一株异养硝化-好氧反硝化菌的研究

恶臭假单胞菌;异养硝化-好氧反硝化;自养硝化;生活污水脱氮;Pseudomonas putida;heterotrophic nitrification-aerobic denitrification;autotrophic nitrification;nitrogen removal for domestic wastewater treatment

有机碳对混合菌群亚硝酸盐氮氧化的影响

自养硝化过程在自然界氮素循环和污水处理系统脱氮过程中起着关键作用。因此，了解有机碳对硝化的影响和硝化菌与异养菌之间的竞争对微生物生态学和污水处理系统设计都很重要。目前对氨氧化到硝酸盐氮过程的研究文献很多，但对亚硝酸盐氧化过程在异养菌的存在下如何受到有机碳影响的研究甚少。本文从生理生化指标、基因组学、蛋白组学三方面考察了在实验室条件下有机碳（乙酸钠）对硝化细菌和异养菌组成的混合菌群的硝化性能、菌群结构及代谢功能的变化的影响。 全文分为两大部分： 第一部分为乙酸钠对游离态硝化混合菌群的硝化性能和菌群结构的短期影响。混合菌株先在自养条件下进行连续培养，两个月后硝化速率达到20 mg N/(L·d)；而后离心收集菌体进行批式实验。在批式反应器中，初始亚硝氮均为126mg N/ L，乙酸钠-C 与亚硝酸盐-N 的比分别为0，0.44，0.88，4.41，8.82。结果表明：在低C/N 比（0.44 和0.88）时，亚硝酸盐去除速率比C/N=0 下高，细菌呈现一次生长；而在高C/N 比（4.41 和8.82）时，出现连续的硝化反硝化，亚硝酸盐去除率仍比对照下高，细菌呈现二次生长。不同C/N 比下微生物群落明显不同，优势菌群从自养和寡营养细菌体系（包括亚硝酸盐氧化菌，拟杆菌门,α-变形菌纲,浮霉菌门和绿色非硫细菌下的一些菌株）过渡到异养和反硝化菌体系 （γ-变形菌纲的菌株尤其是反硝化菌Pseudomonas stutzeri 和P. nitroreducens 占主导）。 第二部分为乙酸钠对硝化混合菌群生物膜的硝化性能和菌群结构的长期影响。接种富集的硝化混合菌群于装有组合式填料的三角瓶中，于摇床中自养培养；两个月后填料上形成生物膜的硝化速率达到20 mg N/ (L·d)；而后进行长期实验，每12 小时更换混合营养培养基（亚硝氮约200 mg N/ L，C/N 比同上）。结果显示：相较于C/N 比=0 时的亚硝酸盐氧化反应来说，低C/N 比出现了部分的反硝化，而高C/N 比则是几乎完全的反硝化。与对照比，C/N=0.44 时亚硝酸盐氧化速率并未受乙酸钠的影响，反而上升了，但C/N=0.88 时亚硝酸盐氧化速率有所下降。菌群结构分析表明自养对照与混合营养下微生物群落的不同；PCR-DGGE未检测出混合营养下硝化杆菌的存在，而显示异养菌尤其是反硝化菌的大量存 在。荧光定量PCR 结果表明随C/N 比上升，硝化杆菌数量从2.42 × 104 下降到1.34× 103 16S rRNA gene copies/ ng DNA，反硝化菌由0 增加至2.51 × 104 nosZgene copies/ ng DNA。SDS-PAGE 的结果表明不同C/N 比下的蛋白组较为复杂且呈现一定的差异性。 有机碳对亚硝氮氧化及微生物群落的影响很复杂，本文分别讨论了对游离态和生物膜固定态两种状态的混合菌群相应的短期和长期影响研究。研究发现，有机碳并非一定带来硝化的负影响，如果控制在适当的C/N 比范围，有机碳是有利于亚硝氮氧化的。这些发现阐明了有机碳和硝化反硝化的关系，填补了硝化微生物生态学上的空白，对污水处理系统中减少异养菌的影响并提高氮去除率有一定理论指导意义。 Nitrification plays a key role in the biological removal of nitrogen in both nature and wastewater treatment plant (WWTP). So, understanding of the effect of organic carbon on nitrification and the competition between nitrifying bacteria and heterotrophic bacteria is important for both microbial ecology and WWTP design and operation. Despite the fact that the nitrification process of ammonia to nitrate has been extensively investigated, it is not known how the process of nitrite oxidization is affected by organic carbon when heterotrophic bacteria are present. By measuring different physiological and biochemical parameters, as well as using genomic DNA and proteome analysis, we investigated the influence of organic (acetate) on nitrite oxidizing performance, community structure and metabolic function of nitrite-oxidizing and heterotrophic bacteria under laboratory conditions. The dissertation involves two parts: Part one deals with the effect of organic matter on functional performance and bacterial community shift of nitrite-oxidizing and heterotrophic bacteria under suspended state. The bacteria were prepared in a continuous-flow stirred reactor under autotrophic condition; after two months, the nitrification rate of the culture reached about 20 mg N/ (L·d); then the bacteria were harvested for the next batch experiments. The initial concentrations of nitrite were 126 ± 6 mg N/ L in all flasks, and sodium acetate (C) to nitrite (N) ratios were 0, 0.44, 0.88, 4.41, and 8.82, respectively. The results showed that at low C/N ratios (0.44 or 0.88), the nitrite removal rate was higher than that obtained under autotrophic condition and the bacteria had single growth phase, while at high C/N ratios (4.41 or 8.82), continuous aerobic nitrification and denitrification occurred besides higher nitrite removal rates, and the bacteria had double growth phases. The community structure of total bacteria strikingly varied with the different C/N ratios; the dominant populations shifted from autotrophic and oligotrophic bacteria (NOB, and some strains of Bacteroidetes, Alphaproteobacteria, Actinobacteria, and green nonsulfur bacteria) to heterotrophic and denitrifying bacteria (strains of Gammaproteobacteria, especially Pseudomonas stutzeri and P. nitroreducens). Part two describes the influence of acetate on nitrite oxidizing performance, community structure and metabolic function of nitrite-oxidizing and heterotrophic bacteria in biofilms. Bacterial enrichments was transferred into flasks with polypropylene carriers and cultured under agitated and autotrophic condition. After two month, the biofilms grown on the carriers had a nitrification rate of about 20 mg N/ (L·h); then the biofilms were refreshed with mixotrophic medium (nitrite were 200 mg N/ L in all flasks, and C/N ratios was the same as above) every 12 h. the results show: normal nitrite oxidization reactions were performed when C/N = 0, but nitrite oxidization and partial denitrification occurred with low C/N ratios (0.44 or 0.88). At high C/N ratios (4.41 or 8.82), we mainly observed denitrification. In contrast to C/N = 0, the nitrite oxidization rate was unaffected when C/N = 0.44, but decreased with C/N = 0.88. The structure of bacterial communities varied significantly between autotrophic and mixotrophic conditions. Nitrobacter was hard to detect by PCR-DGGE while heterotrophs and especially denitrifiers were in the majority under mixotrophic conditions. Real-time PCR indicated that the Nitrobacter population decreased from 2.42 × 104 to 1.34 × 103 16S rRNA gene copies/ ng DNA, while the quantity of denitrifiers obviously increased from 0 to 2.51×104 nosZ gene copies/ ng DNA with an increasing C/N ratio. SDS-PAGE indicated the complexity of and a certain difference between the proteome of nitrite-oxidizing and heterotrophic bacteria at different C/N ratios. We conclude that the influence of organic matter on nitrite oxidation and the community structure of NOB and heterotrophic bacteria is complex. In this dissertation, we focused on how sodium acetate influenced the system both under suspended state and in biofilms. We observed that acetate did not necessarily have a negative impact on nitrification. Instead, an appropriate amount of acetate benefited both nitrite oxidization and denitrification. These findings provide a greater understanding about the relationship between organics and nitrification; they fill the gaps in the field of microbial ecology of nitrifying bacteria; they also provide insight into how to minimize the negative impact of heterotrophic bacteria and maximize the benefit of nitrogen removal in biological treatment systems.

ISOLATION AND CHARACTERIZATION OF CHLORELLA SOROKINIANA GXNN01 (CHLOROPHYTA) WITH THE PROPERTIES OF HETEROTROPHIC AND MICROAEROBIC GROWTH

Heterotrophic and anaerobic microalgae are of significance in both basic research and industrial application. A microalga strain was isolated from a wastewater treatment pond and identified as Chlorella sorokiniana Shihira et W. R. Krauss GXNN01 in terms of morphology, physiology, and phylogeny. The strain grows rapidly in heterotrophic or mixotrophic conditions with addition of various carbon sources, and even in anaerobic conditions. The maximum growth rate reached 0.28 d(-1) when using D,L-malate as the carbon source, and the protein content of the microalgae was 75.32% in cell dry weight. The strain was shown to be capable of (1) utilizing D, L-malate only with light, (2) inhibiting photosynthesis in mixotrophic growth, and (3) growing in anaerobic conditions with regular photosynthesis and producing oxygen internally. This study demonstrates the influence of oxygen (aerobic vs. anaerobic) and metabolic regime (autotrophy, mixotrophy, heterotrophy) on the physiological state of the cell.

Extracellular hydrolytic enzyme screening of culturable heterotrophic bacteria from deep-sea sediments of the Southern Okinawa Trough

The Southern Okinawa Trough is an area of focused sedimentation due to particulate matter export from the shelf of the East China Sea and the island of Taiwan. In order to understand the geomicrobiological characteristics of this unique sedimentary environment, bacterial cultivations were carried out for an 8.61 m CASQ core sediment sample. A total of 98 heterotrophic bacterial isolates were characterized based on 16S rRNA gene phylogenetic analysis. These isolates can be grouped into four bacterial divisions, including 13 genera and more than 20 species. Bacteria of the gamma-Proteobacteria lineage, especially those from the Halomonas ( 27 isolates) and Psychrobacter ( 20 isolates) groups, dominate in the culturable bacteria assemblage. They also have the broadest distribution along the depth of the sediment. More than 72.4% of the isolates showed extracellular hydrolytic enzyme activities, such as amylases, proteases, lipases and Dnases, and nearly 59.2% were cold-adapted exoenzyme-producers. Several Halomonas strains show almost all the tested hydrolases activities. The wide distribution of exoenzyme activities in the isolates may indicate their important ecological role of element biogeochemical cycling in the studied deep-sea sedimentary environment.

Spatial variability in biomass and production of heterotrophic bacteria in the East China Sea and the Yellow Sea

The distributions of heterotrophic bacterial abundance and production were investigated in the East China Sea and the Yellow Sea during the autumn of 2000 and spring of 2001. Bacterial abundance varied in the range 3.2-15.7 (averaging 5.7) x 10(5) and 2.3-13.6 (averaging 6.2) x 10(5) cells cm(-3) in the spring and autumn, respectively. During autumn, bacterial production (BP) (0.27-7.77 mg C m(-3) day(-1)) was on average 3 fold that in spring (0.001-2.04 mg C m(-3) day(-1)). Bacterial average turnover rate (ratio of bacterial production:bacterial biomass, mu=0.21 day(-1)) in autumn was 3 times as high as in spring (0.07 day(-1)). The ratio of integrated bacterial biomass to integrated phytoplankton biomass in the euphotic zone ranged from 4 to 101% (averaging 35%) in spring and 24 to 556% (averaging 121%) in autumn. The results indicate that the distributions of heterotrophic bacteria were controlled generally by temperature in spring and additionally by substrate supply in autumn. (C) 2010 Elsevier Ltd. All rights reserved.

Spatial variations of size-fractionated chlorophyll, cyanobacteria and heterotrophic bacteria in the Central and Western Pacific

Geographic and vertical variations of size-fractionated (0.2-1 mu m, 1-10 mu m, and >10 mu m) Chlorophyll a (Chl.a) concentration, cyanobacteria abundance and heterotrophic bacteria abundance were investigated at 13 stations from 4 degrees S, 160 degrees W to 30 degrees N, 140 degrees E in November 1993. The results indicated a geographic distribution pattern of these parameters with instances of high values occurring in the equatorial region and offshore areas, and with instance of low values occurring in the oligotrophic regions where nutrients were almost undetectable. Cyanobacteria showed the highest geographic variation (ranging from 27x10(3) to 16,582x10(3) cell l(-1)), followed by Chl.a (ranging from 0.048 to 0.178 mu g l(-1)), and heterotrophic bacteria (ranging from 2.84x10(3) to 6.50 x 10(5) cell l(-1)). Positive correlations were observed between nutrients and Chl.a abundance. Correspondences of cyanobacteria and heterotrophic bacteria abundances to nutrients were less significant than that of Chl.a. The total Chl.a was accounted for 1.0-30.9%, 35.9-53.7%, and 28.1-57.3% by the >10 mu m, 1-10 mu m and 0.2-1 mu m fractions respectively. Correlation between size-fractionated Chl.a and nutrients suggest that the larger the cell size, the more nutrient-dependent growth and production of the organism. The ratio of pheophytin to chlorophyll implys that more than half of the > 10 mu m and about one third of the 1-10 mu m pigment-containing particles in the oligotrophic region were non-living fragments, while most of the 1-10 mu m fraction was living cells. In the depth profiles, cyanobacteria were distributed mainly in the surface layer, whereas heterotrophic bacteria were abundant from surface to below the euphotic zone. Chl.a peaked at the surface layer (0-20 m) in the equatorial area and at the nitracline (75-100 m) in the oligotrophic regions. Cyanobacteria were not the principle component of the picoplankton. The carbon biomass ratio of heterotroph to phytoplankton was greater than 1 in the eutrophic area and lower than 1 in oligotrophic waters.

Direct and Indirect Effects of Dissolved Organic Matter Source and Concentration on Denitrification in Northern Florida Rivers

Using a natural gradient of dissolved organic carbon (DOC) source and concentration in rivers of northern Florida, we investigated how terrestrially-derived DOC affects denitrification rates in river sediments. Specifically, we examined if the higher concentrations of DOC in blackwater rivers stimulate denitrification, or whether such terrestrially-derived DOC supports lower denitrification rates because (1) it is less labile than DOC from aquatic primary production; whether (2) terrestrial DOC directly inhibits denitrification via biochemical mechanisms; and/or whether (3) terrestrial DOC indirectly inhibits denitrification via reduced light availability to-and thus DOC exudation by-aquatic primary producers. We differentiated among these mechanisms using laboratory denitrification assays that subjected river sediments to factorial amendments of NO3- and dextrose, humic acid dosing, and cross-incubations of sediments and water from different river sources. DOC from terrestrial sources neither depressed nor stimulated denitrification rates, indicating low lability of this DOC but no direct inhibition; humic acid additions similarly did not affect denitrification rates. However, responses to addition of labile C increased with long-term average DOC concentration, which supports the hypothesis that terrestrial DOC indirectly inhibits denitrification via decreased autochthonous production. Observed and future changes in DOC concentration may therefore reduce the ability of inland waterways to remove reactive nitrogen. © 2013 Springer Science+Business Media New York.

Denitrification and inference of nitrogen sources in the karstic Floridan Aquifer

Aquifer denitrification is among the most poorly constrained fluxes in global and regional nitrogen budgets. The few direct measurements of denitrification in groundwaters provide limited information about its spatial and temporal variability, particularly at the scale of whole aquifers. Uncertainty in estimates of denitrification may also lead to underestimates of its effect on isotopic signatures of inorganic N, and thereby confound the inference of N source from these data. In this study, our objectives are to quantify the magnitude and variability of denitrification in the Upper Floridan Aquifer (UFA) and evaluate its effect on N isotopic signatures at the regional scale. Using dual noble gas tracers (Ne, Ar) to generate physical predictions of N2 gas concentrations for 112 observations from 61 UFA springs, we show that excess (i.e. denitrification-derived) N2 is highly variable in space and inversely correlated with dissolved oxygen (O2). Negative relationships between O2 and δ15N NO3 across a larger dataset of 113 springs, well-constrained isotopic fractionation coefficients, and strong 15N:18O covariation further support inferences of denitrification in this uniquely organic-matter-poor system. Despite relatively low average rates, denitrification accounted for 32 % of estimated aquifer N inputs across all sampled UFA springs. Back-calculations of source δ15N NO3 based on denitrification progression suggest that isotopically-enriched nitrate (NO3-) in many springs of the UFA reflects groundwater denitrification rather than urban- or animal-derived inputs. © Author(s) 2012.

Aquatic Distribution And Heterotrophic Degradation Of Polycyclic Aromatic-Hydrocarbons (PAH) In The Tamar Estuary

Variations in the concentrations and microheterotrophic degradation rates of selected Polycyclic Aromatic Hydrocarbons (PAH) in the water column of the Tamar Estuary were investigated in relation to the major environmental variables. Concentrations of individual PAH varied typically between i and 50 ng l−1 Based on their observed environmental behaviour the PAH appeared divisible into two groupings: (1) low molecular weight PAH incorporating naphthalene, phenanthrene and anthracence and (a) the larger molecular weight homologues (fluoranthene, pyrene, chrysene, benz(a)anthracene, benzo(b)fluoranthene, benzo(k)fluoranthene, benzo(a)-pyrene). Group 1 PAH showed a complex distribution throughout the estuary with no significant correlations with either salinity or suspended particulates. Based on their relatively low particle affinity and high water solubilities and vapour pressures, volatilization is proposed as an important process in determining their fate. Microheterotrophic turnover times of naphthalene varied between x and 30 days, and were independent of suspended solids with maximum degradation rates located in the central and urban regions of the Estuary. When compared with the flushing times for the Tamar (3–5 days), it is probable that heterotrophic activity is important in the removal of naphthalene (and possibly the other Group 1 PAH) from the estuarine environment. In contrast Group 2 PAH concentrations exhibited highly significant correlations with suspended particulates. Highest concentrations occurred at the turbidity maximum, with a secondary concentration maximum localized to the industrialized portion of the estuary and associated with anthropogenic inputs. Laboratory degradation studies of benzo(a)pyrene in water samples taken from the estuary showed turnover times for the compound of between 2000 and 9000 days. Degradation rates correlated positively with suspended solids. The high particulate affinity and microbial refractivity of Group 2 PAH indicate sediment burial as the principal tate of these PAH in the Tamar Estuary. Estuarine sediments contained typically 50–1500 ng g−1 dry weight of individual PAH which were comparable to the levels of Group 2 PAH associated with the suspended particulates. Highest concentrations occurred at the riverine end of the estuary resulting from unresolved inputs in the catchment. Subsequent dilution by less polluted marine sediments together with slow degradation results in a seaward trend of decreasing concentrations. However, there is a secondary maximum of PAH superimposed on this trend which is associated with urban Plymouth.

Aspects Of Microbial Heterotrophic Production In A Highly Turbid Estuary

The uptake of 14C glucose by natural microbial populations has been studied in the Severn Estuary and Bristol Channel, U.K.; the turbidity (suspended solids) in the estuary varied between < 5 mg · 1−1 at the seaward extremity to >800 mg · 1−1 in the estuary proper. The heterotrophic potential, Vm, was found to correlate with turbidity and particulate organic carbon but there was no correlation between microbial biomass, as assessed by plate counts, and turbidity or Vm; measurement of Vm ranged from 0.9 × 10−4 to 288 × 10−4μgC·1−1·h−1 and turnover time from <2 to >100 h. In 17 out of 42 experiments, the uptake of 14C glucose did not conform to Michaelis kinetics and in five of these experiments the data suggested that there may be a threshold of glucose concentration below which there is no uptake.