Microbial communities biostimulated by ethanol during uranium （VI） bioremediation in contaminated sediment as shown by stable isotope probing

Stable isotope probing （SIP） was used to identify microbes stimulated by ethanol addition in microcosms containing two sediments collected from the bioremediation test zone at the US Department of Energy Oak Ridge site, TN, USA. One sample was highly bioreduced with ethanol while another was less reduced. Microcosms with the respective sediments were amended with ^13C labeled ethanol and incubated for 7 days for SIP. Ethanol was rapidly converted to acetate within 24h accompanied with the reduction of nitrate and sulfate. The accumulation of acetate persisted beyond the 7 d period. Aqueous U did not decline in the microcosm with the reduced sediment due to desorption of U but continuously declined in the less reduced sample. Microbial growth and concomitant 13C-DNA production was detected when ethanol was exhausted and abundant acetate had accumulated in both microcosms. This coincided with U（VI） reduction in the less reduced sample. I3C originating from ethanol was ultimately utilized for growth, either directly or indirectly, by the dominant microbial community members within 7 days of incubation. The microbial community was comprised predominantly of known denitrifiers, sulfate-reducing bacteria and iron （Ⅲ） reducing bacteria including Desulfovibrio, Sphingomonas, Ferribacterium, Rhodanobacter, Geothrix, Thiobacillus and others, including the known U（VI）-redueing bacteria Acidovorax, Anaeromyxobacter, Desulfovibrio, Geobac- ter and Desulfosporosinus. The findings suggest that ethanol biostimulates the U（VI）-reducing microbial com- munity by first serving as an electron donor for nitrate, sulfate, iron （IH） and U（VI） reduction, and acetate which then functions as electron donor for U（VI） reduction and carbon source for microbial growth.

Comparative analysis of DNA-SIP and magnetic-nanoparticle mediated isolation (MMI) on unraveling dimethoate degraders

Microorganisms are crucial in the bioremediation of organophosphorus pesticides. However, most functional microorganisms (> 99%) are yet to be cultivated. This study applied two cultivation-independent approaches, DNA-SIP and magnetic-nanoparticle mediated isolation (MMI), to identify the functional microorganisms in degrading dimethoate in agricultural soils. MMI identified five dimethoate degraders: Pseudomonas, Bacillus, Ramlibacter, Arthrobacter, and Rhodococcus, whereas DNA-SIP identified three dimethoate degraders: Ramlibacter, Arthrobacter, and Rhodococcus. Also, MMI showed higher resolution than DNA-SIP in identifying functional microorganisms. Two organic phosphohydrolase (OPH) genes: ophC2 and ophB, were involved in dimethoate metabolism, as revealed by DNA-SIP and MMI. The degradation products of dimethoate include omethoate, O,O,S-trimethyl thiophosphorothioate, N-methyl-2-sulfanylacetamide, O,O-diethyl S-hydrogen phosphorodithioate, O,O,O-trimethyl thiophosphate, O,O,S-trimethyl thiophosphorodithioate, and O,O,O-trimethyl phosphoric. This study emphasizes the feasibility of using SIP and MMI to explore the functional dimethoate degraders, expanding our knowledge of microbial resources with cultivation-independent approaches.

Culture-free identification of fast-growing cyanobacteria cells by Raman-activated gravity-driven encapsulation and sequencing

By directly converting solar energy and carbon dioxide into biobased products,cyanobacteria are promising chassis for photosynthetic biosynthesis.To make cyanobacterial photosynthetic biosynthesis technology economically feasible on industrial scales,exploring and engineering cyanobacterial chassis and cell factories with fast growth rates and carbon fixation activities facing environmental stresses are of great significance.To simplify and accelerate the screening for fast-growing cyanobacteria strains,a method called Individual Cyanobacteria Vitality Tests and Screening(iCyanVS)was established.We show that the ^(13)C incorporation ratio of carotenoids can be used to measure differences in cell growth and carbon fixation rates in individual cyanobacterial cells of distinct genotypes that differ in growth rates in bulk cultivations,thus greatly accelerating the process screening for fastest-growing cells.The feasibility of this approach is further demonstrated by phenotypically and then genotypically identifying individual cyanobacterial cells with higher salt tolerance from an artificial mutant library via Raman-activated gravity-driven encapsulation and sequencing.Therefore,this method should find broad applications in growth rate or carbon intake rate based screening of cyanobacteria and other photosynthetic cell factories.

Soil microbial community structure and activity in a 100-year-old fertilization and crop rotation experiment

Aims Nitrogen(N)fertilization and lime addition may affect soil micro-bial and nematode communities and ecosystem functions through changing environmental conditions,such as soil pH and soil organic carbon.The objectives of this experiment were to examine the impact of N input and liming on soil microbial and nematode communities and to identify the key environmental determinant of community composition in a century-old fertilization and crop rota-tion experiment.Methods The field experiment consisting of a 3-year crop rotation regime was established in 1911 in southeastern USA.Four treatments,(i)no-input control,(ii)NPK with winter legume,(iii)PK with legume and lime and(iv)NPK with legume and lime,were included in this study.soil samples collected at the 0-5 cm depth were used to determine the bacterial growth rate by the 3H-thymidine incorporation technique.Incorporation of 13C into neutral lipids,glycolipids and phospholipid fatty acids(PlFas)was measured after incubation of soil with 13C-labeled acetate for 24 h.Free-living nematodes in fresh soil were extracted using a density sucrose centrifugal flotation method and identified to trophic group level.Important Findingsliming resulted in a 10-fold increase in bacterial growth rates compared with the no-input control,whereas N fertilization had no significant effect.multivariate analysis of PlFa profiles showed that soil microbial community composition was different among the four treatments;the difference was primarily driven by soil pH.PlFas indicative of gram-negative bacteria covaried with soil pH,but not those of fungi and actinobacteria.liming enhanced 13C incorpora-tion into neutral lipids,glycolipids and phospholipids by 2-15 times.In addition,13C incorporation into 16:0,16:1ω9,18:1ω9,18:1ω7 and 18:2ω6 were greater than other PlFas,suggesting that gram-negative bacteria and fungi were more active and sensitive to simple C input.bacterivorous nematodes were the dominant trophic group in the soil,but no significant differences in nematode communities were found among the treatments.our results suggest that soil pH had a greater impact than N fertilization on soil microbial community composition and activity in a crop rotation system including legumes.

Compositional variations of active autotrophic bacteria in paddy soils with elevated CO_(2) and temperature

Global warming is an increasingly serious ecological problem,we examined how the active autotrophic microbes in paddy soils respond to the elevated CO_(2) and temperature.Here we employed stable isotope probing(SIP)to label the active bacteria using the soil samples from a fully factorial Simulated Climate Change(SCC)field experiment where soils were exposed to ambient CO_(2) and temperature,elevated temperature,elevated CO_(2),and both elevated CO_(2) and temperature.Around 28.9% of active OTUs belonged to ammonia-oxidizing bacteria(AOB)and nitrite-oxidizing bacteria(NOB).Nitrosospira taxa was dominant in all soils and 80.4% of carbon-fixing bacteria under elevated temperature were classified as Nitrosomonas nitrosa.While no labeled NOBs were detected when temperature or CO_(2) were elevated independently,diverse NOBs were detected in the ambient conditions.We found that elevated CO_(2) and temperature had contrasting effects on microbial community composition,while relatively small changes were observed when CO_(2) and temperature were elevated simultaneously.Summarily these results suggest that carbon-fixing bacteria can respond positively to elevated CO_(2) concentrations,but when it’s accompanied with increase in the temperature this positive response could be weakened.Multiple abiotic factors thus need to be considered when predicting how microbial communities will respond to multiple climatic factors.