate cultivation, the results indicated that heating and alkali treatment of the ground palm cake gave no further improvement in enzyme production. The optimal N-source was 2% urea. Optimal initial moisture contents for xylanase and CMCase activities were 60% and 50% respectively, with temperature optima of 30°C and 35°C, respectively. The optimal inoculum size was 1× 108 spores/g palm cake with an initial pH of 4.5–5.0. The maximum activities of xylanase (282.9U/g) and CMCase (23.8U/g) were obtained under the optimum conditions. Solid-substrate cultivation was a better method for the production of enzyme, particularly xylanase, from A. niger ATCC 6275. The application of these enzymes to decanter effluent showed the separation of oil and grease and suspended solids from the effluent. This is comparable to the result achieved from using the commercial xylase preparation Meicelase and superior to the effect of Sumyzyme.

Expression and applications of recombinant crustacean hyperglycemic hormone from eyestalks of white shrimp (Litopenaeus vannamei) against bacterial infection

Crustacean hyperglycemic hormone (CHH) has many functions to regulate carbohydrate metabolism, ecdysis and reproduction including ion transport in crustaceans. The cDNA encoding CHH peptides containing 369 bp open reading frame encoding 122 amino acids was cloned from eyestalk of white shrimp (Litopenaeus vannamei) and was produced by a bacterial expression system. The biological activity of recombinant L. vannamei crustacean hyperglycemic hormone (rLV-CHH) was tested. The hemolymph glucose level of shrimp increased two-fold at 1h after the rLV-CHH injection and then returned to normal after 3h. In addition to the effect of rLV-CHH administration (25 μg/shrimp) on immunological responses of white shrimp against pathogenic bacteria, Vibrio harveyi was studied. Results showed that the blood parameters of shrimp injected with rLV-CHH; the THC, PO activity, serum protein level and clearance ability to V. harveyi, were also higher than those of Neg-protein and PBS-injected shrimp. The survival of shrimp injected with rLV-CHH was significantly higher (66.0%) than shrimp that injected with Neg-protein (33.3%) and PBS (28.9%) after 14 days. It is possible that the administration of rLV-CHH in L. vannamei exhibited a higher immune response related to resistance against V. harveyi infection.

Characterization of a hemocyte intracellular fatty acid-binding protein from crayfish (Pacifastacus leniusculus) and shrimp (Penaeus monodon).

Intracellular fatty acid‐binding proteins (FABPs) are small members of the superfamily of lipid‐binding proteins, which occur in invertebrates and vertebrates. Included in this superfamily are the cellular retinoic acid‐binding proteins and retinol‐binding proteins, which seem to be restricted to vertebrates. Here, we report the cDNA cloning and characterization of two FABPs from hemocytes of the freshwater crayfish Pacifastacus leniusculus and the shrimp Penaeus monodon. In both these proteins, the binding triad residues involved in interaction with ligand carboxylate groups are present. From the sequence and homology modeling, the proteins are probably FABPs and not retinoic acid‐binding proteins. The crayfish transcript (plFABP) was detected at high level in hemocytes, hepatopancreas, intestine and ovary and at low level in hematopoietic tissue and testis. Its expression in hematopoietic cells varied depending on the state of the crayfish from which it was isolated. Expression was 10–15 times higher in cultures isolated from crayfish with red colored plasma, in which hemocyte synthesis was high, if retinoic acid was added to the culture medium. In normal colored crayfish, with normal levels of hemocytes, no increase in expression of p1FABP was detected. Two other putative plFABP ligands, stearic acid and oleic acid, did not have any effect on plFABP expression in hematopoietic cells. These results suggest that retinoic acid‐dependent signaling may be present in crustaceans.

Isolation, identification and growth conditions of photosynthetic bacteria found in seafood processing wastewater.

Four photosynthetic bacteria, isolated from 14 samples taken from seafood processing plants, were identified as species of Rhodocyclus gelatinosus, belonging to the purple, non-sulphur bacteria of the family Rhodospirillaceae. Cultivation in synthetic medium under four different conditions indicated that all four strains gave maximum carotenoid and bacteriochlorophyll synthesis under anaerobic conditions in the light, with values of 11 to 12.6 and 102 to 108 mg/g dry cell wt, respectively. These values are 87% higher than the pigment content obtained from aerobic cultivation, although the cell biomass of all strains (1.7 to 2.3 g/l) was 22 to 38% higher under aerobic conditions. Protein content was always between 32 and 43%. The specific growth rates of all isolates in aerobic cultivation (0.04 to 0.06 h-1) were twice those in anaerobic conditions in the light. No growth occurred in anaerobic conditions in the dark.

Evaluation of Strains of Metarhizium anisopliae and Beauveria bassiana against Spodoptera litura on the Basis of Their Virulence, Germination Rate, Conidia Production, Radial Growth and Enzyme Activity.

Ten strains of the entomopathogenic fungi Metarhizium anisopliae and Beauveria bassiana were evaluated to find the most effective strain for optimization studies. The first criterion tested for strain selection was the mortality (> 50%) of Spodoptera litura larvae after inoculation of the fungus for 4 days. Results on several bioassays revealed that B. bassiana BNBCRC showed the most virulence on mortality S. litura larvae (80% mortality). B. bassiana BNBCRC also showed the highest germination rate (72.22%). However, its conidia yield (7.2 × 108 conidia/mL) was lower than those of B. bassiana B 14841 (8.3 × 108 conidia/mL) and M. anisopliae M6 (8.2 × 108 conidia/mL). The highest accumulative radial growth was obtained from the strain B14841 (37.10 mm/day) while the strain BNBCRC showed moderate radial growth (24.40 mm/day). M. anisopliae M6 possessed the highest protease activity (145.00 mU/mL) while M. anisopliae M8 possessed the highest chitinase activity (20.00 mU/mL) during 96~144 hr cultivation. Amongst these criteria, selection based on virulence and germination rate lead to the selection of B. bassiana BNBCRC. B. bassiana B14841 would be selected if based on growth rate while M. anisopliae M6 and M8 possessed the highest enzyme activities.

Production of biomass and extracellular 5-aminolevulinic acid by Rhodopseudomonas palustris KG31 under light and dark conditions using volatile fatty acid.

Kinetic parameters for growth and extracellular 5-aminolevulinic acid (ALA) production of Rhodopseudomonas palustris KG31 under light and dark conditions in a medium containing volatile fatty acids (VFA) as the carbon sources were estimated using a Gompertz model. The lag phase for growth and the maximum specific growth rate under microaerobic-light cultivations were 7.29-12.49 h and 0.038-0.094 h(-1), respectively, whereas under aerobic-dark cultivations, they were 2.03-14.25 h and 0.016-0.022 h(-1), respectively. The lag phase for extracellular ALA production and the maximum specific extracellular ALA production rate under microaerobic-light cultivations (15.72-24.74 h and 0.222-0.299 h(-1), respectively) were better than those obtained under aerobic-dark cultivations (24.57-44.84 h and 0.103-0.215 h(-1), respectively). The biomass and the extracellular ALA yields of 39.66-56.25 gDCW/l/mol C, and 148.47-245.75 μM/mol C, respectively, under microaerobic-light cultivations were higher than of those obtained under aerobic-dark conditions. An enhancement of extracellular ALA production under aerobic-dark conditions revealed that the ALA yield was markedly increased 8-fold (48.36 μM) by the addition of 10mM succinate, 4.5mM glycine, and 15 mM levulinic acid (LA). By controlling dissolved oxygen (DO) and pH values, a maximum extracellular ALA yield of 66.38 μM was found. The degradation rate of ALA in the culture broth was closely related to the pH value.