The application of fluorescence in situ hybridization (FISH) technique for studying the microbial communities in intestinal tissues of white shrimp (Penaeus vannamei)

Fluorescence in situ hybridization technique is very useful for the evaluation of microbial communities in various environments. It is possible to apply this technique to study the intestinal microflora in white shrimp (Penaeus vannamei). Different fixatives and storage temperature were tested in this technique. It was found that fixation with 10% buffered formalin for 12 hours and changed to 70% ethanol shown positive results when compared to the fixation with Davidson's fixative or RF fixative. The best signaling was obtainedfrom the samples which were stored in -20ºC. By using the DNA probe targeted to the Eubacteria domain (EUB338 probe, 5′-GCT GCC TCC CGT AGG AGT-3′) labeled with fluorescein as a hybridizing probe, it was found that most intestinal microflora were aggregated with the intestinal contents, or dispersed in the lumen. There was not evidence of the attachment of the microflora with the intestinal epithelium in this study

Biological nutrient removal efficiency in treatment of saline wastewater

There is very little known about the effectiveness of wastewater treatment systems for saline wastewater generated by seafood processing industries, aquaculture and tourism activities. In particular, the effect of salinity on nitrogen and phosphorus removal in wastewater treatment processes is not well understood. Therefore we devised experiments to examine the treatment of highly saline wastewater, by using artificial seafood processing wastewater, for removal of nitrogen and phosphorus. Lab scale sequencing batch reactors (SBR) were initially operated at low, and then at increasing salt levels, to determine the overall effects of salinity on the nutrient removal performance. The microbial populations during these experiments were monitored to determine the specific effect of salinity on the various bacterial groups responsible for nutrient removal. The methods used were whole cell probing with fluorescently labelled RNA-directed oligonucleotide probes. Experimental data showed that the SBRs achieved good biological nutrient removal (BNR) when salinity levels in the influent were low (0.03% to 0.2% NaCl) but showed difficulties with biological phosphorus removal at salinity levels of 0.5%. It was found that there was a dominance of Grampositive bacteria with a high mol% G+C in their DNA in the SBR treating wastewater with NaCl at 0.03% to 0.2%. The addition of acetate to improve BNR performance increased the proportion of bacteria from the beta Proteobacterial subclass. (C) 1999 IAWQ Published by Elsevier Science Ltd. All rights reserved

Optimization of ethanol production from food waste hydrolysate by co-culture of Zymomonas mobilis and Candida shehatae under non-sterile condition

A complete conversion of the hexose and pentose sugars in the food wastes hydrolysates (FWH) to ethanol is a prerequisite for maximizing the profitability of an industrial process for bioethanol production. Response surface methodology (RSM) was employed to optimize the effects of nitrogen source [(NH4)2SO4], phosphorus source (KH2PO4), yeast extract and inoculum size on ethanol production from FWH by co-culture of Zymomonas mobilis and Candida shehatae under non-sterile condition. The optimal conditions for ethanol production were 1.15 g/L of (NH4)2SO4, 0.95 g/L of KH2PO4, 1.38 g/L of yeast extract and 14.75%v/v of inoculum. The results indicate that the most significant parameters affecting ethanol production from FWH by co-culture under non-sterile condition was yeast extract. Ethanol production of 77.6 g/L obtained under optimized condition was 56% increased as compared with the use of raw FWH (34 g/L) and was in good agreement with the value predicted by quadratic model (79.98 g/L), thereby confirming its validity. Ethanol yield of FWH in batch fermentation by co-culture was 0.15 g-ethanol/g-food waste (77.6 g/L), which was 94.6% of the theoretical yield while Z. mobilis alone yielded 0.11 g-ethanol/g-food waste (54.2 g/L) and C. shehatae alone yielded 0.09 gethanol/ g-food waste (48 g/L). Ethanol production from FWH in 1-L fermentor by co-culture also gave ethanol yield of 0.16 g-ethanol/g-food waste (78.8 g/L) which was 96% of the theoretical yield. Despite of being a waste, an ethanol yield of 0.16 g-ethanol/g-food waste demonstrated the potential of food waste as a promising biomass resource for ethanol production.Keywords: Co-culture, food waste hydrolysates, non-sterile fermentation, response surface methodology, optimization

Bacterial community from gut of white shrimp, Penaeus vannamei, cultured in earthen ponds

The Fluorescent in situ hybridization (FISH) technique and conventional method were used to analyse the bacterial community in the gut of white shrimp cultured in earthen ponds. Samples were collectedfrom three parts, hepatopancreas, anterior intestine and posterior intestine. Gut bacterial community was enumerated by 15 probes in FISH and 3 bacterial culture technique media. The results showed that bacteriaspecific probes determined bacterial community and Eubacteria as the dominant group of microbial community in the studied gut portions. β-Proteobacteria group (29.53±5.39%) and γ-Proteobacteria group (26.18±6.88%) were major groups of bacterial flora in the hepatopancreas. In contrast, low G+C gram positive bacteria group (LGC) was the most abundant group detected in anterior intestine (36.40±3.53%) andposterior intestine (30.32±4.63%). Vibrio spp. were detected very less in hepatopancreas (0.25±0.43%) and were present in 3 of 9 samples. In the case of bacterial detection using cultivation method, the number ofbacterial groups verified by TSA, TCBS and MRS showed high variation in every part of the studied digestive tract portions; however, no vibrio or lactic acid bacteria were present in the hepatopancreas ofhealthy shrimp. This study reveals the proportion of bacterial community in the digestive tract of white shrimp which can be used as important database for studying the change of the bacterial community in an abnormal condition including the efficiency of probiotics in the gut (in vivo) of white shrimp

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 percent and 92 percent for the 7 d HRT and the 3.5 d HRT, respectively. The ammonia removals of microbial consortia from the commercial seed were 71 percent and 83 percent 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 percent and 30 ± 2 percent, 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