Assessment of microbial diversity in effluent treatment plants by culture dependent and culture independent approaches

Microbial community structure of two distinct effluent treatment plants (ETPs) of pesticide and pharmaceutical industries was assessed and defined by (i) culture dependent and culture independent approaches on the basis of 16S rRNA gene sequencing, (ii) diversity index analysis - operational taxonomic units (OTUs). A total of 38 and 44 bacterial OTUs having 85-99% similarity with the closest match in the database were detected among pharmaceutical and pesticide sludge samples, respectively. Fifty percent of the OTUs were related to uncultured bacteria. These OTUs had a Shannon diversity index value of 2.09-2.33 for culturables and in the range of 3.25-3.38 for unculturables. The high species evenness values of 0.86 and 0.95 indicated the vastness of microbial diversity retrieved by these approaches. The dominant cultured bacteria indicative of microbial diversity in functional ETPs were Alcaligenes, Bacillus and Pseudomonas. Brevundimonas, Citrobacter, Pandoraea and Stenotrophomonas were specific to pesticide ETP and Agrobacterium, Brevibacterium, Micrococcus, Microbacterium, Paracoccus and Rhodococcus were specific to pharmaceutical ETP. These microbes can thus be maintained and exploited for efficient functioning and maintenance of ETPs.     

Characterization of diverse Acinetobacter isolates for utilization of multiple aromatic compounds

This study demonstrates the multiple catabolic capacities of lab isolates belonging to the genus Acinetobacter. Thirty-one Acinetobacter strains were screened initially for their capacity to utilize ten substrates that includes monocyclic, heterocyclic and polycyclic aromatic compounds. These bacteria were isolated from activated biomass of different effluent treatment plants (ETPs) treating wastewater generated at different industries and selected based on partial sequence data of the 16S rRNA gene. Of these 31 isolates, preliminary plate assay results showed eleven isolates that could utilize multiple substrates. Analytical studies demonstrated multiple degradation of hydrocarbons dibenzothiophene, fluorene, dibenzofuran, benzyl sulfide, and sodium benzoate by two isolates, HPC311 and HPC159.     

A <Emphasis Type="Italic">Bacillus subtilis</Emphasis> strain HPC248 from an effluent treatment plant with antimicrobial activity

This study reports the identification and demonstration of an organism with antimicrobial activity isolated from activated biomass of an effluent treatment plant (ETP) treating wastewater containing pesticides. While assessing the heterotrophic diversity of biomass collected from ETP, clear zones were observed on Luria Bertani plates. The bacterial isolate producing the zone as well as the bacterial cells surrounding the zone were isolated and purified by sub-culturing. Both isolates were identified by partial sequencing of the 16S rDNA clone. Presence of antimicrobial activity was demonstrated against various laboratory strains, isolated from different treatment plants and also against waterborne pathogens. The isolate that produced antimicrobial activity was identified as Bacillus subtilis strain HPC248 and the sensitive strain was identified as Bacillus sphaericus strain HPC249.     

Genetic and functional diversity of Bacillus strains in the soils long-term irrigated with paper and pulp mill effluent

The genetic and functional diversity of Bacillus and Bacillus-derived genera was analyzed in soil samples collected from three different fields near Century Paper Mill, Lal Kuan, Uttarakhand, India. Two had been subjected to concentrated and diluted effluent irrigation for the past 25 years and were designated as a concentrated effluent irrigated field (CEIF) and a dilute effluent irrigated field (DEIF), respectively. The field irrigated with fresh water was designated as a water irrigated field (WIF). Increase in pH, Na and Zn content and decrease in Fe content was observed due to effluent irrigation. The population count of Bacillus and Bacillus-derived was maximum in DEIF followed by WIF and CEIF. Variations in plant growth-promoting traits and extracellular enzymes were observed among the isolates from the three different field soils. Based on the amplified ribosomal DNA restriction analysis (ARDRA) with three restriction enzymes, all the selected 104 isolates were clustered into 14 groups. The sequencing of the representative isolates revealed that the majority belonged to the genus Bacillus, while three isolates belonged to Paenibacillus, Lysinibacillus and Orthinibacillus. There were a few species like Orthinibacillus contaminans, B. oleronius, B. safensis, B. methylotrophicus, B. stratosphericus, B. aryabhattai, B. asahii and B. bataviensis that were prevalent only in DEIF and CEIF but not in WIF field soil. The diversity index parameters like the Shannon Index, indices of species richness and species evenness based on biochemical profiling and ARDRA profiling revealed that Bacillaceae members in the fresh water irrigated field were diverse.     

Diversity of culturable actinobacteria isolated from marine sponge <Emphasis Type="Italic">Haliclona</Emphasis> sp.

This study describes actinobacteria isolated from the marine sponge Haliclona sp. collected in shallow water of the South China Sea. A total of 54 actinobacteria were isolated using media selective for actinobacteria. Species diversity and natural product diversity of isolates from marine sponge Haliclona sp. were analysed. Twenty-four isolates were selected on the basis of their morphology on different media and assigned to the phylum Actinobacteria by a combination of 16S rRNA gene based restriction enzymes digestion and 16S rRNA gene sequence analysis. The 16S rRNA genes of 24 isolates were digested by restriction enzymes TaqI and MspI and assigned to different groups according to their restriction enzyme pattern. The phylogenetic analysis based on 16S rRNA gene sequencing showed that the isolates belonged to the genera Streptomyces, Nocardiopsis, Micromonospora and Verrucosispora; one other isolate was recovered that does not belong to known genera based on its unique 16S rRNA gene sequence. To our knowledge, this is the first report of a bacterium classified as Verrucosispora sp. that has been isolated from a marine sponge. The majority of the strains tested belong to the genus Streptomyces and three isolates may be new species. All of the 24 isolates were screened for genes encoding polyketide synthases (PKS) and nonribosomal peptide synthetases (NRPS). PKS and NRPS sequences were detected in more than half of the isolates and the different "PKS-I-PKS-II-NRPS" combinations in different isolates belonging to the same species are indicators of their potential natural product diversity and divergent genetic evolution.     

Effect of Two Plant Species, Flax (Linum usitatissinum L.) and Tomato (Lycopersicon esculentum Mill.), on the Diversity of Soilborne Populations of Fluorescent Pseudomonads

Suppression of soilborne disease by fluorescent pseudomonads may be inconsistent. Inefficient root colonization by the introduced bacteria is often responsible for this inconsistency. To better understand the bacterial traits involved in root colonization, the effect of two plant species, flax (Linum usitatissinum L.) and tomato (Lycopersicon esculentum Mill.), on the diversity of soilborne populations was assessed. Fluorescent pseudomonads were isolated from an uncultivated soil and from rhizosphere, rhizoplane, and root tissue of flax and tomato cultivated in the same soil. Species and biovars were identified by classical biochemical and physiological tests. The ability of bacterial isolates to assimilate 147 different organic compounds and to show three different enzyme activities was assessed to determine their intraspecific phenotypic diversity. Numerical analysis of these characteristics allowed the clustering of isolates showing a high level (87.8%) of similarity. On the whole, the populations isolated from soil were different from those isolated from plants with respect to their phenotypic characteristics. The difference in bacteria isolated from uncultivated soil and from root tissue of flax was particularly marked. The intensity of plant selection was more strongly expressed with flax than with tomato plants. The selection was, at least partly, plant specific. The use of 10 different substrates allowed us to discriminate between flax and tomato isolates. Pseudomonas fluorescens biovars II, III, and V and Pseudomonas putida biovar A and intermediate type were well distributed among the isolates from soil, rhizosphere, and rhizoplane. Most isolates from root tissue of flax and tomato belonged to P. putida bv. A and to P. fluorescens bv. II, respectively. Phenotypic characterization of bacterial isolates was well correlated with genotypic characterization based on repetitive extragenic palindromic PCR fingerprinting.     

Study of biodiversity of <Emphasis Type="Italic">Klebsiella </Emphasis>sp.

Microorganisms provide the largest gamut of genetic and physiological diversity in nature. The determination of the biodiversity of microbes is necessary for the exploitation of their inestimable population, which remains unexplored in the environment. In our study, we have isolated 20 different Klebsiella sp. from contaminated soil and effluent treatment plants and determined their genetic diversity using molecular tools. The phylogenetic result based on 16S rDNA sequencing, shows genetic variation among the strains, which was further confirmed by Randomly Amplified Polymorphic DNA (RAPD) analysis. The occurrence of a broad spectrum of Klebsiella sp. variants in polluted and stress environments portrays the ability of this genus to survive abundantly under stress conditions.     

The isolation and characterisation of bacteria capable of accumulating silver

Summary The isolation of silver-resistant, silver-accumulating bacteria is reported. Following the screening of a number of environmental sources, silver-resistant Enterobacteriaceae were isolated from both sewage and photographic processing effluent. The level of resistance to silver and other heavy metals was determined for a selection of these isolates and, together with preliminary accumulation data derived from batch culture studies, one isolate, a strain of Citrobacter intermedius, was selected for further examination. The effect of silver concentration on batch culture growth of this organism was also investigated.     

Intraspecies diversity of Macrophomina phaseolina in Iran

To study the pathogenic and genetic diversity of the Macrophomina phaseolina in Iran, 52 isolates of the fungus were isolated from 24 host plants across the 14 Iranian provinces. All isolates were confirmed to the species based on the species-specific primers. The aggressiveness of M. phaseolina isolates was evaluated on the common bean. Based on the pathogenicity tests, M. phaseolina isolates from the different hosts displayed different levels of aggressiveness on the common beans. The results showed that there was significant variation in the aggressiveness of the pathogen; however, there was no distinct pattern of differentiation based on the host or geographical origin linked to the virulence of the isolates, as frequently theisolates from the same host or geographical origin had different levels of aggressiveness. Inter-simple sequence repeat (ISSR) markers were used to assess the genetic diversity of the fungus. The unweighted pair-group method, using arithmetic mean clustering of data, showed that isolates did not clearly differentiate to the specific group according to the host or geographical origins; however, usually the isolates from the same host or the same geographical origin tend to group nearly. Our results did not show a correlation between the genetic diversity based on the ISSR and pathogenic patterns on common bean in the greenhouse. Similar to the M. phaseolina populations in the other countries, the Iranian isolates were highly diverse based on the pathogenic and genotypic characteristics.     

Different portions of the maize root system host Burkholderia cepacia populations with different degrees of genetic polymorphism

In order to acquire a better understanding of the spatial and temporal variations of genetic diversity of Burkholderia cepacia populations in the rhizosphere of Zea mays , 161 strains were isolated from three portions of the maize root system at different soil depths and at three distinct plant growth stages. The genetic diversity among B. cepacia isolates was analysed by means of the random amplified polymorphic DNA (RAPD) technique. A number of diversity indices (richness, Shannon diversity, evenness and mean genetic distance) were calculated for each bacterial population isolated from the different root system portions. Moreover, the analysis of molecular variance ( amova ) method was applied to estimate the genetic differences among the various bacterial populations. Our results showed that, in young plants, B. cepacia colonized preferentially the upper part of the root system, whereas in mature plants, B. cepacia was mostly recovered from the terminal part of the root system. This uneven distribution of B. cepacia cells among different root system portions partially reflected marked genetic differences among the B. cepacia populations isolated along maize roots on three distinct sampling occasions. In fact, all the diversity indices calculated indicated that genetic diversity increased during plant development and that the highest diversity values were found in mature maize plants, in particular in the middle and terminal portions of the root system. Moreover, the analysis of RAPD patterns by means of the amova method revealed highly significant divergences in the degree of genetic polymorphism among the various B. cepacia populations.     

Diversity of cultivable endophytic fungi in two Rubia plants and their potential for production of anti-tumour Rubiaceae-type cyclopeptides

Rubia plants are one of the most important plant resources possessing significant commercial and medicinal values. Plant endophytes could benefit their host plants in different ways. Rubiaceae-type cyclopeptides (RAs), mainly isolated from Rubia plants, have attracted considerable attentions for their distinctive bicyclic structures and significant antitumor activities, but their contents in plants are low. The aim of this study is to investigate the diversity of endophytic fungi in Rubia plants and their potential for production of RAs. In this work, 143 endophytic fungi isolates were obtained from two Rubia plants. Phylogenetic analysis was performed based on the ITS rDNA sequences, and the isolates were classified into 29 genera. Among them, four endophytic fungal strains were found to produce anti-tumour RAs by LC-MS/MS analysis. This work successfully provides valuable knowledges of endophytic fungi microbiome in Rubia plants for agricultural and industrial applications, and exploits a new environmental-friendly resource of RAs.     

Diversity of endophytic fungal community associated with Piper hispidum (Piperaceae) leaves

Tropical and subtropical plants are rich in endophytic community diversity. Endophytes, mainly fungi and bacteria, inhabit the healthy plant tissues without causing any damage to the hosts. These fungi can be useful for biological control of pathogens and plant growth promotion. Some plants of the genus Piper are hosts of endophytic microorganisms; however, there is little information about endophytes on Piper hispidum, a medicinal shrub used as an insecticide, astringent, diuretic, stimulant, liver treatment, and for stopping hemorrhages. We isolated the fungal endophyte community associated with P. hispidum leaves from plants in a Brazilian forest remnant. The endophytic diversity was examined based on sequencing of the ITS1-5.8S-ITS2 region of rDNA. A high colonization frequency was obtained, as expected for tropical angiosperms. Isolated endophytes were divided into 66 morphogroups, demonstrating considerable diversity. We identified 21 isolates, belonging to 11 genera (Alternaria, Bipolaris, Colletotrichum, Glomerella, Guignardia, Lasiodiplodia, Marasmius, Phlebia, Phoma, Phomopsis, and Schizophyllum); one isolate was identified only to the order level (Diaporthales). Bipolaris was the most frequent genus among the identified endophytes. Phylogenetic analysis confirmed the molecular identification of some isolates to genus level while for others it was confirmed at the species level.     

The diversity of Escherichia coli serotypes and biotypes in cattle faeces

AIM: To study the diversity of commensal Escherichia coli populations shed in faeces of cattle fed on different diets. METHODS AND RESULTS: Thirty Brahman-cross steers were initially fed a high grain (80%) diet and then randomly allocated into three dietary treatment groups, fed 80% grain, roughage, or roughage + 50% molasses. Up to eight different E. coli isolates were selected from primary isolation plates of faecal samples from each animal. Fifty-two distinct serotypes, including nine different VTEC strains, were identified from a total of 474 E. coli isolates. Cattle fed a roughage + molasses diet had greater serotype diversity (30 serotypes identified) than cattle fed roughage or grain (21 and 17 serotypes identified respectively). Cluster analysis showed that serotypes isolated from cattle fed roughage and roughage + molasses diets were more closely associated than serotypes isolated from cattle fed grain. Resistance to one or more of 11 antimicrobial agents was detected among isolates from 20 different serotypes. Whilst only 2.3% of E. coli isolates produced enterohaemolysin, 25% were found to produce alpha-haemolysin. CONCLUSIONS: Diverse non-VTEC populations of E. coli serotypes are shed in cattle faeces and diet may affect population diversity. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides new information on the serotype diversity and phenotypic traits of predominant E. coli populations in cattle faeces, which could be sources of environmental contamination.     

A Study on the Prevalence of Bacteria that Occupy Nodules within Single Peanut Plants

In this study, bacteria hosted in root nodules of single plants of legume Arachis hypogaea L. (peanut) cv Tegua Runner growing at field were isolated. The collection of nodule isolates included both fast and slow growing strains. Their genetic diversity was assessed in order to identify the more frequently rhizobial strain associated to nodules from single plants. Molecular fingerprinting of 213 nodular isolates indicated heterogeneity, absence of a dominant genotype and, therefore, of a unique strains highly competitive. Efficient nitrogen-fixing isolates were identified as Bradyrhizobium sp. by phylogenetic analysis of the sequences of their 16S rRNA genes. The genetic diversity of 68 peanut nodulating isolates from all the collected plants was also analyzed. Considering their ERIC-PCR profiles, they were grouped in eighteen different OTUs for 60% similarity cut-off. Results obtained in this study indicate that the genetic diversity of rhizobia occupying nodules from single plant is very high, without the presence of a dominant strain. Therefore, the identification of useful peanut rhizobia for agricultural purposes requires strongly the selection, among the diverse population, of a very competitive genotype in combination with a high-symbiotic performance.     

Multiresistance, beta-lactamase-encoding genes and bacterial diversity in hospital wastewater in Rio de Janeiro, Brazil

Aims: To investigate the bacterial diversity, antimicrobial resistance patterns and types of beta‐lactamase genes in Gram‐negative bacteria isolated from a hospital sewage treatment plant. Methods and Results: Between July and December 2008, we collected samples from influent, clarifier tank effluent and chlorine contact tank effluent from a sewage treatment plant service of a hospital located in the city of Rio de Janeiro, Brazil. Of the 221 isolates identified, 40% were characterized as extended‐spectrum beta‐lactamase (ESBL) producers. Nonpathogenic micro‐organisms and some pathogenic genera were quantified. The most common ESBL‐producing isolates were Klebsiella pneumoniae, Enterobacter cloacae and Escherichia coli. The bla_TEM, bla_SHV and bla_CTX‐M genes were detected in 82, 48 and 67% of bacterial isolates, respectively. Conclusions: Results showed that hospital wastewater treatment plant is not suitable systems for the removal of all antibiotic‐resistant micro‐organisms present in hospital wastewaters. Significance and Impact of the Study: This study provides evidence that bacteria resistant to multiple antibiotics and their resistance genes that are usually present in the hospital can reach the environment, even after the use of hospital wastewater treatment plants.     

Characterization of Fusarium oxysporum Isolates from Common Bean and Sugar Beet Using Pathogenicity Assays and Random-amplified Polymorphic DNA Markers

Fusarium wilt is an economically important fungal disease of common bean and sugar beet in the Central High Plains (CHP) region of the USA, with yield losses approaching 30% under appropriate environmental conditions. The objective of this study was to characterize genetic diversity and pathogenicity of isolates of Fusarium oxysporum obtained from common bean and sugar beet plants in the CHP that exhibited Fusarium wilt symptoms. A total of 166 isolates of F. oxysporum isolated from diseased common bean plants were screened for pathogenicity on the universal susceptible common bean cultivar ‘UI 114’. Only four of 166 isolates were pathogenic and were designated F. oxysporum f.sp. phaseoli (Fop). A set of 34 isolates, including pathogenic Fop, F. oxysporum f.sp. betae (Fob) isolates pathogenic on sugar beet, and non‐pathogenic (Fo) isolates, were selected for random‐amplified polymorphic DNA (RAPD) analysis. A total of 12 RAPD primers, which generated 105 polymorphic bands, were used to construct an unweighted paired group method with arithmetic averages dendrogram based on Jaccard's coefficient of similarity. All CHP Fop isolates had identical RAPD banding patterns, suggesting low genetic diversity for Fop in this region. CHP Fob isolates showed a greater degree of diversity, but in general clustered together in a grouping distinct from Fop isolates. As RAPD markers revealed such a high level of genetic diversity across all isolates examined, we conclude that RAPD markers had only limited usefulness in correlating pathogenicity among the isolates and races in this study.     

Molecular epidemiological survey of Listeria monocytogenes in seafoods and seafood-processing plants

To evaluate the role of seafoods in the epidemiology of human listeriosis and the role of the processing environment as a source of Listeria monocytogenes in seafood products, 305 L. monocytogenes isolates were characterized by multilocus enzyme electrophoresis using 21 genetic loci and restriction enzyme analysis of total DNA. Forty-four isolates were recovered from patients in Norway; 93 were isolated from seafoods, seafood-processing environments, and seawater from 55 different producers; and the remaining 168 isolates originated from six seafood-processing plants and one transport terminal examined in detail for L. monocytogenes. The patient isolates fell into 11 electrophoretic types, with four of them being responsible for 77% of the listeriosis cases in 1992 to 1996. Isolates from Norwegian seafoods and processing environments showed great genetic diversity, indicating that seafoods and seafood-processing environments do not offer a niche for specific L. monocytogenes strains. On the other hand, isolates from individual processing plants were genetically more homogenous, showing that plants are likely to be colonized with specific subclones of L. monocytogenes. The isolation of identical subclones of L. monocytogenes from both human patients and seafoods, including ready-to-eat products, suggests that such products may have been possible sources for listeriosis cases in Norway.     

Correlation of functional instability and community dynamics in denitrifying dispersed-growth reactors

Understanding the relationship between microbial community dynamics and functional instability is an important step towards designing reliable biological water treatment systems. In this study, the community dynamics of two dispersed-growth denitrifying reactors were examined during periods of functional stability and instability. In both reactors during the period of functional instability, the effluent chemistry changed over time, with periods of high nitrate concentrations followed by periods of fluctuating nitrite concentrations. Community structure was examined by clone library analysis of the 16S rRNA gene. Community dynamics were investigated with terminal restriction fragment (T-RF) length polymorphism, and the functional diversity represented by T-RFs was assessed through nitrate reduction assays of representative isolates. During the period of functional instability, the community structure changed considerably, and the dynamics correlated significantly with effluent chemistry. The nitrite concentration was significantly correlated with the relative abundances of the nitrate-reducing Delftia- and Achromobacter-like T-RFs. The isolate representing the Acidovorax-like T-RF reduced nitrate directly to nitrogen in batch assays without the accumulation of any intermediates. The Acidovorax-like T-RF relative abundance was significantly negatively correlated with nitrite concentration, indicating that it was associated with good functional performance. The results of this study reveal a clear relationship between community dynamics and functional instability and the importance of diversity among nitrate-reducing populations within a denitrifying community.     

Proteomic assessment of host-associated microevolution in the fungus Thielaviopsis basicola

Thielaviopsis basicola, a soil-borne pathogen with a broad host range and a cosmopolitan distribution, is emerging as a major risk to sustainable cotton production in Australia. Previous studies suggested that host specialization has occurred making T. basicola an ideal model for a comparative proteomic analysis of strains isolated from different hosts. Elucidation of the genomic diversity and investigation of the functional differences in the Australian population could provide valuable information towards disease control. In this study, isolates of T. basicola were investigated for genomic (internal transcribed spacers region), proteomic and cotton virulence level variations. Internal transcribed spacers sequence analysis revealed that isolates are grouped based on host of origin irrespective of geographical origin. At the proteome level a degree of diversity was apparent and hierarchical clustering analysis of the data also demonstrated a close correlation between the proteome and the host of origin. LC-MS/MS analysis and identification using cross-species similarity searching and de novo sequencing of host-specific differentially expressed proteins and the virulence-correlated proteome allowed successful identification of 43 spots. The majority were found to be involved in metabolism. Spots that were correlated with host and virulence differences included a hypothetical protein with a Rossman-fold NAD(P)(+)-binding protein domain, glyceraldehyde-3-phosphate dehydrogenase, arginase and tetrahydroxynaphthalene reductase.     

南京小龙山钾矿区植物根际可培养细菌的遗传多样性分析

摘要:【目的】矿区优势植物可培养细菌生物多样性研究将有助于了解植物根际细菌与矿物,植物根系相互作用及对矿物风化和土壤形成的重要影响。【方法】采用纯培养法分离南京小龙山废钾矿区野生植物野塘蒿,千金子和栽培植物甘薯根内与根周围土壤的可培养细菌, 通过16S rDNA限制性酶切多态性分析(amplified rDNA restriction analysis, ARDRA)和16S rDNA序列分析研究了可培养细菌的多样性。【结果】分离纯化到60株具不同菌落形态的可培养细菌, 在60%相似水平上可分为18个OTU. 19株代表菌株分别属于3个门, 10个科, 11个属。多数菌株属于变形菌门(α-proteobacteria, 4株, 21.1%; β-proteobacteria, 2株, 10.5%; γ-proteobacteria, 6株, 31.6%)。假单胞菌属(Pseudomonas), 泛菌属(Pantoea)和根瘤菌属(Rhizobium)为优势种群。【结论】小龙山废矿区优势植物根围具有丰富的微生物种群多样性。