Haihan Zhang

The Response of Dark Septate Endophytes (DSE) to Heavy Metals in Pure Culture

Dark septate endophytes (DSE) occur widely in association with plants exposed to heavy metal stress. However, little is known about the response of DSE exposed to heavy metals. In this study, five DSE were isolated from the roots of Astragalus adsurgens Pall. seedlings growing on lead-zinc mine tailings in China. Based on morphological characteristics and DNA sequence analyses, the isolates were identified as Gaeumannomyces cylindrosporus, Paraphoma chrysanthemicola, Phialophora mustea, Exophiala salmonis, and Cladosporium cladosporioides. G. cylindrosporus was selected to explore responses to Pb stress. Scanning electron microscopic observations of G. cylindrosporus grown on solid medium revealed curling of hyphae and formation of hyphal coils in response to Pb. In contrast, in liquid medium, hyphae became thick and swollen with an increase in Pb (II) concentration. We interpret that these changes are related to the variation in cell wall components. We also demonstrated that fungal melanin content increased with the addition of Pb(II). Melanin, as an important component in the cell wall, is known to be an essential antioxidant responsible for decreasing heavy metal toxicity. We also measured the total soluble protein content and glutathione (GSH) concentrations in G. cylindrosporus and found that they initially increased and then decreased with the increase of Pb(II) concentrations. The antioxidant enzyme activities were also examined, and the results showed that superoxide dismutase (SOD) activity was significantly positively correlated with Pb(II) concentrations (r = 0.957, P<0.001). Collectively, our observations indicate that the intracellular antioxidant systems, especially fungal melanin, play an important role in abating the hazards of heavy metals.

Characteristics of nitrogen transformation and microbial community in an aerobic composting reactor under two typical temperatures

Batch experiments were conducted for feces composting using an aerobic composting reactor with sawdust as bulky matrix. In the 14-day composting processes at 35±2 and 55±2°C, compost samples were collected daily and chemical analyses and PCR-DGGE were carried out for investigating the influence of composting temperature on organic decomposition, nitrogen transformation, and microbial communities. At 55±2°C, in addition to a slightly higher COD removal, nitrogen loss was greatly restrained. As organic nitrogen took about 85% of the total nitrogen originated from human feces, the suppression of ammonification process under thermophilic environment might be the main reason for less nitrogen loss at 55±2°C. By PCR-DGGE analysis, the microbial community was found to undergo successions differently at 35±2 and 55±2°C. Certain sequences identified from the compost at 55±2°C represented the microbial species which could perform nitrogen-fixation or sustain a lower pH in the compost so that gaseous ammonia emission was suppressed.

Nitrogen Removal Characteristics of a Newly Isolated Indigenous Aerobic Denitrifier from Oligotrophic Drinking Water Reservoir, Zoogloea sp. N299

Nitrogen is considered to be one of the most widespread pollutants leading to eutrophication of freshwater ecosystems, especially in drinking water reservoirs. In this study, an oligotrophic aerobic denitrifier was isolated from drinking water reservoir sediment. Nitrogen removal performance was explored. The strain was identified by 16S rRNA gene sequence analysis as Zoogloea sp. N299. This species exhibits a periplasmic nitrate reductase gene (napA). Its specific growth rate was 0.22 h−1. Obvious denitrification and perfect nitrogen removal performances occurred when cultured in nitrate and nitrite mediums, at rates of 75.53% ± 1.69% and 58.65% ± 0.61%, respectively. The ammonia removal rate reached 44.12% ± 1.61% in ammonia medium. Zoogloea sp. N299 was inoculated into sterilized and unsterilized reservoir source waters with a dissolved oxygen level of 5–9 mg/L, pH 8–9, and C/N 1.14:1. The total nitrogen removal rate reached 46.41% ± 3.17% (sterilized) and 44.88% ± 4.31% (unsterilized). The cell optical density suggested the strain could survive in oligotrophic drinking water reservoir water conditions and perform nitrogen removal. Sodium acetate was the most favorable carbon source for nitrogen removal by strain N299 (p < 0.05). High C/N was beneficial for nitrate reduction (p < 0.05). The nitrate removal efficiencies showed no significant differences among the tested inoculums dosage (p > 0.05). Furthermore, strain N299 could efficiently remove nitrate at neutral and slightly alkaline and low temperature conditions. These results, therefore, demonstrate that Zoogloea sp. N299 has high removal characteristics, and can be used as a nitrogen removal microbial inoculum with simultaneous aerobic nitrification and denitrification in a micro-polluted reservoir water ecosystem.

Nitrogen-removal efficiency of a novel aerobic denitrifying bacterium, Pseudomonas stutzeri strain ZF31, isolated from a drinking-water reservoir.

An aerobic denitrifier, identified as Pseudomonas stutzeri strain ZF31, was isolated from the Zhoucun drinking-water reservoir. Strain ZF31 removed 97% of nitrate nitrogen after 16h, without nitrite accumulation. Sequence amplification indicated the presence of the denitrification genes napA, nirS, norB, and nosZ. Nitrogen balance analysis revealed that approximately 75% of the initial nitrogen was removed as gas products. Response surface methodology (RSM) experiments showed that maximum removal of total nitrogen (TN) occurred at pH 8.23, a C/N ratio of 6.68, temperature of 27.72°C, and with shaking at 54.15rpm. The TN removal rate at low C/N ratio (i.e., 3) and low temperature (i.e., 10°C) was 73.30% and 60.08%, respectively. These results suggest that strain ZF31 has potential applications for the bioremediation of slightly polluted drinking-water reservoirs.

Ignored sediment fungal populations in water supply reservoirs are revealed by quantitative PCR and 454 pyrosequencing

BackgroundThe sediment hosts a variety of fungal species in water supply reservoirs; however, the taxonomically and functionally diverse fungal populations have remained vastly unexplored. Here, quantitative PCR (qPCR) and recently developed high-throughput 454 GS FLX pyrosequencing were combined to investigate the abundance and diversity of sediment fungal communities in three water supply reservoirs.ResultsThese results revealed 1991, 2473, and 2610 copies of the 18S rRNA gene in the sediments from the ZC, SBY, and JP reservoirs, respectively. The fungal abundance in JP reservoir was 1.31 times higher than that of the ZC reservoir. In general, 43123 reads were recovered, corresponding to 945 distinct molecular operational taxonomic units (OTUs, 97% similarity cut-off level). The majority of the fungal nuclear ribosomal internal transcribed spacer (ITS) region sequences were affiliated with Ascomycota, Chytridiomycota, Basidiomycota, Glomeromycota, and Mucoromycotina. The highest Chao 1 index (962) was observed in the JP reservoir, and this value was 5.66 times greater than that of the SBY reservoir. Heat map analysis showed that Rhizophydium (relative frequency 30.98%), Placidium (20.20%), Apophysomyces (8.43%), Allomyces (6.26%), and Rhodotorula (6.01%) were the dominant genera in the JP reservoir, while Elaphomyces (20.0%) was the dominant genus in the ZC reservoir and Rhizophydium (30.98%) and Mattirolomyces (39.40%) were the most abundant in the JP and SBY reservoirs. Glomus sp. was only found in the JP reservoir. Furthermore, the larger proportions of “unassigned fungi” call for crafting International Nucleotide Sequence Database. Principle component analysis (PCA) and network analysis also suggested that tremendously diverse functional fungal populations were resident in the sediments of the three water supply reservoirs.ConclusionsThus, the results of this research suggest that the combination of high-throughput Roche 454 GS FLX pyrosequencing and qPCR is successfully employed to decrypt reservoir sediment fungal communities. Diverse fungi occur widely in the sediments of water supply reservoirs. These findings will undoubtedly broaden our understanding of reservoir sediment fungal species harbored in this freshwater stressful environmental condition. Future research should be conducted to determine the potential for fungi to degrade complex pollutants and their secondary metabolites related to the water quality.

Nitrogen removal characteristics of enhanced in situ indigenous aerobic denitrification bacteria for micro-polluted reservoir source water.

Indigenous oligotrophic aerobic denitrifiers nitrogen removal characteristics, community metabolic activity and functional genes were analyzed in a micro-polluted reservoir. The results showed that the nitrate in the enhanced system decreased from 1.71±0.01 to 0.80±0.06mg/L, while the control system did little to remove and there was no nitrite accumulation. The total nitrogen (TN) removal rate of the enhanced system reached 38.33±1.50% and the TN removal rate of surface sediment in the enhanced system reached 23.85±2.52%. TN removal in the control system experienced an 85.48±2.37% increase. The densities of aerobic denitrifiers in the enhanced system ranged from 2.24×10(5) to 8.13×10(7)cfu/mL. The abundance of nirS and nirK genes in the enhanced system were higher than those of in the control system. These results suggest that the enhanced in situ indigenous aerobic denitrifiers have potential applications for the bioremediation of micro-polluted reservoir system.

Effects of rainfall patterns on water quality in a stratified reservoir subject to eutrophication: Implications for management.

The seasonal variation of hydrological conditions caused by shifting rainfall patterns observed in recent years has significant effects on water quality. High-volume inflows following heavy rainfall events that significantly disturb stratification lead to increased dissolved oxygen (DO) at the bottom of the reservoir, inhibiting the release of nutrients from sediments and causing a rapid reduction of algal biomass in the reservoir. However, the duration and extent of these effects depend not only on the frequency and intensity of heavy rainfall events but also on the period of thermal stratification in the reservoir. The effects of heavy rainfall events on water quality during three typical stratification periods of the reservoir were systematically investigated using extensive field data. The continuous heavy rainfall that occurred in September 2011 (stratification began to diminish) completely mixed the reservoir and produced a high concentration of DO along with a low phytoplankton concentration throughout the reservoir until stratification occurred the following year. Conversely, several days were required for anoxic conditions (in the hypolimnion) and cyanobacterial blooms to reappear after the storm runoff that occurred during the stable period of stratification (August 2012). In addition, the heavy rainfall that occurred in May 2013 accelerated the formation of an anoxic zone at the bottom of the reservoir and promoted cyanobacterial blooms due to the high nutrient input and the increased water temperature after the storm runoff ended. Water-lifting aerators (WLAs) were employed in the Shibianyu Reservoir to inhibit algal growth and to control the release of nutrients. Based on our field observations and theoretical analyses, optimized management strategies are recommended to improve water quality in the reservoir under different rainfall patterns at a reduced cost.

Microbial community functional diversity and enzymatic activity in the sediments of drinking water reservoirs, Northwest China

AbstractSediment microbial communities act as an important role in the aquatic environmental conditions by influencing nutrient cycling, organic matter metabolism, heavy metal transportation, and organic pollutant transformation. In this study, microbial community functional diversity and enzyme activity in the sediments of two drinking water reservoirs were examined. Sediment bacterial and fungal communities’ functional diversity were determined using substrate utilization profiling (BIOLOG) method. Sucrase, dehydrogenase, urease, alkaline phosphatase activities were measured spectrophotometrically. The results showed that sucrase activity in Tang Yu (TY) reservoir was 329.77 mg glucose/g 24 h, which is significant higher than that of Shi Bian Yu (SBY) (p   0.05). Average well color...

Sediment enzyme activities and microbial community diversity in an oligotrophic drinking water reservoir, eastern China.

Drinking water reservoir plays a vital role in the security of urban water supply, yet little is known about microbial community diversity harbored in the sediment of this oligotrophic freshwater environmental ecosystem. In the present study, integrating community level physiological profiles (CLPPs), nested polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE) and clone sequence technologies, we examined the sediment urease and protease activities, bacterial community functional diversity, genetic diversity of bacterial and fungal communities in sediments from six sampling sites of Zhou cun drinking water reservoir, eastern China. The results showed that sediment urease activity was markedly distinct along the sites, ranged from 2.48 to 11.81 mg NH3-N/(g·24h). The highest average well color development (AWCD) was found in site C, indicating the highest metabolic activity of heterotrophic bacterial community. Principal component analysis (PCA) revealed tremendous differences in the functional (metabolic) diversity patterns of the sediment bacterial communities from different sites. Meanwhile, DGGE fingerprints also indicated spatial changes of genetic diversity of sediment bacterial and fungal communities. The sequence BLAST analysis of all the sediment samples found that Comamonas sp. was the dominant bacterial species harbored in site A. Alternaria alternate, Allomyces macrogynus and Rhizophydium sp. were most commonly detected fungal species in sediments of the Zhou cun drinking water reservoir. The results from this work provide new insights about the heterogeneity of sediment microbial community metabolic activity and genetic diversity in the oligotrophic drinking water reservoir.

Nitrogen Removal from Micro-Polluted Reservoir Water by Indigenous Aerobic Denitrifiers

Treatment of micro-polluted source water is receiving increasing attention because of environmental awareness on a global level. We isolated and identified aerobic denitrifying bacteria Zoogloea sp. N299, Acinetobacter sp. G107, and Acinetobacter sp. 81Y and used these to remediate samples of their native source water. We first domesticated the isolated strains in the source water, and the 48-h nitrate removal rates of strains N299, G107, and 81Y reached 33.69%, 28.28%, and 22.86%, respectively, with no nitrite accumulation. We then conducted a source-water remediation experiment and cultured the domesticated strains (each at a dry cell weight concentration of 0.4 ppm) together in a sample of source water at 20–26 °C and a dissolved oxygen concentration of 3–7 mg/L for 60 days. The nitrate concentration of the system decreased from 1.57 ± 0.02 to 0.42 ± 0.01 mg/L and that of a control system decreased from 1.63 ± 0.02 to 1.30 ± 0.01 mg/L, each with no nitrite accumulation. Total nitrogen of the bacterial system changed from 2.31 ± 0.12 to 1.09 ± 0.01 mg/L, while that of the control system changed from 2.51 ± 0.13 to 1.72 ± 0.06 mg/L. The densities of aerobic denitrification bacteria in the experimental and control systems ranged from 2.8 × 104 to 2 × 107 cfu/mL and from 7.75 × 103 to 5.5 × 105 cfu/mL, respectively. The permanganate index in the experimental and control systems decreased from 5.94 ± 0.12 to 3.10 ± 0.08 mg/L and from 6.02 ± 0.13 to 3.61 ± 0.11 mg/L, respectively, over the course of the experiment. Next, we supplemented samples of the experimental and control systems with additional bacteria or additional source water and cultivated the systems for another 35 days. The additional bacteria did little to improve the water quality. The additional source water provided supplemental carbon and brought the nitrate removal rate in the experimental system to 16.97%, while that in the control system reached only 3.01%, with no nitrite accumulation in either system. Our results show that aerobic denitrifying bacteria remain highly active after domestication and demonstrate the applicability of such organisms in the bioremediation of oligotrophic ecosystems.