Soil microbial community analysis using terminal restriction fragment length polymorphisms

Terminal restriction fragment length polymorphism (T-RFLP) analysis is a polymerase chain reaction (PCR)-fingerprinting method that is commonly used for comparative microbial community analysis. The method can be used to analyze communities of bacteria, archaea, fungi, other phylogenetic groups or subgroups, as well as functional genes. The method is rapid, highly reproducible, and often yields a higher number of operational taxonomic units than other, commonly used PCR-fingerprinting methods. Sizing of terminal restriction fragments (T-RFs) can now be done using capillary sequencing technology allowing samples contained in 96- or 384-well plates to be sized in an overnight run. Many multivariate statistical approaches have been used to interpret and compare T-RFLP fingerprints derived from different communities. Detrended correspondence analysis and the additive main effects with multiplicative interaction model are particularly useful for revealing trends in T-RFLP data. Due to biases inherent in the method, linking the size of T-RFs derived from complex communities to existing sequence databases to infer their taxonomic position is not very robust. This approach has been used successfully, however, to identify and follow the dynamics of members within very simple or model communities. The T-RFLP approach has been used successfully to analyze the composition of microbial communities in soil, water, marine, and lacustrine sediments, biofilms, feces, in and on plant tissues, and in the digestive tracts of insects and mammals. The T-RFLP method is a user-friendly molecular approach to microbial community analysis that is adding significant information to studies of microbial populations in many environments.

Stable Isotope Probing:Linking Functional Activity to Specific Members of Microbial Communities

Linking organisms or groups of organisms to specific functions within natural environments is a fundamental challenge in microbial ecology. Advances in technology for manipulating and analyzing nucleic acids have made it possible to characterize the members of microbial communities without the intervention of laboratory culturing. Results from such studies have shown that the vast majority of soil organisms have never been cultured, highlighting the risks of culture-based approaches in community analysis. The development of culture-independent techniques for following the flow of substrates through microbial communities therefore represents an important advance. These techniques, collectively known as stable isotope probing (SIP), involve introducing a stable isotope-labeled substrate into a microbial community and following the fate of the substrate by extracting diagnostic molecular species such as fatty acids and nucleic acids from the community and determining which specific molecules have incorporated the isotope. The molecules in which the isotope label appears provide identifying information about the organism that incorporated the substrate. Stable isotope probing allows direct observations of substrate assimilation in minimally disturbed communities, and thus represents an exciting new tool for linking microbial identity and function. The use of lipids or nucleic acids as the diagnostic molecule brings different strengths and weaknesses to the experimental approach, and necessitates the use of significantly different instrumentation and analytical techniques. This short review provides an overview of the lipid and nucleic acid approaches, discusses their strengths and weaknesses, gives examples of applications in various settings, and looks at prospects for the future of SIP technology.

Co-immobilized microbial biosensor for BOD estimation based on sol-gel derived composite material

A novel type of biochemical oxygen demand (BOD) biosensor was developed for water monitor, based on co-immobilizing of Trichosporon cutaneum and Bacillus subtilis in the sol-gel derived composite material which is composed of silica and the grafting copolymer of poly (vinyl alcohol) and 4-vinylpyridine (PVA-g-P(4-VP)). Factors that influence the performance of the resulting biosensor were examined. The biodegradable substrate spectrum could be expanded by the co-immobilized microorganisms. The biosensor prepared also exhibited good reproducibility and long-term stability. Good agreement was obtained between the results of the sensor BOD measurement and those obtained from conventional BOD5 method for water samples.

Enrichment of the hydrogen-producing microbial community from marine intertidal sludge by different pretreatment methods

To determine the effects of pretreatment on hydrogen production and the hydrogen-producing microbial community, we treated the sludge from the intertidal zone of a bathing beach in Tianjin with four different pretreatment methods, including acid treatment, heat-shock, base treatment as well as freezing and thawing. The results showed that acid pretreatment significantly promoted the hydrogen production by sludge and provided the highest efficiency of hydrogen production among the four methods. The efficiency of the hydrogen production of the acid-pretreated sludge was 0.86 +/- 0.07 mol H-2/mol glucose (mean +/- S.E.), whereas that of the sludge treated with heat-shock, freezing and thawing, base method and control was 0.41 +/- 0.03 mol H-2/mol glucose, 0.17 +/- 0.01 mol H-2/mol glucose, 0.11 +/- 0.01 mol H-2/mol glucose and 0.20 +/- 0.04 mol H-2/mol glucose, respectively. The result of denaturing gradient gel electrophoresis (DGGE) showed that pretreatment methods altered the composition of the microbial community that accounts for hydrogen production. Acid and heat pretreatments were favorable to enrich the dominant hydrogen-producing bacterium, i.e. Clostridium sp., Enterococcus sp. and Bacillus sp., However, besides hydrogen-producing bacteria, much non-hydrogen-producing Lactobacillus sp. was also found in the sludge pretreated with base, freezing and thawing methods. Therefore, based on our results, we concluded that, among the four pretreatment methods using acid, heat-shock, base or freezing and thawing, acid pretreatment was the most effective method for promoting hydrogen production of microbial community. (C) 2009 Professor T. Nejat Veziroglu. Published by Elsevier Ltd. All rights reserved.

Concurrence of cat and tet genes in multiple antibiotic-resistant bacteria isolated from a sea cucumber and sea urchin mariculture farm in China

A basic understanding of abundance and diversity of antibiotic-resistant microbes and their genetic determinants is necessary for finding a way to prevent and control the spread of antibiotic resistance. For this purpose, chloramphenicol and multiple antibiotic-resistant bacteria were screened from a mariculture farm in northern China. Both sea cucumber and sea urchin rearing ponds were populated with abundant antibiotic-resistant bacteria, especially marine vibrios. Sixty-five percent chloramphenicol-resistant isolates from sea cucumber harbored a cat gene, either cat IV or cat II, whereas 35% sea urchin isolates harbored a cat gene, actually cat II. The predominant resistance determinant cat IV gene mainly occurred in isolates related to Vibrio tasmaniensis or Pseudoalteromonas atlantica, and the cat II gene mainly occurred in Vibrio splendidus-like isolates. All the cat-positive isolates also harbored one or two of the tet genes, tet(D), tet(B), or tet(A). As no chloramphenicol-related antibiotic was ever used, coselection of the cat genes by other antibiotics, especially oxytetracycline, might be the cause of the high incidence of cat genes in the mariculture farm studied.

Microbial modulation in the biomass and toxin production of a red-tide causing alga

The effect of S-10, a strain of marine bacteria isolated from sediment in the Western Xiamen Sea, on the growth and paralytic shellfish poison (PSP) production in the alga Alexandrium tamarense (A. tamarense) was studied under controlled experimental conditions. The results of these experiments have shown that the growth of A. tamarense is obviously inhibited by S-10 at high concentrations, however no evident effect on its growth was observed at low concentrations. Its PSP production was also inhibited by S 10 at different concentrations, especially at low concentrations. The toxicity of this strain of A. tamarense is about (0.9512.14) x 10(-6) MU/cell, a peak toxicity value of 12.14 x 10(-6) MU/cell appeared on the 14th day, after which levels decreased gradually. The alga grew well in conditions of pH 6-8 and salinities of 20-34 parts per thousand. The toxicity of the alga varied markedly at different pH and salinity levels. Toxicity decreased as pH increased, while it increased with salinity and reached a peak value at a salinity of 30 parts per thousand, after which it declined gradually. S-10 at a concentration of 1.02 x 10(9) cells/ml inhibited growth and the PSP production of A. tamarense at different pH and salinity levels. S-10 had the strongest inhibitory function on the growth of A. tamarense under conditions of pH 7 and a salinity of 34 parts per thousand. The best inhibitory effect on PSP production by A. tamarense was at pH 7, this inhibitory effect on PSP production did not relate to salinity. Interactions between marine bacteria and A. tamarense were also investigated using the flow cytometer technique (FCM) as well as direct microscope counting. S-10 was identitied as being a member of the genus Bacillus, the difference in 16S rDNA between S-10 and Bacillus halmapalus was only 2%. The mechanism involved in the inhibition of growth and PSP production of A. tamarense by this strain of marine bacteria, and the prospect of using it and other marine bacteria in the biocontrol of red-tides was discussed. (c) 2005 Elsevier Ltd. All rights reserved.

An unidentified haplosporidian parasite of bay scallop Argopecten irradians cultured in the Shandong and Liaoning provinces of China

Since 1988 growers of bay scallop Argopecten irradians in China have been experiencing mortality in their cultured stocks. Although poorly documented, mortality apparently began near Qingdao and has since spread to other areas of Shandong and Liaoning provinces. Samples of cultured scallops were collected from several growing areas in these provinces and analyzed by histological methods for pathogens. An unidentified haplosporidian parasite was observed in a high proportion of scallops from two of the stocks examined. Most infections were of low intensity, but one heavy infection was also observed. Only plasmodia stages were observed; they occurred intercellularly in connective tissues throughout the scallops. Plasmodia were spherical to oval, varied from 4.0 to 17.0 mu m in diameter and contained from 2 to 18 nuclei. Absence of spores prevented generic assignment of the parasite. The source and pathogenicity of the haplosporidian could not be assessed without additional research. No other microbial parasites (i.e. rickettsia-like, chlamydia-like or kidney coccidia) were observed in any of the scallops examined.