Nanoscale zero-valent iron particles (nZVI), with sizes smaller than 100 nm, are promising for environmental remediation of polluted water, soil and sediments. nZVI particles have high potential for migration in the environment and are likely to interact not only with pollutant chemicals but also with living organisms. For these reasons, an environmental concern is rising with respect to unintended effects that need to be weighed against the benefits of remediation. The nZVI particles have a tendency to release electrons and Fe2+. The Fe2+ can convert less reactive hydrogen peroxide to more reactive oxygen species, particularly hydroxyl radicals, via the Fenton reaction. Hydroxyl radicals show strong biochemical activity and can react directly with membrane lipids, proteins and DNA. Reactive oxygen species are normally scavenged by antioxidants and various enzymes; however, elevated concentrations of ROS in microbial cells can result in oxidative stress. Cells under severe oxidative stress show various dysfunctions of membrane lipids, proteins and DNA. This review focuses on the processes resulting in oxidative stress and on up-to-date studies of nZVI-induced intracellular changes leading to such stress in microorganisms.
Nitrogen is generally the most limiting nutrient for rice production. In rice paddy soils, various biochemical processes can occur regarding N cycling, including nitrification, denitrification, and nitrogen fixation. Since its discovery in the 1930s, the nitrification-denitrification process has been extensively studied in Japan. It may cause N loss from rice paddy soils, while it can also reduce environmental pollutions such as nitrate leaching and emission of nitrous oxide (N2O). In this review article, we first summarize the early and important findings regarding nitrification-denitrification in rice paddy soils, and then update recent findings regarding key players in denitrification and N2O reduction. In addition, we also discuss the potential occurrence of other newly found reactions in the N cycle, such as archaeal ammonia oxidization, fungal denitrification, anaerobic methane oxidation coupled with denitrification, and anaerobic ammonium oxidation.
The use of mixed microbial cultures enriched for biological mercury removal is explored in this paper, focusing on the ecological shifts occurring throughout acclimatization to mercury and on the long-term stability of four microbial enrichments. The 16S rRNA genetic profiles obtained by denaturing gradient gel electrophoresis (DGGE) revealed that the glucose and ethanol cultures had similar profiles, whereas the acetate cultures diverged into a totally dissimilar cluster. Quantification of the merA gene copies in each enrichment showed higher values for the glucose culture, followed by the ethanol and then the acetate cultures, which was consistent with the mercury removal performance throughout the study. Isolates were obtained from the four cultures and analyzed with respect to their genetic (16S rRNA) and functional (merA) phylogenies in order to identify mercury-resistant species enriched with different carbon sources. All mercury-resistant isolates obtained from the glucose and ethanol cultures belonged to the Gammaproteobacteria, whereas acetate cultures also contained members of other phyla, with differences in merA sequences. Higher phylogenetic than functional diversity of the isolates, together with increasing merA copies even after culture stabilisation, highlight the role of horizontal gene transfer in the acclimatization process.
We developed a simple, less laborious method to cultivate and isolate obligate anaerobic microorganisms using a six-well plate together with the AnaeroPack System, designated as the six-well plate method. The cultivation efficiency of this method, based on colony-forming units, colony formation time, and colony size, was evaluated with four authentic obligate anaerobes (two methanogenic archaea and two sulfate-reducing bacteria). The method was found to be comparable to or even better than the roll tube method, a technique that is commonly used at present for the cultivation of obligate anaerobes. Further experiments using 21 representative obligate anaerobes demonstrated that all examined anaerobes (11 methanogens, 5 sulfate- or thiosulfate-reducing bacteria, and 5 syntrophs) could form visible colonies on the six-well plate and that these colonies could be successfully subcultured in fresh liquid media. Using this method, an unidentified sulfate-reducing bacterium was successfully isolated from an environmental sample.
The effects of soil and fertilizer types on archaeal communities were evaluated by real-time PCR and PCR-denaturing gradient gel electrophoresis (DGGE) targeting the 16S rRNA gene of total DNA directly extracted from upland field soils. Twelve experimental upland field plots containing four different soil types, i.e., Cumulic Andosol, Low-humic Andosol, Yellow Soil and Gray Lowland Soil, were maintained under three different fertilizer management systems for 8 years (chemical fertilizer, rice husks and cow manure, and pig manure, respectively). Two-way ANOVA and RDA analyses showed that the copy number and PCR-DGGE profile of archaeal 16S rRNA gene were affected mainly by soil type, especially between Andosol and non-Andosol, but were also influenced by fertilizer type. Among several soil chemical properties, total N content showed a significant correlation to archaeal community. Sequence analyses showed that most of the major DGGE bands corresponded to uncultured Crenarchaeota of Group I.1b that contained ammonia-oxidizing archaea (AOA). These sequences were separated into two clusters in the phylogenetic tree and each lineage showed a different response to total N content.
A new extremely thermophilic, anaerobic, gram-negative bacterium, strain NTOU1, was enriched and isolated from acidic marine hydrothermal fluids off Gueishandao island in Taiwan with 0.5% starch and 0.5% maltose as carbon sources. This strain was capable of growth utilizing various sugars found in lignocellulosic biomass as well as xylan and cellulose, and produced ethanol, lactate, acetate, and CO2 as fermentation products. The results of a 16S rRNA gene sequence analysis (1,520 bp) revealed NTOU1 to belong to the genus Thermoanaerobacterium. When tested for the ability to grow and produce ethanol from xylose or rice straw hemicellulosic hydrolysate at 70°C, the strain showed the highest levels of ethanol production (1.65 mol ethanol mol xylose-1) in a medium containing 0.5% xylose plus 0.5% yeast extract. Maximum ethanol production from the rice straw hemicellulose was 0.509 g g-1, equivalent to 98.8% theoretical conversion efficiency. Low concentrations of inhibitors (derived from dilute acid hydrolysis) in the rice straw hemicellulose hydrolysate did not affect the ethanol yield. Thus, Thermoanaerobacterium strain NTOU1 has the potential to be used for ethanol production from hemicellulose.
Forest disturbance often results in changes in soil properties and microbial communities. In the present study, we characterized a soil bacterial community subjected to disturbance using 16S rRNA gene clone libraries. The community was from a disturbed broad-leaved, low mountain forest ecosystem at Huoshaoliao (HSL) located in northern Taiwan. This locality receives more than 4,000 mm annual precipitation, one of the highest precipitations in Taiwan. Based on the Shannon diversity index, Chao1 estimator, richness and rarefaction curve analysis, the bacterial community in HSL forest soils was more diverse than those previously investigated in natural and disturbed forest soils with colder or less humid weather conditions. Analysis of molecular variance also revealed that the bacterial community in disturbed soils significantly differed from natural forest soils. Most of the abundant operational taxonomic units (OTUs) in the disturbed soil community at HSL were less abundant or absent in other soils. The disturbances influenced the composition of bacterial communities in natural and disturbed forests and increased the diversity of the disturbed forest soil community. Furthermore, the warmer and humid weather conditions could also increase community diversity in HSL soils.
A comparison was drawn between the effect of glyphosate (Roundup Plus), a post-emergence applied herbicide, and Harness GTZ, a pre-emergence applied herbicide, on the culturable fraction of the rhizobacterial communities of genetically modified NK603 glyphosate-tolerant maize. Two different non-selective rich media were used to grow fast-growing culturable bacteria, BHI and NB, as a more accurate estimation of the soil fast-growing culturable bacterial population would be obtained from the results of cultivating in more than one medium. The potential effect was monitored by direct amplification, cloning and sequencing of bacterial DNA encoding 16S rRNA, and high-throughput DNA pyrosequencing of the bacterial DNA coding for the 16S rRNA hypervariable V6 region from bacterial communities grown in the two different media. The estimated relative composition of the culturable maize rhizobacterial population varied considerably in accordance with the growth medium used. Both herbicides do, in fact, affect the maize rhizobacterial communities, glyphosate being, to a great extent, the less aggressive herbicide, regardless of the cultivation medium used. The pyrosequencing analysis of the fast-growing bacterial populations from the different soils represents a useful and invaluable tool to estimate the bacterial biodiversity of the culturable rhizobacteria of agricultural soils.
Using bromodeoxyuridine (BrdU) magnetic beads immunocapture and a PCR-denaturing gradient gel electrophoresis (DGGE) technique (BUMP-DGGE), we determined seasonal variations in the community structures of actively growing bacteria in the neritic waters of Hiroshima Bay, western Japan. The community structures of actively growing bacteria were separated into two clusters, corresponding to the timing of phytoplankton blooms in the autumn-winter and spring-summer seasons. The trigger for changes in bacterial community structure was related to organic matter supply from phytoplankton blooms. We identified 23 phylotypes of actively growing bacteria, belonging to Alphaproteobacteria (Roseobacter group, 9 phylotypes), Gammaproteobacteria (2 phylotypes), Bacteroidetes (8 phylotypes), and Actinobacteria (4 phylotypes). The Roseobacter group and Bacteroidetes were dominant in actively growing bacterial communities every month, and together accounted for more than 70% of the total DGGE bands. We revealed that community structures of actively growing bacteria shifted markedly in the wake of phytoplankton blooms in the neritic waters of Hiroshima Bay.
Changes in the acetogenic population were investigated in an experimental laboratory-scale biogas reactor (37°C) subjected to gradually elevated ammonia levels (0.8 to 6.9 g NH4+-N L-1). A shift from aceticlastic acetate degradation to syntrophic acetate oxidation had previously been confirmed in this reactor. In a parallel control reactor, operating at constant ammonia levels (0.65-0.90 g NH4+-N L-1), acetate degradation proceeded via the aceticlastic pathway throughout the operating period (660 d). The acetogenic populations in the reactors were analysed using degenerated primers designed to target the functional gene encoding a key enzyme of the acetyl-CoA pathway, 10-formyltetrahydrofolate synthetase (FTHFS). The analysis consisted of terminal restriction fragment length polymorphism (T-RFLP) analysis coupled with the construction of clone libraries, and quantitative PCR (qPCR) analysis. The T-RFLP data obtained were statistically analysed by non-metric multidimensional scaling. The most abundant FTHFS genes recovered in the clone libraries were assigned to terminal restriction fragments of the T-RFLP profile. The results of the investigation clearly indicated that increased ammonia concentration substantially influenced the putative acetogenic population structure and caused two distinct shifts of the most abundant members; however, the identity of the dominating species remains unknown, as none of the genes had been identified previously. Despite the shifts in the population, the qPCR analysis revealed a relatively stable abundance of the acetogenic population throughout the operation.
The guts of Tenebrio Molitor Linnaeus (yellow mealworm) were used as inocula to isolate reducing sugar-producing bacteria during bioconversion of lignocellulose to reducing sugars in this study. Three carbon sources, i.e., carboxymethyl cellulose (CMC), filter paper (FP), and lignocellulosic waste (LIG), were specifically selected; and two types of culturing media (M1 and M2) were used. After 6 months of sequential cultivation, lignocellulose (i.e., polysaccharides) degradation of enrichments M1-CMC (47.5%), M1-FP (73.3%), M1-LIG (70.4%), M2-CMC (55.7%), M2-FP (73.1%) and M2-LIG (71.7%) was achieved, respectively, with incubation for 48 h. Furthermore, seven bacterial strains were successfully isolated corresponding to most of the major bands detected by denaturing gradient gel electrophoresis analysis. The maximum reducing sugars yield by the combination of Agromyces sp. C42 and Stenotrophomonas sp. A10b was 56.7 mg g·LIG-1 of 48 h, which is approximate 2-5 times higher than the original enrichments and individual microbial strains. These findings suggest that bioconversion by microorganisms from mealworm guts has great application potential for lignocellulose hydrolysis.
The Yarqon is the largest urban river in Israel, and is a slow-flowing stream whose water originates mostly from wastewater treatment plants. Thus, its microbial community is expected to be heavily impacted both by anthropogenic factors and by seasonal temporal variation. In order to identify the main factors that influence the bacterial community, and their spatial-temporal variation, 50 samples were collected representing five different time points and eleven locations. Samples were analyzed for biotic and a-biotic parameters and the bacterial populations were analyzed by Automated Ribosomal Intergenic Spacer Analysis (ARISA). Bacterial richness and diversity were calculated and compared across samples. Canonical Correspondence Analysis (CCA) showed that ARISA clustered the samples according to temporal variation. Molecular fingerprinting analysis provided a snapshot of the microbial community and showed good correlation with geochemical parameters, despite the rapid changes of the Mediterranean environment and the anthropogenic impact. Molecular fingerprinting methods based on natural fragment length polymorphisms may therefore represent a supplementary approach for stream monitoring, alongside physico-chemical measurements.
Several fungi in the Aspergillus section Flavi have been widely used for fermentative food production, while some related species in the section are known to produce mycotoxin(s) that causes mycotic diseases. Common evolutionary markers, such as rRNA gene sequences and their internal transcribed spacers, cannot differentiate these non-aflatoxin-producing species from aflatoxin producers. Multilocus sequence analysis (MLSA) based on four aflatoxin biosynthetic genes encoding aflR, aflT, norA, and vbs, which are more variable nucleotide sequences than rRNA genes, can distinguish safe koji molds, A. oryzae and A. sojae, from aflatoxin-producing strains, A. flavus, A. toxicarius and A. parasiticus.