Isolation Of Novel Bacteria Research Articles

Approaches for attaining clean bacterial fractions from complex environmental samples

Marine bacteria have been targeted by industry and pharmaceutics as genetic resources for highly active enzymes or novel lead compounds. Although numerous techniques have been introduced to isolate useful bacteria from the environment, we are still highly dependent on the conventional direct cultivation method to attain pure cultures. However, efficient bacterial isolation is hindered by several factors, including the presence of impurities. In this work, to demonstrate the significance of removing impurities and their impact on bacterial isolation, we employed two approaches: dielectrophoresis (DEP) and fluorescent D-amino acids (FDAA). We successfully attained clean bacterial fractions applicable for downstream processing using these approaches, uniquely designed to identify bacteria based on their characteristics and features. The diversity of bacteria attained by both approaches was investigated using 16S rRNA sequencing and compared to that attained by the standard differential centrifugation method. In addition, the viability of the isolates was also determined via direct cultivation. As a result, the separation of bacteria from impurities allowed for the identification of novel and useful bacteria unique to each approach. Successful cultivation also suggested that both approaches were applicable for attaining viable bacteria. In conclusion, removing impurities to attain clean bacterial fractions promotes the isolation of novel bacteria and thus could aid in the successful isolation of useful bacteria within complex environmental samples.

Isolation of Novel Bacteria Including Rarely Cultivated Phyla, Acidobacteria and Verrucomicrobia, from the Roots of Emergent Plants by Simple Culturing Method.

A number of novel bacteria including members of rarely cultivated phyla, Acidobacteria and Verrucomicrobia, were successfully isolated from the roots of two emergent plants, Iris pseudacorus and Scirpus juncoides, by a simple culturing method. A total of 47.1% (66 strains) for I. pseudacorus and 42.1% (59 strains) for S. juncoides of all isolates (140 strains from each sample) were phylogenetically novel. Furthermore, Acidobacteria and Verrucomicrobia occupied 10.7% (15 strains) and 2.9% (4 strains) of I. pseudacorus isolates, and 2.1% (3 strains) and 3.6% (5 strains) of S. juncoides isolates, respectively, indicating that plant roots are attractive sources for isolating rarely cultivated microbes.

Isolation and Characterisation of Lignolytic Bacteria for Bioethanol Production

In the present century, usage of non-renewable hydrocarbon fuels for sufficing energy demands are not considered environmentally and ecologically sustainable. Second generation biofuel production as lignocellulosic bioethanol is considered a sustainable way for generating fuel. Bioethanol production is considered as an option among other thermo-chemical processes for providing energy in form of ethanol, preserving the ecosystem by reducing the pollutants emitted during fossil fuel combustion. The release of fermentable sugars had been a challenging issue due to the presence of lignin in plant cell walls. This paper focused its study on isolation of novel lignin degrading enzymes from bacteria present in the soil of Western Ghats of India as the region is unhabituated followed by its application for biodegradation of lignocellulosic biomass for bioethanol production. Much has been done for lignin degrading fungi but still there is not a single method of choice which can be applicable on industrial scale successfully. Hence, the present study focuses on the isolation of novel bacteria and their enzymes with lignolytic potential and their application in bioconversion of lignocellulosic biomass into ethanol.

Screening for genes coding for putative antitumor compounds, antimicrobial and enzymatic activities from haloalkalitolerant and haloalkaliphilic bacteria strains of Algerian Sahara Soils.

Extreme environments may often contain unusual bacterial groups whose physiology is distinct from those of normal environments. To satisfy the need for new bioactive pharmaceuticals compounds and enzymes, we report here the isolation of novel bacteria from an extreme environment. Thirteen selected haloalkalitolerant and haloalkaliphilic bacteria were isolated from Algerian Sahara Desert soils. These isolates were screened for the presence of genes coding for putative antitumor compounds using PCR based methods. Enzymatic, antibacterial, and antifungal activities were determined by using cultural dependant methods. Several of these isolates are typical of desert and alkaline saline soils, but, in addition, we report for the first time the presence of a potential new member of the genus Nocardia with particular activity against the yeast Saccharomyces cerevisiae. In addition to their haloalkali character, the presence of genes coding for putative antitumor compounds, combined with the antimicrobial activity against a broad range of indicator strains and their enzymatic potential, makes them suitable for biotechnology applications.

Effects of rhizobial inoculation, cropping systems and growth stages on endophytic bacterial community of soybean roots

The effects of rhizobial inoculation, soybean-maize intercropping and plant growth stages on the diversity of soybean root endophytic bacteria were evaluated. PCR-based terminal restriction fragment length polymorphism (T-RFLP) of 16S rRNA genes and analyses of cloned partial 16S rRNA gene libraries were used to study the endophytic bacterial communities. The root endophytic bacterial populations during the whole growing season was significantly affected by the three factors analyzed, with plant growth and rhizobial inoculation showing the highest and lowest impact. The alphaproteobacteria and betaproteobacteria were the most predominant endophytes of soybean roots. Some undefined bacterial lineages were also found, indicating that the root endophytic populations might constitute an important source for the isolation of novel bacteria. Bradyrhizobium liaoningense (used as inoculant) occupied most of the soybean nodules formed and Sinorhizobium americanum-related bacteria were detected as the main root endophytes. Results also suggest that bacteria might be transferred from nodules to roots (as endophytic bacteria) when the seeds become mature, which might be a possible process in the life cycle of rhizobia. Rhizobial inoculation and intercropping practices could increase the diversity of the soybean root endophytes, which might have a positive influence on the growth of soybean plants.

Improving the culturability of freshwater bacteria using FW70, a low-nutrient solid medium amended with sodium pyruvate

Bacterial culture based on the use of plate media is an effective method for investigating bacterial populations in the environment. To improve the culturability of bacteria from freshwater lakes, we developed a new medium, FW70, which contains sodium pyruvate, casamino acids, and lake water and is solidified using gellan gum. To test the importance of these components, we prepared a series of media in which one or more of the components was absent. Water was sampled 31 times from 3 Japanese lakes and was passed through a membrane filter (pore size = 0.45 microm) to remove fast-growing microbes before the water was spread onto the plates. In most cases, significantly larger numbers of bacterial colonies were detected on FW70 than on other media. Furthermore, to test the practicality of FW70, we compared it with standard nutrient agar and R2A agar. In all cases, the culturability was significantly greater on FW70 than on standard nutrient agar or R2A agar. Some isolates recovered by means of FW70 belonged to bacteria that had not previously been classified. These results suggest that FW70 improves the culturability of freshwater bacteria and can be used for the isolation of novel bacteria as a result of the filtration step.

Isolation of novel bacteria within the Chloroflexi capable of reductive dechlorination of 1,2,3‐trichloropropane

Two strictly anaerobic bacterial strains were isolated from contaminated groundwater at a Superfund site located near Baton Rouge, LA, USA. These strains represent the first isolates reported to reductively dehalogenate 1,2,3-trichloropropane. Allyl chloride (3-chloro-1-propene), which is chemically unstable, was produced from 1,2,3-trichloropropane, and it was hydrolysed abiotically to allyl alcohol and also reacted with the sulfide- and cysteine-reducing agents in the medium to form various allyl sulfides. Both isolates also dehalogenated a variety of other vicinally chlorinated alkanes (1,2-dichloropropane, 1,2-dichloroethane, 1,1,2-trichloroethane, 1,1,2,2- tetrachloroethane) via dichloroelimination reactions. A quantitative real-time PCR (qPCR) approach targeting 16S rRNA genes indicated that both strains couple reductive dechlorination to cell growth. Growth was not observed in the absence of hydrogen (H2) as an electron donor and a polychlorinated alkane as an electron acceptor. Alkanes containing only a single chlorine substituent (1-chloropropane, 2-chloropropane), chlorinated alkenes (tetrachlorothene, trichlorothene, cisdichloroethene, trans-dichloroethene, vinyl chloride) and chlorinated benzenes (1-chlorobenzene and 1,2- dichlorobenzene) were not dechlorinated. Phylogenetic analysis based on 16S rRNA gene sequence data showed these isolates to represent a new lineage within the Chloroflexi. Their closest previously cultured relatives are 'Dehalococcoides' strains, with 16S rRNA gene sequence similarities of only 90%.

Linking sequence to function in soil bacteria: sequence-directed isolation of novel bacteria contributing to soilborne plant disease suppression.

Microbial community profiling of samples differing in a specific ecological function, i.e., soilborne plant disease suppression, can be used to mark, recover, and ultimately identify the bacteria responsible for that specific function. Previously, several terminal restriction fragments (TRF) of 16S rRNA genes were statistically associated with damping-off disease suppression. This work presents the development of sequence-based TRF length polymorphism (T-RFLP)-derived molecular markers to direct the identification and isolation of novel bacteria involved in damping-off pathogen suppression. Multiple sequences matching TRF M139 and M141 were cloned and displayed identity to multiple database entries in the genera incertae sedis of the Burkholderiales. Sequences matching TRF M148, in contrast, displayed greater sequence diversity. A sequence-directed culturing strategy was developed using M139- and M141-derived markers and media reported to be selective for the genera identified within this group. Using this approach, we isolated and identified novel Mitsuaria and Burkholderia species with high levels of sequence similarity to the targeted M139 and M141 TRF, respectively. As predicted, these Mitsuaria and Burkholderia isolates displayed the targeted function by reducing fungal and oomycete plant pathogen growth in vitro and reducing disease severity in infected tomato and soybean seedlings. This work represents the first successful example of the use of T-RFLP-derived markers to direct the isolation of microbes with pathogen-suppressing activities, and it establishes the power of low-cost molecular screening to identify and direct the recovery of functionally important microbes, such as these novel biocontrol strains.

Isolation of novel bacteria, including a candidate division, from geothermal soils in New Zealand

We examined bacterial diversity of three geothermal soils in the Taupo Volcanic Zone of New Zealand. Phylogenetic analysis of 16S rRNA genes recovered directly from soils indicated that the bacterial communities differed in composition and richness, and were dominated by previously uncultured species of the phyla Actinobacteria, Acidobacteria, Chloroflexi, Proteobacteria and candidate division OP10. Aerobic, thermophilic, organotrophic bacteria were isolated using cultivation protocols that involved extended incubation times, low-pH media and gellan as a replacement gelling agent to agar. Isolates represented previously uncultured species, genera, classes, and even a new phylum of bacteria. They included members of the commonly cultivated phyla Proteobacteria, Firmicutes, Thermus/Deinococcus, Actinobacteria and Bacteroidetes, as well as more-difficult-to-cultivate groups. Isolates possessing < 85% 16S rRNA gene sequence identity to any cultivated species were obtained from the phyla Acidobacteria, Chloroflexi and the previously uncultured candidate division OP10. Several isolates were prevalent in 16S rRNA gene clone libraries constructed directly from the soils. A key factor facilitating isolation was the use of gellan-solidified plates, where the gellan itself served as an energy source for certain bacteria. The results indicate that geothermal soils are a rich potential source of novel bacteria, and that relatively simple cultivation techniques are practical for isolating bacteria from these habitats.

Isolation of novel bacteria and actinomycetes using soil-extract agar medium

Novel bacteria were discovered using an isolation technique consisting of (i) selection of microorganisms that grew on soil-extract agar medium, but not on conventional media, and (ii) detection of small microbial colonies with a microscope. Three bacterial strains thus isolated were provisionally designated Shinshu-th1, -th2, -th 3, and five actinomycete strains, Shinshu-MS-01, -02, -03, -04, -05, respectively. Sequence analysis of their 16S rDNA showed that th1 had 95--96% homology with three unculturable bacteria, and th2 had 96% similarity to Bradyrhizobium sp., one unculturable and one unidentified bacterial strain. A phylogenetic study indicated that both strains were alpha-Proteobacteria belonging to the order Rhizobiales and the family Bradyrhizobiaceae. Since they had low homology (96%) with their close relatives, it is possible that th1 and th2 belong to a new genus. The actinomycetes Shinshu-MS-02 and -03 had 95--96% homology with four strains of Actinomadura, -04 had 95--96% similarity to Streptosporangium and Microbispora, and -05 had 97--98% homology with three strains of Acrocarpospora, Herbidospora and Planotetraspora. According to the phylogenetic study, both 02 and 03 are possibly new species of Actinomadura, -04 of Streptosporangium, and -05 of Acrocarpospora. Shinshu-th 3 and -MS-01 were identified as Mycobacterium cookii and Frankia sp., respectively, having 99% homology with these species.