An untapped bacterial cellulolytic community enriched from coastal marine sediment under anaerobic and thermophilic conditions

A bacterial community with strong cellulose [filter paper (FP) and microcrystalline cellulose] degradation ability was isolated from the coastal marine environment. They were isolated under thermophilic (60?°C) and anaerobic cultivation conditions. The library of 16S rRNA gene clones revealed a total of 16 operational taxonomic units after 50 clones were surveyed. Sixty percent of the clones were most related to the type strain of Clostridium thermocellum with 16S rRNA gene identity around 87-89%. All of them showed extremely low sequence similarities and were novel at least in species level. The gene clone libraries of glycosyl hydrolase family 48 showed low gene and amino acid sequence similarities around 70-72%. The results indicated that the cellulose degradation systems in the specific environment have not been well studied. The enrichment could disrupt FP within 3?days in a basal medium. The cellulase activity of the community was comparable to that of C.?thermocellum LQR1. The main fermentation products were ethanol, acetic acid and butyric acid. This work identified a novel microbial resource with a potential in lignocellulose conversion and biofuel production.     

Biodegradation of olive washing wastewater pollutants by highly efficient phenol-degrading strains selected from adapted bacterial community

The bacterial community of an olive washing water (OWW) storage basin was characterized, by both cultivation and cultivation-independent methods. PCR-TGGE fingerprints analysis of different samples, taken along the olive harvesting season, revealed important variations of the bacterial community structure showing rapid establishment of prevalent bacterial populations. Several bacteria, isolated from OWW, were cultivated, in media containing increasing amounts of polyphenols, in order to select high phenol-degrading strains for the effluent pollutants reduction. Strains PM3 and PM15, affiliated to Raoultella terrigena and Pantoea agglomerans by 16S rRNA gene sequencing, were selected and used for OWW biological treatment under batch conditions in shake flasks cultures. The OWW content of phenols, BOD₅, COD and colour, was reduced by 93, 91, 89 and 62%, respectively, permitting effluent disposal and/or reuse with no additional treatments.     

Effect of 2,4‐dinitrotoluene on the anaerobic bacterial community in marine sediment

Aims: To study the impact of added 2,4‐dinitrotoluene (DNT) on the anaerobic bacterial community in marine sediment collected from an unexploded ordnance dumping site in Halifax Harbour. Methods and Results: Marine sediment was spiked with 2,4‐DNT and incubated under anaerobic conditions in the presence and absence of lactate. Indigenous bacteria in the sediment removed 2,4‐DNT with subsequent formation of its mono‐ and diamino‐derivatives under both conditions. PCR–DGGE and nucleotide sequencing were used to monitor the change in the bacterial population in sediment caused by the presence of 2,4‐DNT. The results showed that denaturing gradient gel electrophoresis banding patterns of sediment microcosms treated with 2,4‐DNT were different from controls that did not receive 2,4‐DNT. Bacteroidetes, Firmicutes and δ‐Proteobacteria were present in sediment incubated in the absence of 2,4‐DNT. However, several γ‐Proteobacteria became dominant in sediment in the presence of 2,4‐DNT, two of which were 99% similar to Shewanella canadensis and Shewanella sediminis. In the presence of both 2,4‐DNT and lactate, two additional δ‐Proteobacteria were enriched, one closely related (98% similarity) to Desulfofrigus fragile and the other affiliated (96% similarity) to Desulfovibrio sp. In contrast, none of the above four Proteobacteria were enriched in sediment incubated with lactate alone. Conclusions: Presence of 2,4‐DNT led to a significant change in bacterial population of marine sediment with the enrichment of several γ‐ and δ‐Proteobacteria. Significance and Impact of the Study: Our results provided the first evidence on the impact of the pollutant 2,4‐DNT on the indigenous bacterial community in marine sediment, and provided an insight into the composition of bacterial community that degrade 2,4‐DNT.     

Interaction of neomycin with other antibiotics on selected bacterial strains

Antimicrobial combinations are used most frequently to provide broad-spectrum empirical coverage in the treatment of bacterial infections. However, combination of two antibiotics may not influence their activity, may lead to synergy or antagonism in the activity. Neomycin may be combined with one of the following antibiotics: ampicillin, procaine penicillin, gramicidin, bacitracin, polymyxin B, lincomycin, oxytetracycline, and erythromycin in some human and veterinary multiantibiotic drugs distributed in Poland. The checkerboard method has been one of the traditional assays for the measurement of antibiotic interactions. The aim of this study was to analyse the activity interaction of neomycin with second antibiotic in multiantibiotic drugs distributed in Poland on standards and clinical bacterial strains. Checkerboard results for all strains demonstrated synergism for 2.5% of combinations, only for standards strains. In one case Salmonella Enteritidis, in combination of neomycin with bacitracin, inhibition effect was observed. Additive effects were predominant--49%. In 18% neutral effects were shown, but in 26% of combinations FIC indexes were not possible to calculate, because of the resistance of clinical strains to the highest concentration of at least one antibiotic. In combination of aminoglycoside (neomycin) with beta-lactams antibiotics (ampicillin, procaine penicillin) in vitro, no synergy was observed for all examined strains. The best results were achieved for combinations of neomycin with peptide antibiotics (polymyxin, gramicidin and bacitracin)--5 for all 6 synergy effect observed.     

Volcanic ash supports a diverse bacterial community in a marine mesocosm

Shallow‐water coral reef ecosystems, particularly those already impaired by anthropogenic pressures, may be highly sensitive to disturbances from natural catastrophic events, such as volcanic eruptions. Explosive volcanic eruptions expel large quantities of silicate ash particles into the atmosphere, which can disperse across millions of square kilometres and deposit into coral reef ecosystems. Following heavy ash deposition, mass mortality of reef biota is expected, but little is known about the recovery of post‐burial reef ecosystems. Reef regeneration depends partly upon the capacity of the ash deposit to be colonised by waterborne bacterial communities and may be influenced to an unknown extent by the physiochemical properties of the ash substrate itself. To determine the potential for volcanic ash to support pioneer bacterial colonisation, we exposed five well‐characterised volcanic and coral reef substrates to a marine aquarium under low light conditions for 3 months: volcanic ash, synthetic volcanic glass, carbonate reef sand, calcite sand and quartz sand. Multivariate statistical analysis of Automated Ribosomal Intergenic Spacer Analysis (ARISA) fingerprinting data demonstrates clear segregation of volcanic substrates from the quartz and coral reef substrates over 3 months of bacterial colonisation. Overall bacterial diversity showed shared and substrate‐specific bacterial communities; however, the volcanic ash substrate supported the most diverse bacterial community. These data suggest a significant influence of substrate properties (composition, granulometry and colour) on bacterial settlement. Our findings provide first insights into physicochemical controls on pioneer bacterial colonisation of volcanic ash and highlight the potential for volcanic ash deposits to support bacterial diversity in the aftermath of reef burial, on timescales that could permit cascading effects on larval settlement.     

Kinetics of amino acids mineralization by a pond sediment bacterial community

Bacterial degradation of a high concentration of amino acids (up to 150 mg l^–1), released by quail manure pellets, was studied in a fertilized fish pond. Aspartic acid, Glutamic acid and Alanine were major components of the free amino acid pool (respectively 14.6%, 16.4% and 11.1%). Laboratory experiments showed a fast decomposition of these three amino acids. The degradation was a zero order reaction. Activation energy, Ea, was of the same order for the three amino acids (from 64.2 to 70.3 kg-mol^–1). There was no significant difference between the behaviour of aspartic acid in the presence or not of other carbon and nitrogen supply, but stimulation of degradation of glutamic acid and of Alanine by NH₄ ⁺ and C, was marked. This process may be important in the initial mineralisation of the fertilizer in fish ponds.     

Genetic relationships among strains of Xylella fastidiosa from RAPD-PCR data

Genetic relationships among 11 Xylella fastidiosa strains isolated from mulberry, almond, ragweed, grape, plum, elm, and citrus were determined by random amplified polymorphic DNA (RAPD). Twenty-two 10-base primers amplified a total of 77 discrete polymorphic bands. Phenetic analysis based on a similarity matrix corresponded well with previous reports on X. fastidiosa RFLP-based similarity relationships, indicating that RAPD-PCR amplification products can be used as a reliable indicator of genetic distance in X. fastidiosa. Cladistic analysis suggests the existence of five groups of X. fastidiosa: the citrus group, the plum-elm group, the grape-ragweed group, the almond group, and the mulberry group.     

三种益生菌混合培养及共生机理研究

确定酪酸菌、肠膜芽孢杆菌与粪肠球菌三种菌混合工艺.研究了三种菌混合培养的生长曲线,混合批式传49代、混合连续培养考察三种菌的共存稳定性.三种菌无细胞上清液彼此之间均有促进生长作用,混合培养液的菌数较单独培养增加,在稀释率0.042/h,填充床连续培养11d,三种菌可稳定共存,但批式传49代过程中,肠膜芽孢杆菌有消失现象,而酪酸菌、粪肠球菌均较传代前增加了100倍.平板打孔生长圈法、点种法实验分别表明酪酸菌、肠膜芽孢杆菌对粪肠球菌有显著的促进作用,连续培养较批式传代可更好的研究菌际关系.并得到简单易行的复方益生菌剂组方方法.     

Stable coexistence of five bacterial strains as a cellulose-degrading community

A cellulose-degrading defined mixed culture (designated SF356) consisting of five bacterial strains (Clostridium straminisolvens CSK1, Clostridium sp. strain FG4, Pseudoxanthomonas sp. strain M1-3, Brevibacillus sp. strain M1-5, and Bordetella sp. strain M1-6) exhibited both functional and structural stability; namely, no change in cellulose-degrading efficiency was observed, and all members stably coexisted through 20 subcultures. In order to investigate the mechanisms responsible for the observed stability, "knockout communities" in which one of the members was eliminated from SF356 were constructed. The dynamics of the community structure and the cellulose degradation profiles of these mixed cultures were determined in order to evaluate the roles played by each eliminated member in situ and its impact on the other members of the community. Integration of each result gave the following estimates of the bacterial relationships. Synergistic relationships between an anaerobic cellulolytic bacterium (C. straminisolvens CSK1) and two strains of aerobic bacteria (Pseudoxanthomonas sp. strain M1-3 and Brevibacillus sp. strain M1-5) were observed; the aerobes introduced anaerobic conditions, and C. straminisolvens CSK1 supplied metabolites (acetate and glucose). In addition, there were negative relationships, such as the inhibition of cellulose degradation by producing excess amounts of acetic acid by Clostridium sp. strain FG4, and growth suppression of Bordetella sp. strain M1-6 by Brevibacillus sp. strain M1-5. The balance of the various types of relationships (both positive and negative) is thus considered to be essential for the stable coexistence of the members of this mixed culture.     

Interaction of aminoglycosides and the other antibiotic on selected bacterial strains

The aim of this study was to analyse the activity interaction of aminoglycosides (gentamicin, kanamycin, streptomycin and dihydrostreptomycin) when combined with other antibiotics (lincomycin, benzylpenicillin, amoxicillin, cephalexin, spectinomycin and erythromycin), on selected clinical bacterial strains. The checkerboard method has been selected from the traditional assays for the measurement of antibiotic interaction. Checkerboard results for all strains demonstrated synergism for nine cases (9/112--8%). Additive effects were predominant--73/112--65.2%. In 12.5% neutral effects were shown, but in 11.6% of combinations FIC indexes were not possible to calculate, because of the resistance of clinical strains to the highest concentration of at least one antibiotic. The best results were achieved for combinations of dihydrostreptomycin with procaine penicillin because of higher number of cases synergy effect was observed. Antagonism of aminoglycosides and beta-lactams in case of gentamicin and amoxicillin for E. coli and E. cloacea strains were shown. Potential activity for combination of streptomycin and erythromycin was shown.     

再生水补水对河道底泥细菌群落结构的影响

以北京市永定河麻峪湿地再生水补水口附近河道底泥为研究对象,应用末端限制性片段长度多态性(Terminal Restriction Fragment Length Polymorphism,T-RFLP)技术探究再生水补水口附近底泥细菌群落结构的空间差异特征以及环境因子所产生的影响,并借助典范对应分析(Canonical Correspondence Analysis,CCA)方法分析麻峪湿地底泥细菌群落结构空间差异特征的形成原因。分析结果表明:河道底泥微生物群落对再生水的净化主要发生在距补水口1200m的范围内,随着再生水与上游来水径向混合净化渐变过程,在补水口2000m处河道底泥微生物群落结构与河道再生水补水口上游趋于相似。基于样点聚类结果的群落结构多样性分析表明随着再生水净化过程的实现微生物综合多样性指数呈现下降的趋势,从均匀度指数的变化趋势看,再生水补水口具有最高均匀度指数,补水口下游1200米处由于非优势菌群所占相对丰度最低以及偶见菌群缺失,导致群落结构比较单一、群落多样性和均匀度指数都最低。CCA方面分析结果表明再生水补水口上游细菌群落与因累积效应形成的重金属有密切关系,补水口出口底泥细菌群落则主要受到总磷和总有机碳的影响较大,而再生水补水与上游来水汇水径向渐变过程可能与氨氮净化具密切关系。研究区的主要优势菌种为假单胞菌属、贪食菌属和芽孢杆菌属,是与再生水中有机碳、氮降解具有密切关系的菌属。     

Naphthalene and biphenyl oxidation by two marine Pseudomonas strains isolated from Venice Lagoon sediment

A sediment sample from Venice Lagoon was found to be contaminated with 475 mg Kg⁻¹ polycyclic aromatic hydrocarbons (PAHs). Naphthalene was the principal pollutant at 26% of total PAHs. Two strains of Pseudomonas SN1 and SB1 were isolated from sediment amended with 2% naphthalene. 16S rRNA gene sequence analysis indicated that the two strains have about 99% nucleotide identity with strains of the genus Pseudomonas, and are very close to Pseudomonas stutzeri. Their metabolic profiles showed significant nutritional differences, the most significant of which was that SN1 grows in marine mineral medium spiked with naphthalene and SB1 grows with biphenyl as sole carbon and energy sources. Pseudomonas sp. SN1 had a doubling time of 3.1 h with 2% naphthalene and SB1 had a doubling time of 19.5 h with 2% biphenyl. Strain SN1 oxidised naphthalene at 564±32 mg O₂ l⁻¹ d⁻¹ and SB1 oxidised biphenyl at 426±25 mg O₂ l⁻¹ d⁻¹ in respirometry reaction vessels under controlled conditions. Screening of the two strains for dioxygenase genes involved in the first step of the two hydrocarbon degradation pathways, by polymerase chain reaction, showed naphthalene dioxygenase in SN1 and biphenyl dioxygenase in SB1. The strains each have a different catechol 2,3-dioxygenase responsible for cleavage of the aromatic ring.     

环境因素对东平湖沉积物细菌群落结构的影响

【目的】探讨环境因素对东平湖沉积物细菌群落结构的影响。【方法】应用T-RFLP(Terminal RestrictionFragment Length Polymorphism)技术分析和比较了6个不同位置的东平湖沉积物在丰水期和枯水期的细菌多样性,并结合不同样品环境因子的差异,采用主成分分析(PCA)和典型对应分析(CCA),探讨了环境因子对细菌多样性的影响。【结果】不同沉积物样品的T-RFLP图谱具有较高的相似性。除2号样品外,所有枯水期样品的细菌群落具有较高的丰富度、多样性、均匀度和较低的优势度。CCA结果表明,558bp T-RF的丰度与总磷、总氮、总有机碳、铵态氮和硝态氮含量呈负相关而与碳氮比和水深呈正相关;64.5、164、509和543bp T-RFs的丰度与总氮、总有机碳、铵态氮、硝态氮、碳氮比和水深呈正相关;而其它14种主要的T-RFs在不同样品间分布较为稳定受环境因子影响不大。90、136.5、138和488bp等T-RFs可能代表了东平湖沉积物中占优势地位的土著菌群。通过Phylogenetic Assignment Tool在线分析结果推测,东平湖沉积物中的优势菌群可能属于Firmicutes和Proteobacteria门。【结论】环境因素对东平湖沉积物的细菌多样性产生显著影响,但对其土著菌群的影响不大。     

A preliminary report of phylogenetic diversity of bacterial strains isolated from marine creatures

Bacterial diversity among marine creatures, especially molluscs, as a source for searching out novel lineages of bacteria, was studied. Marine creatures were collected at the coasts of the Kanto area in Japan. A total of 116 strains of bacteria were isolated from the intestines of 19 species of marine creatures includings molluscs, pisces and protochordata. Partial sequencing of 16S rDNA revealed that most of the isolates belonged to the gamma subclass of the Proteobacteria and Cytophaga-Flavobacterium-Bacteroides group. The BLAST searches revealed that the complete 16S rDNA sequence of 17 strains out of 116 isolates showed less than 94% similarity with 16S rDNA sequences deposited in the database. Four strains out of the 17 isolates belonged to the Rhodobacter group, 8 strains to the Alteromonas group, and the remaining 5 strains to the Cytophaga-Flavobacterium-Bacteroides group. Phylogenetic positions of 6 strains belonging to the Alteromonas group, which were isolated from different marine creatures, were close to each other, and represented a novel 16S rDNA lineage within the gamma subclass of Proteobacteria. Therefore, it may be inferred that these 6 strains belong to a new genus of Proteobacteria. Phylogenetic positions of the other strains are also independent from neighboring taxa, and they were suggested to respectively form a novel lineage. From these results, it is clear that the biodiversity of bacteria in marine creatures is much wider than was previously thought, and unknown microbiological resources are buried in these organisms.     

A mathematical model for the growth of bacterial microcolonies on marine sediment

Counts of bacterial microcolonies attached to deep-sea sediment particles showed 4-, 8-, 16-, and 32-celled microcolonies to be very rare. This was investigated with a mathematical model in which microcolonies grew from single cells at a constant growth rate (), detached from particles at constant rate (), and reattached as single cells. Terms for attachment of foreign bacteria (a) and death of single cells (d) were also included. The best method of fitting the model to the microcolony counts was a weighted least-squares approach by which(0.83 hour^–1) was estimated to be about 20 times greater than(0.038 hour^–1). This showed that the bacteria were very mobile between sediment particles and this mobility was explained in terms of attachment by reversible sorption. The implications of the results for the frequency of dividing cell method for estimating growth rates of sediment bacteria are discussed. The ratio of and was found to be very robust both in terms of the errors associated with the microcolony counts and the range of microcolony sizes used to obtain the solution.     

Characterization of bacterial strains capable of desulphurisation in soil and sediment samples from Antarctica

The presence of sulphur in fossil fuels and the natural environment justifies the study of sulphur-utilising bacterial species and genes involved in the biodesulphurisation process. Technology has been developed based on the natural ability of microorganisms to remove sulphur from polycyclic aromatic hydrocarbon chains. This biotechnology aims to minimise the emission of sulphur oxides into the atmosphere during combustion and prevent the formation of acid rain. In this study, the isolation and characterization of desulphurising microorganisms in rhizosphere and bulk soil samples from Antarctica that were either contaminated with oil or uncontaminated was described. The growth of selected isolates and their capacity to utilise sulphur based on the formation of the terminal product of desulphurisation via the 4S pathway, 2-hydroxybiphenyl, was analysed. DNA was extracted from the isolates and BOX-PCR and DNA sequencing were performed to obtain a genomic diversity profile of cultivable desulphurising bacterial species. Fifty isolates were obtained showing the ability of utilising dibenzothiophene as a substrate and sulphur source for maintenance and growth when plated on selective media. However, only seven genetically diverse isolates tested positive for sulphur removal using the Gibbs assay. DNA sequencing revealed that these isolates were related to the genera Acinetobacter and Pseudomonas.     

Occurrence of antibiotic and metal resistance and plasmids in Bacillus strains isolated from marine sediment

Eleven hundred Bacillus strains isolated from marine sediment from the Minas Basin, Nova Scotia, Canada, were purified on LB agar supplemented with ampicillin, chloramphenicol, erythromycin, streptomycin, tetracycline, or mercuric chloride. Seventy-seven isolates were examined for plasmid DNA, and for resistance to 11 antibiotics, HgCl2, and phenylmercuric acetate. Minimum inhibitory concentrations of Ag, Cd, Co, Cu, and Zn were also determined. Forty-three percent of antibiotic- and mercury-resistant strains contained one or more plasmids ranging from 1.9 to 210 MDa. Fifty-four percent carried plasmids greater than 20 MDa, and 97% were resistant to two or more metals. There was no correlation between plasmid content and resistance either to antibiotics or to mercurial compounds in these strains. Mercury-resistant isolates were unable to transform Hg2+ to volatile Hg0 by virtue of a mercuric reductase enzyme system (mer). Strains resistant to Hg2+ were investigated for their ability to produce H2S and intracellular acid-labile sulfide when grown in the absence and presence of HgCl2. Lower levels of H2S and intracellular sulfide were detected only in metal-resistant strains grown in the presence of HgCl2, suggesting that cellular sulfides complexed with Hg2+ in these strains.     

Impacts of cultivation of marine diatoms on the associated bacterial community

The composition of bacterial communities associated with four diatom species was monitored during isolation and cultivation of algal cells. Strong shifts in the associated communities, linked with an increase in the numbers of phylotypes belonging to members of the Gammaproteobacteria, were observed during cultivation.