Cultivable Bacterial Diversity Research Articles

Study on seed-borne cultivable bacterial diversity and antibiotic resistance of Poa pratensis L.

In order to study the difference of cultivable seed-borne bacterial diversity between commercial varieties and wild species of Poa pratensis L., and their antibiotic resistance to sulfadiazine, tetracycline, oxytetracycline, ciprofloxacin, gentamicin, oxytetracycline and rifampin. In this study, 60 bacterium isolates were isolated by dilution-coated plate method. Through 16S rRNA sequence analysis, 40 representative isolates with different morphological characteristics were identified and phylogenetic tree was constructed. The results of diversity analysis showed that the seed-borne bacterial diversity of commercial varieties was richer than that of wild species. The antibiotic resistance of the isolated bacterial strains was studied by agar dilution method, and it was concluded that the antibiotic resistance of the seed-borne bacteria carried by commercial varieties was stronger than that of the wild species. Finally, the biofilm formation ability and swimming motility of the bacterial strain were measured, and the correlation between the two and the antibiotic resistance of the bacterial strain was analyzed. The analysis showed that the antibiotic resistance of bacterial strains in Poa pratensis L. was significantly correlated with their swimming motility. In addition, the swimming motility of the bacterial strains was significantly correlated with the biofilm formation ability. It is worth mentioning that this is the first time to study the drug-resistant bacteria distributed in the seed-borne bacteria of Poa pratensis L.

Microencapsulation and in situ incubation methodology for the cultivation of marine bacteria.

Environmental microorganisms are important sources of biotechnology innovations; however, the discovery process is hampered by the inability to culture the overwhelming majority of microbes. To drive the discovery of new biotechnology products from previously unculturable microbes, several methods such as modification of media composition, incubation conditions, single-cell isolation, and in situ incubation, have been employed to improve microbial recovery from environmental samples. To improve microbial recovery, we examined the effect of microencapsulation followed by in situ incubation on the abundance, viability, and diversity of bacteria recovered from marine sediment. Bacteria from marine sediment samples were resuspended or encapsulated in agarose and half of each sample was directly plated on agar and the other half inserted into modified Slyde-A-Lyzer™ dialysis cassettes. The cassettes were incubated in their natural environment (in situ) for a week, after which they were retrieved, and the contents plated. Colony counts indicated that bacterial abundance increased during in situ incubation and that cell density was significantly higher in cassettes containing non-encapsulated sediment bacteria. Assessment of viability indicated that a higher proportion of cells in encapsulated samples were viable at the end of the incubation period, suggesting that agarose encapsulation promoted higher cell viability during in situ incubation. One hundred and 46 isolates were purified from the study (32–38 from each treatment) to assess the effect of the four treatments on cultivable bacterial diversity. In total, 58 operational taxonomic units (OTUs) were identified using a 99% 16S rRNA gene sequence identity threshold. The results indicated that encapsulation recovered greater bacterial diversity from the sediment than simple resuspension (41 vs. 31 OTUs, respectively). While the cultivable bacterial diversity decreased by 43%–48% after in situ incubation, difficult-to-culture (Verrucomicrobia) and obligate marine (Pseudoalteromonas) taxa were only recovered after in situ incubation. These results suggest that agarose encapsulation coupled with in situ incubation in commercially available, low-cost, diffusion chambers facilitates the cultivation and improved recovery of bacteria from marine sediments. This study provides another tool that microbiologists can use to access microbial dark matter for environmental, biotechnology bioprospecting.

Effect of Feedstock Type on Biostimulation Efficiency and Microbial Community Structure during Biochar-Facilitated Remediation of Petroleum Contaminated Soil

This study investigated the effect of plant feedstock– and animal feedstock–derived biochar at 10%w/w and 15%w/w amendment levels on the biostimulation efficiency and the cultivable microbial community in the soil during biochar-facilitated remediation of petroleum contaminated soil using standard techniques. Biostimulation was most effective with the animal-based biochar (ABB) treatment while total petroleum hydrocarbon (TPH) removal was greatest in the plant-based biochar (PBB) amended soil. Observed mean TPH levels on Day 60 ranged from about 7000 mg/kg – 7800 mg/kg for the PBB and 11000 mg/kg – 14000 mg/kg for ABB representing removal levels of roughly 51.0%, 57.7%, 72.4% and 73.7% in 10% ABB, 15% ABB, 10% PBB and 15% PBB amended contaminated soils respectively. The cultivable bacterial diversity for both feedstock types shifted from the combination of Firmicutes, Actinobacteria and Proteobacteria phyla at the onset of the study to predominantly Proteobacteria by the end of the study with a distinct reduction in diversity observed with increasing contact time. The dominant cultivable heterotrophic bacterial isolates were Bacillus spp., Pseudomonas spp. and Staphylococcus aureus for ABB and Pseudomonas spp., Klebsiella pneumoniae and Bacillus spp. for PBB. Amongst the cultivable hydrocarbon utilising bacteria obtained, Klebsiella pneumoniae, Pseudomonas spp. and Enterobacter spp. dominated. There were significant differences in TCHB and CHUB abundance and TPH removal efficiency between PBB and ABB amendments at 95% confidence interval. The study established that application of biochar effectively manages petroleum pollutants in soil by stimulating the proliferation and activities of relevant degradative species.

Psychrotrophic plant beneficial bacteria from the glacial ecosystem of Sikkim Himalaya: Genomic evidence for the cold adaptation and plant growth promotion

Commercial biofertilizers tend to be ineffective in cold mountainous regions due to reduced metabolic activity of the microbial inoculants under low temperatures. Cold-adapted glacier bacteria with plant growth-promoting (PGP) properties may prove significant in developing cold-active biofertilizers for improving mountain agriculture. With this perspective, the cultivable bacterial diversity was documented from the East Rathong glacier ecosystem lying above 3900 masl of Sikkim Himalaya. A total of 120 bacterial isolates affiliated to Gammaproteobacteria (53.33%), Bacteroidetes (16.66%), Actinobacteria (15.83%), Betaproteobacteria (6.66%), Alphaproteobacteria (4.16%), and Firmicutes (3.33%) were recovered. Fifty-two isolates showed many in vitro PGP activities of phosphate solubilization (9–100 µg/mL), siderophore production (0.3–100 psu) and phytohormone indole acetic acid production (0.3–139 µg/mL) at 10 °C. Plant-based bioassays revealed an enhancement of shoot length by 21%, 22%, and 13% in ERGS5:01, ERMR1:04, and ERMR1:05, and root length by 14%, 17%, 11%, and 22% in ERGS4:06, ERGS5:01, ERMR1:04, and ERMR1:05 treated seeds respectively. An increased shoot dry weight of 4–29% in ERMR1:05 and ERMR1:04, and root dry weight of 42–98% was found in all the treatments. Genome analysis of four bacteria from diverse genera predicted many genes involved in the bacterial PGP activity. Comparative genome study highlighted the presence of PGP-associated unique genes for glucose dehydrogenase, siderophore receptor, tryptophan synthase, phosphate metabolism (phoH, P, Q, R, U), nitrate and nitrite reductase, TonB-dependent receptor, spermidine/putrescine ABC transporter etc. in the representative bacteria. The expression levels of seven cold stress-responsive genes in the cold-adapted bacterium ERGS4:06 using real-time quantitative PCR (RT-qPCR) showed an upregulation of all these genes by 6–17% at 10 °C, and by 3–33% during cold-shock, which indicates the cold adaptation strategy of the bacterium. Overall, this study signifies the psychrotrophic bacterial diversity from an extreme glacier environment as a potential tool for improving plant growth under cold environmental stress.

Cultivable bacterial diversity of the canine dental plaque as a potential source of bacterial infections

Dental diseases are a persistent problem, not only in humans, but very often in companion animals as well. Aetiological agents of these diseases are the dental plaque bacteria. In the present study, we focused on identifying cultivable bacteria living in the dental plaque of dogs, specifically dogs suffering from the early stages of periodontal disease. Canine oral bacteria pose a risk to humans that get bitten by the dog, but they also have a zoonotic potential. Dental plaque samples were taken from five dogs of small breeds. Samples were cultured under aerobic and anaerobic conditions on several types of microbiological agars. All obtained and selected bacterial colonies were identified by PCR with universal primers for the 16S rRNA gene and the sequences of the 16S rRNA gene were compared with the sequences available in the GenBank database using BLASTn analysis. A total of 75 bacteria belonging to five phyla, predominantly to Firmicutes and Proteobacteria, were identified. The most frequent species was Pasteurella canis which was detected in all samples. In addition, representatives of the genera Actinomyces, Bacillus, Bacteroides, Corynebacterium, Frederiksenia, Fusobacterium, Haemophilus, Lactobacillus, Leucobacter, Neisseria, Ottowia, Porphyromonas, Pseudomonas, Staphylococcus, Stenotrophomonas and Streptococcus were detected in the samples. In the present study, a broad spectrum of bacteria in dental plaque samples, including canine periodontal pathogens such as Porphyromonas gulae or Porphyromonas macacae were identified. In addition, highly pathogenic bacteria, specifically Actinomyces hordeovulneris, Bacillus circulans, and Bacteroides pyogenes, which pose a serious risk to human health, were detected in samples.

Cultivable bacterial diversity associated with bromeliad roots from ironstone outcrops in central Brazil.

Studies on the bacterial diversity associated with wild plants are rare, especially on those that grow in association with bromeliads. In the present study, we isolated and identified epiphytic and endophytic bacteria from the roots of the bromeliads Dyckia excelsa, Dyckia leptostachya and Deuterocohnia meziana occurring in the "cangas" in the Pantanal from Mato Grosso do Sul State, Brazil. The epiphytic bacteria were isolated from washed roots, while the endophytic bacteria were isolated from surface disinfested roots. Bacterial representatives corresponding to each BOX-PCR fingerprint, as well as those that did not result in amplicons, were selected for 16S rDNA gene sequence analysis. The BOX-PCR data showed intrageneric and intraspecific diversity and could discriminate strains and identify their phenotypic characteristics. The 16S rDNA gene sequence and phylogeny analysis showed a higher occurrence of strains belonging to the genus Bacillus than Mycobacterium and Brevibacterium, which were found in lower numbers. Species from the Bacillus genus are well known for their sporulation capacity and longer survival in arid locations, such as the "cangas". This study clearly showed that the bromeliad species represent a vast reservoir of bacterial community diversity, and the cultivable strains represent a new source for biotechnological prospecting.

Clinical characteristics influence cultivable-bacteria composition in the meconium of Indonesian neonates

BackgroundMicrobial colonization of a neonate's gastrointestinal tract has significant perinatal and lifetime health consequences. However, information regarding the profile of meconium microbiota in neonates and the influence of clinical parameters are lacking in the Indonesian population. This study aimed to preliminary investigate the profile of cultivable bacterial diversity of meconium isolated from neonates born at Cipto Mangunkusumo Hospital (CMH), Jakarta. The cultivable bacteria were isolated from meconium samples and were then processed for cultivation and molecular identification. ResultsFourteen neonates were enrolled as described, i.e., seven hyperbilirubinemia (Hyp) and seven non-Hyp with ten neonates delivered by cesarean section (CS) and four others by vaginal route (VR), and with five exclusive breastfeeding (Ebf), four formula milk, and five combinations. Microbiological identification, molecular 16S rDNA PCR-Sanger sequencing, and PCA analysis of cultivable bacteria isolated from meconium showed Firmicutes' predominance (84.41%), with an abundant population of Staphylococcus, which consist of S. hominis, S. epidermidis, and S. haemolyticus. The influence of mode of delivery showed a lower diversity than the CS populates the VR, but their composition was similar. Concurrently, between feeding patterns, the genera profile did not show much difference; in the non-Ebf group, the total amount of Staphylococcus and Bacillus showed a higher amount but a less diverse. Interestingly, the non-Hyp group showed more abundant and diverse Staphylococcus than that of the Hyp group. In contrast, neonates diagnosed with NEC and proven sepsis showed the same pattern of Staphylococcus domination. ConclusionStaphylococcus predominated the composition of cultivable bacteria in neonates meconium. Due to the small sample size, only the hyperbilirubinemia parameter significantly influenced the profile, i.e., Staphylococcus's proportion (p = 0.037).

Bacterial profile of meconium of neonates born at National Referral Hospital Cipto Mangunkusumo, Jakarta, Indonesia

Microbial colonization of a neonate’s gastrointestinal tract has significant perinatal and lifetime health consequences, with some clinical outcomes that have been linked to differences in the diversity and composition of gut microbiota. The effort to engineer intestinal ecosystem has led us to preserve the cultivable commensal microbiota. Here we investigated the association of cultivable bacterial diversity of neonates meconium from Indonesian National Referral Hospital Cipto Mangunkusumo (NRHCM) with mode of delivery and feeding patterns, as well as hyperbilirubinemia. We performed a cross-sectional study of meconium and clinical data collected from 14 Indonesian neonates born at NRHCM. Culture-dependent identification of bacterial isolates was conducted by performing simultaneous microbiological and molecular 16S rDNA PCR-Sanger sequencing methods. Phylogenetic tree and principal components analysis were employed to determine the bacterial profile and their association with clinical characteristics and outcomes. Cultivable bacterial profile indicates the predominance of Firmicutes (84,41%), with an abundant population of Staphylococcus (53.24%) with top three most significant population present are, i.e. S. hominis (12.99%), S. epidermidis (11.68%), and S. haemolyticus (10.39%). Bacterial diversity was associated with mode of delivery which showed that vaginal route populated by lower diversity of cultivable bacteria but by fewer opportunistics one than that of cesarean, with Staphylococcus hominis dominates the population, whereas with feeding patterns showed that the exclusive breast-fed was most populated by Staphylococcus, whereas non-exclusive one shared the same proportion of Staphylococcus and Bacterioides. While, non-hyperbilirubinemia group showed more abundant and diverse Staphylococcus than that of the opposite group.

Cultivable bacterial diversity, physicochemical profiles, and toxicity determination of car wash effluents.

Carwash effluents contain potentially toxic chemical and microbiological pollutants which may pose public health and ecotoxicological threats if directly discharged into surface waters. This work was aimed at determining the microbiological, physicochemical, and toxicological parameters of carwash effluents. Toxicity assays were determined using whole effluent toxicity (WET) using Danio rerio and Daphnia pulex. For microbiological analysis, sample aliquots were spread plated onto R2A Agar for the isolation of heterotrophic bacteria followed by DNA extraction from axenic cultures for sequencing analysis. The pH of effluent samples lay in the alkaline range, and ranged from pH7 to pH10. Sample salinity ranged from 0.2 to 0.3g/Kg. Electrical conductivity values ranged from 274 to 554μS/cm. Concentrations of Co, Pb, and Ni were < 1mg/L in all samples while the concentrations of Cu ranged from 0.94 to 3.8mg/L and Zn from 1.15 to 3mg/L. Oil and grease concentrations ranged from 5 to 24mg/L. The concentrations of TPH-GRO were low at < 1mg/L in all samples. All the carwash effluents were categorised as acutely toxic, with ≥ 75% mortality recorded for both test organisms within the first 24h of exposure to the test solutions. Heterotrophic bacteria counts ranged from 2800 to 4600CFU/100ml. Sequencing analysis revealed that 57% of the isolates were closely related to Aeromonas species, with 43% closely related to Pseudomonas species. We conclude that carwash effluents are veritable sources of microbiological contaminants and potentially toxic chemical pollutants of public health and ecotoxicological concern.

Cultivable bacterial diversity and early plant growth promotion by the traditional organic formulations prepared using organic waste materials

Traditional organic formulations are widely used as a plant growth promoters; however, the knowledge on the microbial aspect of traditional organic formulations is still limited. The aim of this study was to illustrate the cultivable bacterial diversity of various traditional organic formulations and their potential for early plant growth promotion. Five different traditional organic formulations such as 100 % panchagavya, 33 % panchagavya, plant extract with native microorganisms, commercial organic fertilizer extract with two percent leaf soil extract and commercial organic fertilizer extract with 2 % yogurt were prepared and used in this study. The liquid fraction of these traditional organic formulations were used to analyze the beneficial effect on plant growth by seed treatment and foliar applications. Bacterial 16SrDNA analysis revealed that the isolates fell into forty-three different genera, which can be grouped into seven different classes, such as Alphaproteobacteria, Actinobacteria, Bacilli (or Firmibacteria), Betaproteobacteria, Cytophagia, Flavobacteriia and Gammaproteobacteria. Higher bacterial diversity was observed in cow dung followed by 33 and 100 % panchagavya. Radish and Chinese cabbage seed germination and growth were significantly improved by traditional organic formulations compared to control. The results of this study showed that the bacterial diversity changes depend on the type and concentration of ingredients used in traditional organic formulations. Substantial increase in plant growth by the traditional organic formulations indicates the suitability of using these organic preparations in eco-friendly agriculture.

Cultivable bacterial diversity and amylase production in two typical light-flavor Daqus of Chinese spirits

Culture-dependent methods and molecular techniques were used to simultaneously investigate the cultivable bacterial diversity and amylase production in two typical light-flavor Daqus of Chinese spirits. Eight bacteria were identified from Niulanshan Daqu (Bacillus licheniformis, Bacillus subtilis, Bacillus cereus, Bacillus sonorensis, Streptomyces albus, Bacillus atrophaeus, Bacillus tequilensis and Bacillus megaterium) and eight from Hongxing Daqu (B. licheniformis, B. subtilis, B. cereus, Bacillus thuringiensis, Bacillus altitudinis, Bacillus pumilus, Geobacillus stearothermophilus and Bacillus amyloliquefaciens). All of the bacterial isolates from the Hongxing Daqu could produce extracellular α-amylase with a maximum yield of 25.3 U/ml by B. cereus H17, whereas B. licheniformis H55 could produce a maximum glucoamylase yield of 41.6 U/ml. Some of the bacterial isolates from the Niulanshan Daqu could also produce extracellular α-amylase and glucoamylase. The maximum yield of 27.6 U/ml α-amylase was achieved by B. subtilis N3, and the maximum yield of 58.1 U/ml glucoamylase was achieved by B. cereus N25. Bacillus licheniformis, B. subtilis and B. cereus were not only the dominant bacteria, but also possessed high α-amylase and glucoamylase activities, which may play very important roles during fermentation.

ASSESSING THE POTENTIAL BACTERIAL ORIGIN OF THE CHEMICAL DIVERSITY IN CALCAREOUS SPONGES

The chemodiversity and cultivable bacterial diversity of temperate calcareous sponges were investigated in a time series of collection of two sponges, Leuconia johnstoni (Baerida, Calcaronea) collected from the northeastern Atlantic Ocean and Clathrina clathrus (Clathrinida, Calcinea) collected from the northwestern Mediterranean Sea, using combined chemical and microbiological approaches.Bacteria were visualized in tissue sections of these sponges with Gram staining and in situ hybridization. The sponge crude extracts revealed annually persistent biological activities against reference human pathogen strains: L. johnstoni extracts displayed antimicrobial activity against a Gram positive Staphylococcus aureus strain; C. clathrus extracts displayed a broad spectrum of antimicrobial activities against Gram positive S. aureus and Gram negative Escherichia coli strains as well as against the yeast Candida albicans. In order to determine the sponge or bacterial origin of the secondary metabolites isolated from the sponge holobiont, sponge cells and bacteria were separated via differential centrifugation of dissociated sponge suspensions and each fraction was screened for biological activity. The aerobic cultivable bacteria associated with these calcareous sponges were isolated, amounting to about 105 − 5 × 106 bacteria per sponge wet weight. Based on 16S rRNA sequence comparison, the bacterial community was found in both species to be composed mostly of Gamma-proteobacteria (60-80%) and Alpha-proteobacteria (15%), with a few Firmicutes, Actinobacteria and Bacteroidetes. A range of biologically active compounds were purified from crude extracts of sponge or from culture broths of bacterial isolates. Ecological implications for the host sponge are being discussed based on localization and abundance of the producing bacteria in situ in the sponge tissue and on the co-detection of the chemical fingerprint of the bacterial metabolites in the host extracts. Some bacterial compounds were shown to have a role as chemical mediators of interactions within the sponge-associated bacterial compartment.

Cultivable bacterial diversity and amylase production in three typical Daqus of Chinese spirits

SummaryBoth culture‐dependent method and molecular technique were firstly used to simultaneously investigate the cultivable bacterial diversity and amylase production in three typical Daqus of Chinese spirits. The results showed that both cultivable bacterial diversity and amylase production were obviously different. The species of nine bacteria from Deshan, nine from Baisha and six from Wuling Daqus were identified. The total bacterial strains of 17, 15 and 14, and 9, 16 and 10 could produce α‐amylase and glucoamylase, respectively, from the Daqus, and the enzyme yields were different. Bacillus licheniformis, Bacillus subtilis and Bacillus amyloliquefaciens not only were dominant bacteria in the Daqus, but also possessed high activities of α‐amylase and glucoamylase. By comparison, Bacillus cereus and Bacillus oleronius were found to be another predominant bacterial species and good producers of α‐amylase and glucoamylase in Deshan and Wuling Daqus, respectively.

Cultivable bacterial diversity along the altitudinal zonation and vegetation range of tropical Eastern Himalaya

The Northeastern part of India sprawls over an area of 262 379km2 in the Eastern Himalayan range. This constitutes a biodiversity hotspot with high levels of biodiversity and endemism; unfortunately, is also a poorly known area, especially on its microbial diversity. In this study, we assessed cultivable soil bacterial diversity and distribution from lowlands to highlands (34 to 3 990m.a.s.l.). Soil physico-chemical parameters and forest types across the different altitudes were characterized and correlated with bacterial distribution and diversity. Microbes from the soil samples were grown in Nutrient, Muller Hinton and Luria-Bertani agar plates and were initially characterized using biochemical methods. Parameters like dehydrogenase and urease activities, temperature, moisture content, pH, carbon content, bulk density of the sampled soil were measured for each site. Representative isolates were also subjected to 16S rDNA sequence analysis. A total of 155 cultivable bacterial isolates were characterized which were analyzed for richness, evenness and diversity indices. The tropical and sub-tropical forests supported higher bacterial diversity compared to temperate pine, temperate conifer, and sub-alpine rhododendron forests. The 16S rRNA phylogenetic analysis revealed that Firmicutes was the most common group followed by Proteobacreria and Bacteroidetes. Species belonging to the genera Bacillus and Pseudomonas were the most abundant. Bacterial CFU showed positive but insignificant correlation with soil parameters like pH (r=0.208), soil temperature (r=0.303), ambient temperature (r=0.443), soil carbon content (r=0.525), soil bulk density (r=0.268), soil urease (r=0.549) and soil dehydrogenase (r=0.492). Altitude (r=-0.561) and soil moisture content (r=-0.051) showed negative correlation. Altitudinal gradient along with the vegetation and soil physico-chemical parameters were found to influence bacterial diversity and distribution. This study points out that this is a biome with a vast reservoir of bacteria which decrease with increasing altitudes, and highlights the microbiological importance of the poorly studied Eastern Himalayan range, justifying efforts to explore the prevalence of novel species in the biome.

Microbial burden and diversity of commercial airline cabin air during short and long durations of travel

Total microbial burden and diversity associated with commercial airliner cabin air was assessed by molecular methods in 125 air samples from the business-class sections of 16 domestic and international flights. Viable microbial burden within these cabin air parcels constituted only 1–10% of the total microbial population and ranged from below detection limits to 1.2 × 104 cells m–3 as determined with a validated ATP-based technology. Cultivable bacterial diversity was almost entirely limited to Gram-positive bacteria such as Staphylococcus and Bacillus. In contrast, cloning and sequencing 16S rRNA gene directly from the samples without cultivation indicated a significantly broader diversity, as sequences representing more than 100 species, and encompassing 12 classes of bacteria, were retrieved in varying abundance. Sequences of proteobacterial and Gram-positive lineage were retrieved most frequently (58% and 31% of all clone sequences, respectively), with Gram-positive and α-proteobacterial sequences dominating international flight samples and β- and γ-proteobacterial sequences comprising the largest portion of those retrieved from domestic flights. Significant differences in bacterial load and diversity were noted between samples obtained on domestic and international flights. The disparities observed in microbial abundance and diversity further underscore the immense value of state-of-the art molecular assays in augmenting traditional culture-based techniques.Supplementary informationThe online version of this article (doi:10.1038/ismej.2008.11) contains supplementary material, which is available to authorized users.

Cultivable Bacterial Community from South China Sea Sponge as Revealed by DGGE Fingerprinting and 16S rDNA Phylogenetic Analysis

The cultivable bacterial communities associated with four South China Sea sponges-Stelletta tenuis, Halichondria rugosa, Dysidea avara, and Craniella australiensis in mixed cultures-were investigated by microbial community DNA-based DGGE fingerprinting and 16S rDNA phylogenetic analysis. Diverse bacteria such as alpha-, gamma-, delta-Proteobacteria, Bacteroidetes, and Firmicutes were cultured, some of which were previously uncultivable bacteria, potential novel strains with less than 95% similarity to their closest relatives and sponge symbionts growing only in the medium with the addition of sponge extract. According to 16S rDNA BLAST analysis, most of the bacteria were cultured from sponge for the first time, although similar phyla of bacteria have been previously recognized. The selective pressure of sponge extract on the cultured bacterial species was suggested, although the effect of sponge extract on bacterial community in high nutrient medium is not significant. Although alpha- and gamma-Proteobacteria appeared to form the majority of the dominant cultivable bacterial communities of the four sponges, the composition of the cultivable bacterial community in the mixed culture was different, depending on the medium and sponge species. Greater bacterial diversity was observed in media C and CS for Stelletta tenuis, in media F and FS for Halichondria rugosa and Craniella australiensis. S. tenuis was found to have the highest cultivable bacterial diversity including alpha-, gamma-, delta-Proteobacteria, Bacteroidetes, and Firmicutes, followed by sponge Dysidea avara without delta-Proteobacteria, sponge Halichondria rugosa with only alpha-, gamma-Proteobacteria and Bacteroidetes, and sponge C. australiensis with only alpha-, gamma-Proteobacteria and Firmicutes. Based on this study, by the strategy of mixed cultivation integrated with microbial community DNA-based DGGE fingerprinting and phylogenetic analysis, the cultivable bacterial community of sponge could be revealed effectively.

Cultivable Bacterial Diversity of Alkaline Lonar Lake, India

Aerobic, alkaliphilic bacteria were isolated and characterized from water and sediment samples collected in the winter season, January 2002 from alkaline Lonar lake, India, having pH 10.5. The total number of microorganisms in the sediment and water samples was found to be 10(2)-10(6) cfu g(-1) and 10(2)-10(4) cfu ml(-1), respectively. One hundred and ninety-six strains were isolated using different enrichment media. To study the bacterial diversity of Lonar lake and to select the bacterial strains for further characterization, screening was done on the basis of pH and salt tolerance of the isolates. Sixty-four isolates were subjected to phenotypic, biochemical characterization and 16S rRNA sequencing. Out of 64, 31 bacterial isolates were selected on the basis of their enzyme profile and further subjected to phylogenetic analysis. Phylogenetic analysis indicated that most of the Lonar lake isolates were related to the phylum Firmicutes, containing Low G+C, Gram-positive bacteria, with different genera: Bacillus, Paenibacillus, Alkalibacillus, Exiguobacterium, Planococcus, Enterococcus and Vagococcus. Seven strains constituted a Gram-negative bacterial group, with different genera: Halomonas, Stenotrophomonas and Providencia affiliated to gamma-Proteobacteria, Alcaligenes to beta-Proteobacteria and Paracoccus to alpha-Proteobacteria. Only five isolates were High G+C, Gram-positive bacteria associated with phylum Actinobacteria, with various genera: Cellulosimicrobium, Dietzia, Arthrobacter and Micrococcus. Despite the alkaline pH of the Lonar lake, most of the strains were alkalitolerant and only two strains were obligate alkaliphilic. Most of the isolates produced biotechnologically important enzymes at alkaline pH, while only two isolates (ARI 351 and ARI 341) showed the presence of polyhydroxyalkcanoate (PHA) and exopolysaccharide (EPS), respectively.

Cultivable bacterial diversity from the human colon

Knowledge of the composition of the colonic microbiota is important for our understanding of how the balance of these microbes is influenced by diet and the environment, and which bacterial groups are important in maintaining gut health or promoting disease. Molecular methodologies have advanced our understanding of the composition and diversity of the colonic microbiota. Importantly, however, it is the continued isolation of bacterial representatives of key groups that offers the best opportunity to conduct detailed metabolic and functional studies. This also permits bacterial genome sequencing which will accelerate the linkage to functionality. Obtaining new human colonic bacterial isolates can be challenging, because most of these are strict anaerobes and many have rather exact nutritional and physical requirements. Despite this many new species are being isolated and described that occupy distinct niches in the colonic microbial community. This review focuses on these under-studied yet important gut anaerobes.

Phylogenetic and functional diversity of the cultivable bacterial community associated with the paralytic shellfish poisoning dinoflagellate Gymnodinium catenatum

Gymnodinium catenatum is one of several dinoflagellates that produce a suite of neurotoxins called the paralytic shellfish toxins (PST), responsible for outbreaks of paralytic shellfish poisoning in temperate and tropical waters. Previous research suggested that the bacteria associated with the surface of the sexual resting stages (cyst) were important to the production of PST by G. catenatum. This study sought to characterise the cultivable bacterial diversity of seven different strains of G. catenatum that produce both high and abnormally low amounts of PST, with the long-term aim of understanding the role the bacterial flora has in bloom development and toxicity of this alga. Sixty-one bacterial isolates were cultured and phylogenetically identified as belonging to the Proteobacteria (70%), Bacteroidetes (26%) or Actinobacteria (3%). The Alphaproteobacteria were the most numerous both in terms of the number of isolates cultured (49%) and were also the most abundant type of bacteria in each G. catenatum culture. Two phenotypic (functional) traits inferred from the phylogenetic data were shown to be a common feature of the bacteria present in each G. catenatum culture: firstly, Alphaproteobacteria capable of aerobic anoxygenic photosynthesis, and secondly, Gammaproteobacteria capable of hydrocarbon utilisation and oligotrophic growth. In relation to reports of autonomous production of PST by dinoflagellate-associated bacteria, PST production by bacterial isolates was investigated, but none were shown to produce any PST-like toxins. Overall, this study has identified a number of emergent trends in the bacterial community of G. catenatum which are mirrored in the bacterial flora of other dinoflagellates, and that are likely to be of especial relevance to the population dynamics of natural and harmful algal blooms.