Production of acylated homoserine lactones by Aeromonas and Pseudomonas strains isolated from municipal activated sludge

Up to now, the production and role of N-acyl homoserine lactones (AHLs) in activated sludge have been poorly understood. In this study, cross-feeding assays with the reporter strains Agrobacterium tumefaciens NTL4 and Chromobacterium violaceum CV026 were used to investigate AHL signal production by municipal activated sludge samples. AHL signal production was consistently detected from municipal activated sludge when different samples were incubated on nutrient media. From one municipal activated sludge sample, 10 strains producing AHL-like auto inducers were isolated by an overlay technique. 16S rDNA-based phylogenetic analysis showed that eight of the isolates belonged to Aeromonas spp. and two to Pseudomonas spp. Box-PCR indicated that six of these Aeromonas isolates were different strains and the two Pseudomonas strains were identical. The production of AHL or AHL-like compounds by these strains was confirmed by thin layer chromatography and biosensor overlays. The six different Aeromonas strains were found to produce the same set of AHLs, including N-hexanoyl-L-homoserine lactone. These results may indicate the possible presence of AHLs in municipal activated sludge. The potential roles of AHL in this eco system are briefly discussed.     

Dynamic modeling of nitrogen removal for a three-stage integrated fixed-film activated sludge process treating municipal wastewater

Bioprocess and Biosystems Engineering - The integrated fixed-film activated sludge (IFAS) process is being increasingly used to enhance nitrogen removal for former activated sludge systems. The aim...     

Production and characterization of PHB from two novel strains of Bacillus spp. isolated from soil and activated sludge

The present study reports two bacteria, designated 87I and 112A, which were isolated from soil and activated sludge samples from Hyderabad, India, and that are capable of producing poly-3-hydroxybutyrate (PHB). Based on phenotypical features and genotypic investigations, these microorganisms were identified as Bacillus spp. Their optimal growth occurred between 28°C and 30°C and pH 7. Bacillus sp. 87I yielded a maximum of 70.04% dry cell weight (DCW) PHB in medium containing glucose as carbon source, followed by 55.5% DCW PHB in lactose-containing medium, whereas Bacillus sp. 112A produced a maximum of 67.73% PHB from glucose, 58.5% PHB from sucrose, followed by 50.5% PHB from starch as carbon substrates. The viscosity average molecular mass (M _v) of the polymers from Bacillus sp. 87I was 513 kDa and from Bacillus sp. 112A was 521 kDa. All the properties of the biopolymers produced by the two strains 87I and 112A were characterized.     

Conjugal transfer of plasmid and chromosomal markers between strains of Thiobacillus versutus

In experiments on conjugation in Thiobacillus versutus with the use of pTAV1-less strains as recipients, we have proved that the derivative of the wild-type T. versutus cryptic plasmid pTAV1 (107 kb) marked with Tn1721 (Tc^r) transposon demonstrates Tra^- phenotype but can be mobilized for transfer by pSa Tra⁺ broad-host-range helper plasmid at a low frequency. The possibility of chromosomal gene exchange between different auxotrophic and drug-resistant T. versutus mutants has been confirmed. The previously assumed participation of plasmid pTAV1 in the above process must be excluded because conjugal transfer of chromosomal markers can be observed even when two pTAV1-free strains are mated. Formation of some classes of transconjugants can be reasonably explained only when two-directional chromosomal DNA transfer (retrotransfer) is considered. At this stage of our studies we can not propose any hypothesis on the mechanism of chromosomal gene transfer. The possible role of the megaplasmids discovered in T. versutus in chromosome mobilization needs to be elucidated.     

Characterization of Thiobacillus thioparus isolated from an activated sludge bioreactor used for hydrogen sulfide treatment

AIMS: To compare Thiobacillus thioparus population dynamics in a control and a test activated sludge (AS) bioreactor, used for hydrogen sulfide (H(2)S) degradation. METHODS AND RESULTS: Denaturing gradient gel electrophoresis (DGGE) was used to confirm the presence of T. thioparus, and real-time PCR was used to quantify the level of this bacterium in the AS samples. The DGGE analysis showed a band for T. thioparus in all samples, with the band being more prominent in the test sample with H(2)S diffusion. It also showed that although a change occurred in the diversity of the microbial population in the test sludge after 6 weeks of H(2)S diffusion, the microbial community structure of the test and control was still similar. Thiobacillus thioparus-specific PCR primers confirmed that 50% of the isolates from both the test and control bioreactors were T. thioparus. The thiobacilli population became more efficient at degrading the diffused H(2)S. This increase in efficiency was confirmed by a significant increase in the number of isolates from the test sludge compared with those from the control sludge, when they were grown in a thiosulfate-rich liquid medium. CONCLUSIONS: It was concluded that the use of AS process for H(2)S removal encourages the population of T. thioparus to increase even at times when the total biomass concentration shows a decrease. SIGNIFICANCE AND IMPACT OF THE STUDY: The research results give an insight into the dynamics of the microbial population in an AS pilot plant used in a dual role, to treat the wastewater and H(2)S.     

Mobilization of Thiobacillus ferrooxidans plasmids among Escherichia coli strains.

Nonconjugative Thiobacillus ferrooxidans plasmids were mobilized at high frequencies among Escherichia coli strains by the IncP plasmid RP4 and at low frequencies by the IncN plasmid R46, but not by the IncW plasmid pSa. The mobilization region of a nonconjugative T. ferrooxidans plasmid was located on a 5.3-kilobase T. ferrooxidans DNA fragment.     

Biofilm producing indigenous bacteria isolated from municipal sludge and their nutrient removal ability in moving bed biofilm reactor from the wastewater

In the present study, improved moving bed biofilm reactor (MBBR) was applied to enhance the nutrient removal ability of the municipal wastewater. A total of 18 indigenous bacterial isolates were screened from the sewage sludge sample and nitrate reductase, nitrite reductase and hydroxylamine oxidase was analyzed. The strains Pseudomonas aeruginosa NU1 and Acinetobacter calcoaceticus K12 produced 0.87 ± 0.05 U/mg and 0.52 ± 0.12 U/mg hydroxylamine oxidase, 1.023 ± 0.062 U/mg and 1.29 ± 0.07 U/mg nitrite reductase, and 0.789 ± 0.031 U/mg and 1.07 ± 0.13 U/mg nitrate reductase. Nitrogen and phosphate removal improved by the addition of nutrient sources and achieved > 80% removal rate. pH and temperature of the medium also affected nutrient removal and improved removal was achieved at optimum level (p < 0.05). MBBR was designed with R1 (aerobic), R2 and R3 (anoxic) reactors. MBBR reactors removed acceptable level phosphorus removal properties up to 7.2 ± 3.8%, 42.4 ± 4.6%, and 84.2 ± 13.1% in the R1, R2, R3 and R4 reactors, respectively. Denitrification rate showed linear relationship at increasing concentrations nitrogen content in the reactor and denitrification rate was 1.43 g NO₂-N /m²/day at 1.5 g NO₂-N /m²/day. Dehydrogenase activity was assayed in all reactors and maximum amount was detected in the aerobic biofilm reactor. Based on the present findings, MBBRs and the selected bacterial strains are useful for the degradation domestic wastewater with minimum working area.     

耐热脱氮菌株的分离鉴定及性能研究

[目的]为了解决高温的煤化工废水生物脱氮效率不高的技术难题。[方法]本研究从上海某能化集团有限公司的煤化工废水处理系统的活性污泥中筛选得到一株耐热氨氧化细菌A1和一株耐热反硝化细菌D1。[结果]通过形态学观察、生理生化特征及16S rRNA基因序列分析,菌株A1初步鉴定为Aquamicrobium ahrensii,菌株D1初步鉴定为Pseudomonas stutzeri。采用单因子优化实验研究发现,菌株A1和D1的最适生长温度分别高达42℃和40℃。在模拟实际废水处理的初始NH₄⁺-N浓度100 mg/L和42℃的条件下,构建了由菌株A1和D1 (W/W,20%/10%)组成的共培养物,探究该共培养物在不同pH和C/N对短程硝化反硝化脱氮及N₂O的释放效应。结果表明,该共培养物在42℃、pH 9.0–10.0和初始C/N为2:1时,处理模拟废水的氮素去除率达>99.0%,最大N₂O得率高达51.3%。[结论]本研究的结果可为高温煤化工废水的生物处理提供技术支撑及菌种储备,同时也为高温污水处理过程中N₂O的释放规律提供理论参考。     

Microbial degradation of nitrobenzene and mono-nitrophenol by bacteria enriched from municipal activated sludge.

Using a mixture of three mono nitrophenols as sole carbon, nitrogen and energy sources, mixed cultures were enriched from municipal activated sludge to degrade both nitrophenols and nitrobenzene. Bacterial growth and degradation rate could be increased by supplementing the medium with 0.1% YE. Microorganisms were isolated from the nitrophenols enrichment, and they were identified as strains of Comamonas testosteroni and Acidovorax delafieldii. These strains showed broad degradation ability toward nitrophenols and nitrobenzene.     

Biogas production from supernatant of hydrothermally treated municipal sludge by upflow anaerobic sludge blanket reactor

The supernatant of hydrothermally treated sludge was treated by an upflow anaerobic sludge blanket (UASB) reactor for a 550-days running test. The hydrothermal parameter was 170 °C for 60 min. An mesophilic 8.6 L UASB reactor was seeded with floc sludge. The final organic loading rate (OLR) could reach 18 kg COD/m³ d. At the initial stage running for 189 days, the feed supernatant was diluted, and the OLR reached 11 kg COD/m³ d. After 218 days, the reactor achieved a high OLR, and the supernatant was pumped into the reactor without dilution. The influent COD fluctuated from 20,000 to 30,000 mg/L and the COD removal rate remained at approximately 70%. After 150 days, granular sludge was observed. The energy balance calculation show that heating 1.0 kg sludge needs 0.34 MJ of energy, whereas biogas energy from the supernatant of the heated sludge is 0.43 MJ.     

Presence of two subunit types in ribulose 1,5-bisphosphate carboxylase from Thiobacillus intermedius.

Ribulose bisphosphate carboxylase (EC 4.1.1.39) has been purified to homogeneity from glutamate-CO2-thiosulfate-grown Thiobacillus intermedius by pelleting the protein from the 93,000 X g supernatant fluid followed by ammonium sulfate fractionation and sedimentation into a discontinuous sucrose density gradient. The molecular weight of the native protein approximated that of the higher plant enzyme (550,000) based on its relative electrophoretic mobility in polyacrylamide disc gels compared with that of standards of known molecular weight, including crystalline tobacco ribulose bisphosphate carboxylase. Sodium dodecyl sulfate electrophoresis in 12% polyacrylamide disc gels and Sephadex G-100 chromatography in the presence of sodium dodecyl sulfate indicated that the purified Thiobacillus protein, like the tobacco enzyme, consisted of two types of nonidentical subunits. The molecular weights of the large and small subunits were estimated to be about 55,000 and 13,000, respectively, by means of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The carboxylase activity of the protein purified from spinach leaves and T. intermedius responded similarly to the effectors reduced nicotinamide adenine dinucleotide phosphate and 6-phosphogluconate. Contrary to a previous report (K. Purohit, B. A. McFadden, and A. L. Cohen, J. Bacteriol. 127:505-515, 1976), these results indicate that ribulose bisphosphate carboxylase purified from Thiobacillus intermedius closely resembles the higher plant enzyme with respect to quaternary structure, molecular weight, and regulatory properties.     

Methane recovery from the anaerobic codigestion of municipal sludge and FOG

The anaerobic biodegradability of a mix of municipal primary sludge (PS), thickened waste activated sludge (TWAS) and fat, oil, and grease (FOG) was assessed using semi-continuous feed, laboratory-scale anaerobic digesters operated at mesophilic (35 °C) and thermophilic (52 °C) temperature. Addition of a large FOG fraction (48% of the total VS load) to a PS + TWAS mix, resulted in 2.95 times larger methane yield, 152 vs. 449 mL methane @ STP/g VS added at 35 °C and 2.6 times larger methane yield, 197 vs. 512 mL methane @ STP/g VS added at 52 °C. The high FOG organic load fraction was not inhibitory to the process. The results of this study demonstrate the benefit of sludge and FOG codigestion.     

ROUTES: innovative solutions for municipal sludge treatment and management

Management of sewage sludge is going to be a big challenge in near future due to rapid urbanization and economic growth, in particular in Europe. The Routes project (ref. # 265156) is addressed to discover new routes in wastewater and sludge treatment which allow: (a) to prepare sludge for agricultural utilization by transforming it in a very clean and stabilized product with respect to hygienic aspects and phytotoxicity; (b) to minimize sludge production by new solutions including the use of innovative processes based on metabolic uncoupling or of innovative reactors like microbial fuel cells or sequencing batch biofilter granular reactor; (c) to promote recovery of valuable materials from anaerobic digestion, i.e. biopolymers as polyhydroxyalkanoates and fertilizers; (d) to set up and prove at practical scale a novel technique for sludge disposal (wet oxidation) as sustainable alternative to the nowadays the most used incineration; (e) to minimize energy pumping by adjusting solid concentration, on a practical installation. The general objective of the Routes proposal is therefore to set up a portfolio of different solutions to be applied in different conditions and circumstances, strictly following the waste hierarchy of the EU Directive 08/98 on waste. The above solutions will be studied either in the laboratory or at practical scale, depending on the maturity of the technology, in order to provide the Commission, the technical and scientific community and end-users with applicable solutions and new routes for sludge management.     

In-vitro screening ofSaccharomyces strains for ochratoxin A removal from liquid medium

The aptitude of twentySaccharomyces sensu stricto strains to remove ochratoxin A from a synthetic medium containing 1.1 ng/mL, about half of the European Community limit, was evaluated using four to six mg of biomass (wet weight)/mL. Seven satins show high levels of ochratoxin A removal, 0.72-1.10 ng/mL, equivalent to 66–100% of the available toxin, and unitary removing ativity of 14.31–27.24 pg/mg of biomass. Further research will be carried out to study the mechanism of OTA removal and to confirm the ability of the most efficacious strains ofSaccharomyces sensu stricto to remove OTA from contaminated wort and grape must during alcoholic fermentation.     

A comparison between two strains from Japan and Europe of Aphidius ervi

A European strain of Aphidius ervishall be introduced into Japan for the control of Macrosiphum euphorbiaeon greenhouse tomatoes. We compared this strain, which is originating from The Netherlands (EVP), with a native strain from Hokkaido, Northern Japan (SPR) in several characteristics. The percentage of parasitism of SPR and EVP was significantly different, when attacking three aphid species: 13% and 30% on M. euphorbiaeon tomato, 68% and 93% on Acyrthosiphon pisumon broad bean and 10% and 42% on Aulacorthum solanion sweet pepper, respectively. SPR and EVP differed morphologically (in the colouration of the petiole) and in the esterase banding patterns. Furthermore, cross mating experiments showed that SPR and EVP are partially reproductively isolated from each other. Complete reproductive isolation is evident in the one direction cross (EVP females and SPR males).     

Isolation of bacteria producing polyhydroxyalkanoates (PHA) from municipal sewage sludge

Bacterial isolates from sludge samples collected at a local municipal sewage treatment plant were screened for bacteria producing polyhydroxyalkanoates (PHA). Initially Sudan black B staining was performed to detect lipid cellular inclusions. Lipid-positive isolates were then grown in a nitrogen limitation E2 medium containing 2% (w/v) glucose to promote accumulation of PHA before the subsequent staining with Nile blue A. The positive isolates were quantified initially with a u.v. spectrophotometer, for a very large number of isolates (105) and among them high PHA-producing isolates (15) were selected and were confirmed by gas chromatographic analysis. The GC analysis showed the polymers produced by 13 of the selected isolates to be polyhydroxybutyrate (PHB), and the remaining two isolates produced polyhydroxybutyrate-co-hydroxyvalerate (PHB-co-HV) copolymer. The proportion of the PHA-positive bacterial isolates showed variability in the number of PHA accumulators during various months. The correlation of PHB production with the cell dry weight (CDW) was found to be statistically significant. The metabolism of PHB in these selected 15 isolates was studied using the Nile blue A staining, which showed an initial increase in the fluorescence followed by a decline, on further incubation. All the selected 15 isolates were classified to genus level by studying their morphological and biochemical characteristics. There were seven Bacillus species, three Pseudomonas species, two Alcaligenes species, two Aeromonas species, and one Chromobacterium species.