Antifungal effect of volatile organic compounds produced by Streptomyces salmonis PSRDC-09 against anthracnose pathogen Colletotrichum gloeosporioides PSU-03 in postharvest chili fruit.

AIMS: Application of volatile organic compounds (VOCs) from Streptomyces salmonis PSRDC-09 (VOCs PSRDC-09) grown on sterile wheat seeds against chili anthracnose pathogen was investigated in vitro and in vivo. METHODS AND RESULTS: Among 10 isolates of Colletotrichum species tested, Colletotrichum gloeosporioides PSU-03 was selected as the most aggressive anthracnose pathogenic strain on chili fruit against VOCs produced by Streptomyces species. Among 11 isolates, the strain PSRDC-09 exhibited the highest antifungal activity and was identified as S. salmonis PSRDC-09. The antagonistic mechanism of the VOCs PSRDC-09 on morphological of C. gloeosporioides PSU-03, observed by scanning electron microscope (SEM), revealed the irregular distortions in the fungal hyphae. The effect of inoculum size and spore concentration of S. salmonis PSRDC-09 prepared as a wheat seed inoculum on the suppression of C. gloeosporioides PSU-03 was studied both in vitro and on chili fruit. The optimum inoculum size (45 g 0·31 l-1 ) and spore concentration (107 spores per ml) of the wheat seed culture of S. salmonis PSRDC-09 exhibited the complete suppression (100% inhibition) on C. gloeosporioides PSU-03. The optimum fumigation period of the VOCs PSRDC-09 (45 g 1·38 l-1 ) was found to be 24 h. Based on gas chromatography-mass spectrometry (GC-MS) analysis, 14 major VOCs (produced by the strain PSRDC-09) were detected and l-linalool was the main volatile component. CONCLUSIONS: The results indicated that the VOCs from S. salmonis PSRDC-09 could effectively control the chili anthracnose disease caused by C. gloeosporioides. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings suggest that S. salmonis PSRDC-09 may have the potential to become a promising biofumigant for biocontrol of chili anthracnose disease in the postharvest system.

Efficacy of volatile compounds from Streptomyces philanthi RL-1-178 as a biofumigant for controlling growth and aflatoxin production of the two aflatoxin-producing fungi on stored soybean seeds.

AIMS: This study aimed to apply the volatile organic compounds from Streptomyces philanthi RL-1-178 (VOCs RL-1-178) as a fumigant to protect soybean seeds against the two aflatoxin-producing fungi in stored soybean seeds. METHODS AND RESULTS: The antifungal bioassay tests on potato dextrose agar (PDA) dishes showed that 30 g l-1 wheat seed inoculum of S. philanthi RL-1-178 exhibited total (100%) inhibition on Aspergillus parasiticus TISTR 3276 and Aspergillus flavus PSRDC-4. Identification of the VOCs RL-1-178 using GC-MS revealed 39 compounds with the most abundant substances being geosmin (13·75%) followed by l-linalool (13·55%), 2-mercaptoethanol (9·71%) and heneicosane (5·96%). Comparison on the efficacy of the VOCs RL-1-178 (at 30 g l-1 wheat seed culture) and their four major components (100 µl l-1 each) on the suppression of the two aflatoxin-producing fungi on PDA plates revealed that the VOCs RL-1-178 as well as geosmin, l-linalool and 2-mercaptoethanol completely inhibited (100%) mycelial growth while heneicosane showed only 70·7% inhibition. Use of the VOCs RL-1-178 (30 g l-1 ) as a biofumigant on stored soybean seeds resulted in complete protection (100%) against the infection as well as complete inhibition on production of aflatoxin (B1 , B2 and G2 ) (analysed by HPLC) by the two aflatoxin-producing fungi. CONCLUSIONS: The VOCs RL-1-178 displayed strong inhibitory effects on A. parasiticus TISTR 3276 and A. flavus PSRDC-4 as well as inhibited aflatoxin (B1 , B2 and G2 ) production. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings suggest that the VOCs RL-1-178 can be applied as a biofumigant to control the two aflatoxin-producing fungi on stored seeds products.

Enhancing the degradation of mixed polycyclic aromatic hydrocarbon and medium-chain-length polyhydroxyalkanoate production by mixed bacterial cultures using modified repeated batch fermentation.

AIMS: To increase the biodegradation of phenanthrene (PHE), pyrene (PYR) and fluoranthene (FLU) through mixed cultures of polycyclic aromatic hydrocarbon (PAH)-degrading bacteria, using modified repeated batch fermentation. METHODS AND RESULTS: Novel bacterial strains of Pseudomonas putida, Pseudomonas sp. and Ralstonia eutropha were cultivated and the biodegradation and conversion of mixed PAH to medium-chain-length polyhydroxyalkanoates (MCL-PHA) was determined. The highest degradation of PAH (100%) and PHA production (50·0%) was obtained in medium containing 30 mmol l-1 of mixed PAH after three cycles of repeated batch fermentation. The concentration of PAH in the reactor was increased from 30 to 90 mmol l-1 with repeated additions of PAH, and bacteria were able to produce PHA at 40% of cell dry mass. The MCL-PHA were identified by gas chromatography/mass spectroscopy, with the 3-hydroxydecanoate (3-HD) monomer higher than 75 mol.%. CONCLUSIONS: This study demonstrated that the biodegradation of PHE, PYR and FLU was enhanced by modified repeated batch fermentation using a mixed culture of bacteria. In addition, this fermentation strategy also increased the production of PHA, with an increase in monomer composition. SIGNIFICANCE AND IMPACT OF THE STUDY: This was the first study to describe the enhancement of the degradation of mixed solutions of PHE, PYR and FLU, and PHA production, using novel mixed bacterial cultures and modified repeated batch fermentation. The MCL-PHA formed had uniquely high 3-HD content.

Screening thermotolerant white-rot fungi for decolorization of wastewaters.

To select a thermotolerant fungal strain for decolorization of wastewaters, ligninolytic enzyme production (lignin peroxidase, manganese peroxidase [MnP], and laccase), decolorization, and removal of total phenol and chemical oxygen demand (COD) were detected. Thirty-eight fungal strains were studied for enzyme production at 35 and 43 degrees C on modified Kirk agar medium including 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and MnCl2. Thirteen strains grew on manganese-containing agar and provided green color on ABTS-containing agar plates under culture at 43 degrees C. Decolorization of wastewater from alcohol distillery (WAD) by these strains was compared under static culture at 43 degrees C, and Pycnoporus coccineus FPF 97091303 showed the highest potential. Thereafter, immobilized mycelia were compared with free mycelia for WAD decolorization under culture conditions of 43 degrees C and 100 rpm. The immobilized mycelia on polyurethane foam enhanced the ligninolytic enzyme production as well as total phenol and color removal. At about the same COD removal, MnP and laccase produced by immobilized mycelia were 2 and 19 times higher than by free mycelia; the simultaneous total phenol and color removal were 3.1 and 1.5 times higher than the latter. Moreover, decolorization of synthesis dye wastewater was carried out at 43 degrees C and 100 rpm. More than 80% of 300 mg/L of reactive blue-5 was decolorized by the immobilized mycelia within 1 to 2 d for four cycles.

In vitro fermentation of mixed linkage glucooligosaccharides produced by Gluconobacter oxydans NCIMB 4943 by the human colonic microflora.

The aim of this study was to develop selectively fermented (prebiotic) carbohydrate molecules which would also result in the generation of butyric acid. Gluco-oligosaccharides produced by Gluconobacter oxydans NCIMB 4943 from various types of maltodextrins were evaluated for their fermentation by mixed cultures of human colonic microflora. The selectivity of growth of desirable bacteria (bifidobacteria, lactobacilli) was studied in stirred pH-controlled (6.8) batch cultures. Bacterial populations were enumerated using fluorescent in situ hybridization (FISH). Gluco-oligosaccharides resulted in significantly (P<0.05) increased numbers of bifidobacteria and lactobacilli within 24 hours. Bacteroides, clostridial and eubacterial populations were slightly decreased at 48 h. There was very little difference in selectivity between the maltodextrin substrates and the products, although maltodextrin displayed a slightly less selective fermentation than the gluco-oligosaccharide products, also stimulating the growth of bacteroides, clostridia and eubacteria. Gluco-oligosaccharides, produced from G19 maltodextrin, resulted in the best prebiotic effect with the highest prebiotic index (PI) of 5.90 at 48 hours. Acetate, propionate and butyrate were all produced from gluco-oligosaccharides, derived from G19 maltodextrin, at 48 hours but no lactate or formate were detected.

In vitro three-stage continuous fermentation of gluco-oligosaccharides produced by Gluconobacter oxydans NCIMB 4943 by the human colonic microflora.

Gluco-oligosaccharides produced by Gluconobacter oxydans NCIMB 4943 from maltodextrin as the source, were evaluated for their fermentability by the human colonic microflora. The selectivity of growth of desirable bacteria in the human colon was studied in a three-stage continuous model of the human large intestine. Populations of bacteria, and their fluctuations as a response to the fermentation, were enumerated using fluorescent in situ hybridization (FISH). The gluco-oligosaccharides resulted in increases in numbers of bifidobacteria and the Lactobacillus/Enterococcus group in all 3 vessels of the system, representing the proximal, transverse and distal colonic areas. The prebiotic indices of the gluco-oligosaccharides were 2.29, 4.23 and 2.74 in V1, V2 and V3 respectively.

Enrichment of nitrifying microbial communities from shrimp farms and commercial inocula.

Nitrifying bacteria were selected from shrimp farm water and sediment ("natural" seed) in Thailand and from commercial seed cultures. The microbial consortia from each source giving the best ammonia removal during batch culture pre-enrichments were used as inocula for two sequencing batch reactors (SBRs). Nitrifiers were cultivated in the SBRs with 100 mg NH4-N/l and artificial wastewater containing 25 ppt salinity. The two SBRs were operated at a 7 d hydraulic retention time (HRT) for 77 d after which the HRT was reduced to 3.5 d. The amounts of ammonia removed from the influent by microorganisms sourced from the natural seed were 85% and 92% for the 7 d HRT and the 3.5 d HRT, respectively. The ammonia removals of microbial consortia from the commercial seed were 71% and 83% for these HRTs respectively. The quantity of ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) was determined in the SBRs using the most probable number (MPN) technique. Both AOB and NOB increased in number over the long-term operation of both SBRs. According to quantitative fluorescence in situ hybridisation (FISH) probing, AOB from the natural seed and commercial seed comprised 21 +/- 2% and 30 +/- 2%, respectively of all bacteria. NOB could not be detected with currently-reported FISH probes, suggesting that novel NOB were enriched from both sources. Taken collectively, the results from this study provide an indication that the nitrifiers from shrimp farm sources are more effective at ammonia removal than those from commercial seed cultures.

Present state of food and feed cycle and accompanying issues around Japan.

Nitrogen (N) and phosphorus (P) cycles of food and feed, and the regulation systems for industrial wastewater are studied. The rate of domestic supply of food in Japan is 41% in 1970, 32% in 1990, and 29% in 1998 for N and 33%, 29%, and 28% for P, excluding grass feed. Among 3 countries, Japan, USA and Thailand, Japan is in the most difficult state to recycle food wastes to farmland. Comparing the estimated load of wastewater from food processing industries with the estimated load according to the budget study for N and P cycles, the tentative criteria of wastewater of food processing for P seem to be loose, and denitrification might be considered in every stage of N cycles. New regulation of industrial wastewater recently proposed in Japan may induce the chance for environmental business to remove N and P.

Screening and characterization of bioflocculant produced by isolated Klebsiella sp.

Sixteen strains of polymer-producing bacteria were isolated from the activated sludge samples taken from two seafood processing plants in Southern Thailand. Their culture broths possessed the ability to flocculate kaolin suspension in the presence of 1% CaCl2. Based on the flocculating activity, the strain S11 was selected and identified to be a Klebsiella sp. using the partial 16S rRNA sequencing method. The growth of the isolated Klebsiella sp. was maximal (1.026 g l-1 dry cell mass) after 1 day cultivation while the highest polymer yield (0.973 g l-1) was achieved after 5 days cultivation. The flocculating activity of the culture broth, however, was highest after 2 days cultivation. The polymer was identified to be an acidic polysaccharide containing neutral sugar and uronic acid as its major and minor components, respectively. Results on the properties of the partially purified polysaccharide from Klebsiella sp. S11 revealed that it consisted of galactose, glucose and mannose in an approximate ratio of 5:2:1. It was soluble in acidic or basic solutions but not in organic solvents. Its molecular mass was greater than 2 x 10(6) Da. Infrared spectra showed the presence of hydroxyl, carboxyl and methoxyl groups in its molecules. Differential scanning calorimetry of the polysaccharide indicated the crystalline melting point (Tm) at 314 degrees C. The optimum dosage of polysaccharide to give the highest flocculating activity was 15 mg l-1 in the presence of 1% CaCl2.

Anaerobic filter treatment of fishery wastewater.

Anaerobic treatment of wastewater from a selected seafood processing plant was conducted at organic loading rates (OLR) ranging from 0.3 to 1.8 kg chemical oxygen demand (COD)/m(3).day and hydraulic retention times (HRT) ranging from 36 to 6 days. COD reduction decreased with increasing OLR. More than 75% COD reduction could be maintained up to an OLR of about 1 kg COD/m(3).day with an HRT of 11 days. An OLR of 1.3 kg COD/m(3).day corresponding to an HRT of 6.6 days gave maximal biogas productivity of 1.5 m(3)/m(3).day or 1.3 m(3) biogas/kg COD with a 65% COD reduction. If the HRT was kept constant at 11 days, an OLR of 1.3 kg COD/m(3).day achieved maximal biogas productivity (1.1 m(3)/m(3).day) and yield (0.75 m(3)/kg COD) and a 60% COD reduction for treatment of tuna condensate.

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.

Optimization for growth of Rhodocyclus gelatinosus in seafood processing effluents.

Tuna condensate was a better substrate than shrimp-blanching water or effluent from a frozen-seafood plant for growing Rhodocyclus gelatinosus under anaerobic conditions in the light. One strain out of four examined, R7, gave the highest biomass (4.0 g/l), cell yield (0.32 g cell/g COD), and COD removal (78%) in 1:10 (v/v) diluted tuna condensate. Shrimp-blanching water added to the tuna condensate further increased growth rate, biomass and COD removal. Optimal growth was at pH 7.0 and 3000 Lux light intensity. Acetate, pyruvate, glucose, glutamate, propionate or malate added to the tuna condensate did not increase cell yield, carotenoid or bacteriochlorophyll content or biomass protein. A maximum cell mass of 5.6 g/l (containing 50% protein) and 86% COD removal were obtained after 5 days' incubation under optimal conditions.

Characterization of proteases produced by newly isolated and identified proteolytic microorganisms from fermented fish (Budu).

Eight different strains ofBacillus were isolated from fermented fish (Budu) and their proteolytic enzyme activities were determined after 18 h cultivation at room temperature (35° C). Four isolates possessed high protease activities. Optimum pH for these enzymes was between 7.0 and 8.0 and the optimal temperature was 55° C. The proteases retained 40% of their original activity after 20 min at 55° C but lost all activity at 65° C. Three of the four isolates were identified asBacillus subtilis, the fourth asBacillus licheniformis.

Production of cellulolytic enzymes from fungi and use in the saccharification of palm cake and palm fibre.

Aspergillus niger ATCC 6275 possesses the highest carboxymethyl-cellulase, xylanase and ß-glucosidase activities under liquid and solid cultivations compared withMyceliophthora thermophila IFO 31843 and an isolate, F11. Palm cake proved to be a better substrate for enzyme production and saccharification than palm fibre. Saccharification of these two substrates, using crude enzyme solutions from three fungi and commercial enzymes, was investigated.

Isolation and selection of cellulolytic fungi from palm oil mill effluent.

Cellulolytic fungi, 34 strains, were isolated from samples taken from palm oil mill residues and effluent, and high cellulase producers selected in comparison with nine known reference strains. Although 13 isolates showed good filter paper distintegration within 14 days, only eight isolates exhibited clearing zones around their colonies on carboxymethylcellulose (CMC) agar medium. Quantitative cellulase activity measurements, using CMC as carbon source, selected three of the eight isolates as potential cellulase producers. Using dried palm oil mill condensate as carbon source, only one of the isolates (F 11) showed similar results on both carbon sources. During media optimization for CMCase production, a four-fold increase from 0.058 to 0.275 U/ml was obtained using a medium, containing 0.1% (v/v) Tween 80 0.02% (w/v) NH4NO3, 0.025% (w/v) proteose-peptone and 0.1% (w/v) CMC dissolved in undiluted condensate from the sterilization of oil palm bunches, with an initial pH of 5.5.