Propionate Formation by Opitutus terrae in Pure Culture and in Mixed Culture with a Hydrogenotrophic Methanogen and Implications for Carbon Fluxes in Anoxic Rice Paddy Soil

Propionate-forming bacteria seem to be abundant in anoxic rice paddy soil, but biogeochemical investigations show that propionate is not a correspondingly important intermediate in carbon flux in this system. Mixed cultures of Opitutus terrae strain PB90-1, a representative propionate-producing bacterium from rice paddy soil, and the hydrogenotrophic Methanospirillum hungatei strain SK maintained hydrogen partial pressures similar to those in the soil. The associated shift away from propionate formation observed in these cultures helps to reconcile the disparity between microbiological and biogeochemical studies.     

稻田土壤关键元素的生物地球化学耦合过程及其微生物调控机制

水稻土是在长期植稻下人为培育的特殊耕作土壤,是我国土壤学的特色,其研究也反映我国土壤学的国际地位。水稻土是研究土壤生物地球化学过程的理想模型。稻田土壤关键元素(碳氮磷硫铁等)的生物地球化学循环过程、耦合机理及其驱动机制研究是土壤生物学研究的核心之一。因此,以国际土壤年为契机,结合中国科学院院士工作局资助的"土壤生物学发展战略研究"项目的部分成果,以稻田关键元素(碳氮磷硫铁等)生物地球化学循环过程及其耦合的微生物驱动机制为核心,重点讨论了稻田土壤基本生物化学特征、稻田土壤碳-氮、碳-氮-磷、碳-氮-铁等多元素耦合过程及其与微生物之间的反馈机制,并由此提出了稻田土壤关键元素生物地球化学循环微生物驱动机制研究的未来重点发展方向为:1)土壤关键元素生物地球化学过程的异质性及其微生物过程的互作机制研究;2)微生物参与机制对土壤关键元素循环过程的响应、反馈机制与调控机制研究;3)土壤关键生物地球化学过程的计量学研究。     

Production, oxidation and emission of methane in rice paddies

Abstract Production and emission of methane from submerged paddy soil was studied in laboratory rice cultures and in Italian paddy fields. Up to 80% of the CH₄ produced in the paddy soil did not reach the atmosphere but was apparently oxidized in the rhizosphere. CH₄ emission through the rice plants was inhibited by an atmosphere of pure O₂ but was stimulated by an atmosphere of pure N₂ or an atmosphere containing 5% acetylene. Gas bubbles taken from the submerged soil contained up to 60% CH₄, but only < 1% CH₄ after the bubbles had passed the soil-water interface or had entered the intercellular gas space system of the rice plants. CH₄ oxidation activities were detected in the oxic surface layer of the submerged paddy soil. Flooding the paddy soil with water containing > 0.15% sea salt (0.01% sulfate) resulted in a strong inhibition of the rates of methanogenesis and a decrease in the rates of CH₄ emission. This result explains the observation of relatively low CH₄ emission rates in rice paddy areas flooded with brackish water.     

Genome sequence of the verrucomicrobium Opitutus terrae PB90-1, an abundant inhabitant of rice paddy soil ecosystems

Bacteria of the deeply branching phylum Verrucomicrobia are rarely cultured yet commonly detected in metagenomic libraries from aquatic, terrestrial, and intestinal environments. We have sequenced the genome of Opitutus terrae PB90-1, a fermentative anaerobe within this phylum, isolated from rice paddy soil and capable of propionate production from plant-derived polysaccharides.     

Novel anaerobic ultramicrobacteria belonging to the Verrucomicrobiales lineage of bacterial descent isolated by dilution culture from anoxic rice paddy soil.

The use of dilution culture techniques to cultivate saccharolytic bacteria present in the anoxic soil of flooded rice microcosms allowed the isolation of three new strains of bacteria, typified by their small cell sizes, with culturable numbers estimated at between 1.2 x 10(5) and 7.3 x 10(5) cells per g of dry soil. The average cell volumes of all three strains were 0.03 to 0.04 microns3, and therefore they can be termed ultramicrobacteria or "dwarf cells." The small cell size is a stable characteristic, even when the organisms grow at high substrate concentrations, and thus is not a starvation response. All three strains have genomic DNA with a mol% G+C ratio of about 63, are gram negative, and are motile by means of a single flagellum. The three new isolates utilized only sugars and some sugar polymers as substrates for growth. The metabolism is strictly fermentative, but the new strains are oxygen tolerant. Sugars are metabolized to acetate, propionate, and succinate. Hydrogen production was not significant. In the presence of 0.2 atm of oxygen, the fermentation end products or ratios did not change. The phylogenetic analysis on the basis of 16S ribosomal DNA (rDNA) sequence comparisons indicates that the new isolates belong to a branch of the Verrucomicrobiales lineage and are closely related to a cloned 16S rDNA sequence (PAD7) recovered from rice paddy field soil from Japan. The isolation of these three strains belonging to the order Verrucomicrobiales from a model rice paddy system, in which rice was grown in soil from an Italian rice field, provides some information on the possible physiology and phenotype of the organism represented by the cloned 16S rDNA sequence PAD7. The new isolates also extend our knowledge on the phenotypic and phylogenetic depths of members of the order Verrucomicrobiales, to date acquired mainly from cloned 16S rDNA sequences from soils and other habitats.     

Isolation and molecular characterization of thiosulfate-oxidizing bacteria from an Italian rice field soil

In rice paddy soils an active cycling of sulfur compounds takes place. To elucidate the diversity of thiosulfate-oxidizing bacteria these organisms were enriched from bulk soil and rice roots by the most probable number method in liquid medium. From the MPN enrichment cultures 21 bacterial strains were isolated on solid mineral medium, and could be further shown to produce sulfate from thiosulfate. These strains were characterized by 16S rDNA analyses. The isolates were affiliated to seven different phylogenetic groups within the alpha- and beta-subclass of Proteobacteria. Two of these phylotypes were already described as S-oxidizers in this environment (Xanthobacter sp. and Bosea sp. related strains), but five groups represented new S-oxidizers in rice field soil. These isolates were closely related to Mesorhizobium loti, to Hydrogenophaga sp., to Delftia sp., to Pandoraea sp. or showed sequence similarity to a strain of Achromobacter sp.     

Isolation and characterization of a motile hydrogenotrophic methanogen from rice paddy field soil in Japan

A hydrogenotrophic motile methanogen was isolated from flooded Japanese paddy field soil. Anaerobic incubation of the paddy soil on H(2)-CO(2) at 20 degrees C led to the enrichment of symmetrically curved motile autofluorescent rods. The methanogenic strain TM20-1 isolated from the culture was halotolerant and utilized H(2)-CO(2), 2-propanol-CO(2), or formate as a sole methanogenic substrate. Based on the 16S rRNA gene sequence similarity (94.8%) with Methanospirillum hungateii, and on the physiological and phenotypic characteristics, TM20-1 was suggested to be a newly identified species belonging to the genus Methanospirillum. This is the first report of isolation of the genus Methanospirillum strain from a rice paddy field.     

稻田硒循环转化与水稻硒营养研究进展

稻田土壤氧化还原电位的交替变化和土壤组分的特殊性决定了硒在稻田环境中的循环转化机制明显有别于旱地土壤,并且影响到土壤硒的有效性及水稻对硒的吸收与积累.深入研究稻田土壤硒的循环转化及水稻硒营养吸收对实现土壤无机硒向有机硒的转化具有重要意义.本文对稻田土壤硒的循环机制及形态转化、水稻体内硒的代谢机制及吸收积累特性进行综述,分析硒在土壤 水稻系统中转化及向水稻体内迁移的研究现状与发展趋势,为土壤硒有效性研究及培育富硒营养稻米提供参考.     

Localization of processes involved in methanogenic degradation of rice straw in anoxic paddy soil

In anoxic paddy soil, rice straw is decomposed to CH(4) and CO(2) by a complex microbial community consisting of hydrolytic, fermenting, syntrophic and methanogenic microorganisms. Here, we investigated which of these microbial groups colonized the rice straw and which were localized in the soil. After incubation of rice straw in anoxic soil slurries for different periods, the straw pieces were removed from the soil, and both slurry and straw were studied separately. Although the potential activities of polysaccharolytic enzymes were higher in the soil slurry than in the straw incubations, the actual release of reducing sugars was higher in the straw incubations. The concentrations of fermentation products, mainly acetate and propionate, increased steadily in the straw incubations, whereas only a little CH(4) was formed. In the soil slurries, on the other hand, fermentation products were low, whereas CH(4) production was more pronounced. The production of CH(4) or of fermentation products in the separated straw and soil incubations accounted in sum for 54-82% of the CH(4) formed when straw was not removed from the soil. Syntrophic propionate degradation to acetate, CO(2) and H(2) was thermodynamically more favourable in the soil than in the straw fraction. These results show that hydrolysis and primary fermentation reactions were mainly localized on the straw pieces, whereas the syntrophic and methanogenic reactions were mainly localized in the soil. The percentage of bacterial relative to total microbial 16S rRNA content was higher on the straw than in the soil, whereas it was the opposite for the archaeal 16S rRNA content. It appears that rice straw is mainly colonized by hydrolytic and fermenting bacteria that release their fermentation products into the soil pore water where they are further degraded to CH(4). Hence, complete methanogenic degradation of straw in rice soil seems to involve compartmentalization.     

水稻土中硫酸盐还原微生物研究进展

硫是水稻必需的营养元素之一.硫酸盐还原是硫元素生物地球化学循环中的关键步骤,在稻田土壤表层和水稻根际都十分活跃.介导硫酸盐还原过程的硫酸盐还原菌(sulfate- reducing bacteria, SRB)是稻田土壤中重要的功能菌群.它们不仅是硫元素生物地球化学循环的重要参与者,也是土壤中有机污染物降解的主要力量之一,发挥着重要的生态和环境功能.综述了稻田土壤中微生物参与的硫酸盐还原过程、SRB的生物多样性以及目前研究稻田土壤SRB主要采用的分子生态学方法,如末端限制性片段长度多样性(T-RFLP)、变性梯度凝胶电泳(DGGE)、实时荧光定量PCR(real-time PCR)、荧光原位杂交(FISH),并对水稻土壤中SRB的分子生态学研究方向进行了展望.     

Fermentation pattern of methanogenic degradation of rice straw in anoxic paddy soil

The anaerobic degradation of different fractions of rice straw in anoxic paddy soil was investigated. Rice straw was divided up into stem, leaf sheath and leaf blade. The different straw fractions were mixed with paddy soil and incubated under anoxic conditions. Fermentation of straw components started immediately and resulted in transient accumulation of acetate, propionate, butyrate, isobutyrate, valerate, isovalerate and caproate with much higher concentrations in the presence than in the absence of straw. Also some unidentified compounds with UV absorption could be detected. The maximum concentrations of these compounds were different when using different straw fractions, suggesting differences in the degradation pathway of these straw fractions during the early phase of incubation, i.e. with Fe(III) and sulfate serving as oxidants. When concentrations of the intermediates decreased to background values, CH(4) production started. Rates of CH(4)unamended soil. During the methanogenic phase, the percentage contribution of fermentation products to CH(4) production was determined by inhibition with 2-bromoethanesulfonate (BES). Acetate (48-83%) and propionate (18-28%) were found to be the main intermediates of the carbon flow to CH(4), irrespective of the fraction of the rice straw or its absence. Mass balance calculations showed that 84-89% of CH(4) was formed via acetate in the various incubations. Radiotracer experiments showed that 11-27% of CH(4) was formed from H(2)/CO(2), thus confirming that acetate contributed 73-89% to methanogenesis. Our results show that the addition of rice straw and the fraction of the straw affected the fermentation pattern only in the early phase of degradation, but had no effect on the degradation pathway during the later methanogenic phase.     

两种植物条件下土壤中矿物油和多环芳烃（PAHs）的生物修复研究

选择苜蓿草和水稻为供试植物,以污染物水平、有机以、专性细菌和真菌为调控因子,进行土壤中矿物油和PAHs的生物修复研究,结果表明,投肥对苜蓿草土壤中矿物油降解有促进作用,但对水稻土壤中矿物油降解无明显作用,投肥均使苜蓿草和水稻土壤中多环芒烃总量（11种列于美国EPA黑名单上的多环芳烃）降解率提高,这一降解促进效果在水稻土壤中好于苜蓿草土壤,有机肥量与苜蓿草根际土著真菌、细菌数量明显呈正相关,但仅与水稻根际土著细菌数量呈明显正相关,两种土壤中实测真菌和细菌总数均与试验投加专性真菌和细菌量无关,水稻土和苜蓿草土壤中3环多环芳烃的降解随投肥量增大而降解率提高,其在水稻土蓑中的效果好于苜蓿草土壤,投肥怪4环多环芳烃的降解并未产生有效作用。     

Effect of rice plants on methane production and rhizospheric metabolism in paddy soil

In order to elucidate the effects of rice plants on CH₄ production, we conducted experiments with soil slurries and planted rice microcosms. Methane production in anoxic paddy soil slurries was stimulated by the addition of rice straw, of unsterile or autoclaved rice roots, and of the culture fluid in which rice plants had axenically been cultivated. The addition of these compounds also increased the concentrations of acetate and H₂, precursors of CH₄ production, in the soil. Planted compared to unplanted paddy soil microcosms exhibited lower porewater CH₄ concentrations but higher CH₄ emission rates. They also exhibited higher sulfate concentrations but similar nitrate concentrations. Concentrations of acetate, lactate and H₂ were not much different between planted and unplanted microcosms. Pulse labeling of rice plants with¹⁴CO₂ resulted during the next 5 days in transient accumulation of radioactive lactate, propionate and acetate, and after the second day of incubation in the emission of¹⁴CH₄. Most of the radioactivity (40–70%) was incorporated into the above-ground biomass of rice plants. However, during a total incubation of 16 days about 3–6% of the applied radioactivity was emitted as¹⁴CH₄, demonstrating that plant-derived carbon was metabolized and significantly contributed to CH₄ production. The sequence of the appearance of radioactive products and their specific radioactivities indicate that CH₄ was produced from root exudates by a microbial community consisting of fermenting and methanogenic bacteria.     

Increased microbial functional diversity under long-term organic and integrated fertilization in a paddy soil

Microbes play key roles in diverse biogeochemical processes including nutrient cycling. However, responses of soil microbial community and functional genes to long-term integrated fertilization (chemical combined with organic fertilization) remain unclear. Here, we used pyrosequencing and a microarray-based GeoChip to explore the shifts of microbial community and functional genes in a paddy soil which received over 21-year fertilization with various regimes, including control (no fertilizer), rice straw (R), rice straw plus chemical fertilizer nitrogen (NR), N and phosphorus (NPR), NP and potassium (NPKR), and reduced rice straw plus reduced NPK (L-NPKR). Significant shifts of the overall soil bacterial composition only occurred in the NPKR and L-NPKR treatments, with enrichment of certain groups including Bradyrhizobiaceae and Rhodospirillaceae families that benefit higher productivity. All fertilization treatments significantly altered the soil microbial functional structure with increased diversity and abundances of genes for carbon and nitrogen cycling, in which NPKR and L-NPKR exhibited the strongest effect, while R exhibited the least. Functional gene structure and abundance were significantly correlated with corresponding soil enzymatic activities and rice yield, respectively, suggesting that the structural shift of the microbial functional community under fertilization might promote soil nutrient turnover and thereby affect yield. Overall, this study indicates that the combined application of rice straw and balanced chemical fertilizers was more pronounced in shifting the bacterial composition and improving the functional diversity toward higher productivity, providing a microbial point of view on applying a cost-effective integrated fertilization regime with rice straw plus reduced chemical fertilizers for sustainable nutrient management.

     

Anaerobic 2-Propanol Degradation in Anoxic Paddy Soil and the Possible Role of Methanogens in Its Degradation

The anaerobic degradation of 2-propanol in anoxic paddy soil was studied with soil cultures and a 2-propanol-utilizing methanogen. Acetone was the first and the major intermediate involved in the methanogenic degradation of 2-propanol. Analyses with a methanogenesis inhibitor, bacteria antibiotics, and the addition of H₂ to the gas phase revealed that 2-propanol oxidation to acetone directly occurred using 2-propanol-utilizing methanogens, but not with H₂-producing syntrophic bacteria, for which the removal of acetone is required for complete 2-propanol oxidation. The 2-propanol-utilizing strain IIE1, which is phylogenetically closely related to Methanoculleus palmolei, was isolated from paddy soil, and the potential role of the strain in 2-propanol degradation was investigated. 2-Propanol is one of the representative fermentation intermediates in anaerobic environments. This is the first report on the anaerobic 2-propanol degradation process.     

Comparative phylogenetic assignment of environmental sequences of genes encoding 16S rRNA and numerically abundant culturable bacteria from an anoxic rice paddy soil.

We used both cultivation and direct recovery of bacterial 16S rRNA gene (rDNA) sequences to investigate the structure of the bacterial community in anoxic rice paddy soil. Isolation and phenotypic characterization of 19 saccharolytic and cellulolytic strains are described in the accompanying paper (K.-J. Chin, D. Hahn, U. Hengstmann, W. Liesack, and P. H. Janssen, Appl. Environ. Microbiol. 65:5042-5049, 1999). Here we describe the phylogenetic positions of these strains in relation to 57 environmental 16S rDNA clone sequences. Close matches between the two data sets were obtained for isolates from the culturable populations determined by the most-probable-number counting method to be large (3 x 10(7) to 2.5 x 10(8) cells per g [dry weight] of soil). This included matches with 16S rDNA similarity values greater than 98% within distinct lineages of the division Verrucomicrobia (strain PB90-1) and the Cytophaga-Flavobacterium-Bacteroides group (strains XB45 and PB90-2), as well as matches with similarity values greater than 95% within distinct lines of descent of clostridial cluster XIVa (strain XB90) and the family Bacillaceae (strain SB45). In addition, close matches with similarity values greater than 95% were obtained for cloned 16S rDNA sequences and bacteria (strains DR1/8 and RPec1) isolated from the same type of rice paddy soil during previous investigations. The correspondence between culture methods and direct recovery of environmental 16S rDNA suggests that the isolates obtained are representative geno- and phenotypes of predominant bacterial groups which account for 5 to 52% of the total cells in the anoxic rice paddy soil. Furthermore, our findings clearly indicate that a dual approach results in a more objective view of the structural and functional composition of a soil bacterial community than either cultivation or direct recovery of 16S rDNA sequences alone.     

Metabolism of position-labelled glucose in anoxic methanogenic paddy soil and lake sediment

Abstract Turnover times of radioactive glucose were shorter in paddy soil (4–16 min) than in Lake Constance sediment (18–62 min). In the paddy soil, 65–75% of the radioactive glucose was converted to soluble metabolites. In the sediment, only about 25% of the radioactive glucose was converted to soluble metabolites, the rest to particulate material. In anoxic paddy soil, the degradation pattern of position-labelled glucose was largely consistent with glucose degradation via the Embden-Meyerhof-Parnas (EMP) pathway followed by methanogenic acetate cleavage: CO₂ mainly originated from C-3,4, whereas CH₄ mainly originated from C-1 and C-6 of glucose. Acetate-carbon originated from C-1, C-2 and C-6 rather than from C-3,4 of glucose. In both paddy soil and Lake Constance sediment acetate and CO₂ were the most important early metabolites of radioactive glucose. Other early products included propionate, ethanol/butyrate, succinate, and lactate, but accounted each for less than 1–8% of the glucose utilized. The labelling of propionate by [3,4-¹⁴C]glucose suggests that it was mainly produced from glucose or lactate rather than from ethanol. Isopropanol and caproate were also detectable in paddy soil, but were not produced from radioactive glucose. Chloroform inhibited methanogenesis, inhibited the further degradation of radioactive acetate and resulted in the accumulation of H₂, however, did not inhibit glucose degradation. Since acetate was the main soluble fermentation product of glucose and was produced at a relatively high molar acetate: CO₂ ratio (2.5:1), homoacetogenesis appeared to be the most important glucose fermentation pathway.     

长期不同施肥模式对南方双季稻田生态系统净碳汇效应和经济收益的影响

施肥措施与稻田生态系统净碳汇效应、经济收益的关系密切。本研究以长期(35年)定位施肥试验田为平台,分析了单独施用化肥(MF)、秸秆还田+化肥(RF)、30%有机肥+70%化肥(OM)和无肥对照(CK)4种不同施肥模式对我国南方双季稻田耕层土壤固碳速率、碳密度、年碳汇平衡和经济收益的影响。研究表明: 不同施肥处理双季稻田耕层土壤碳库变化范围为216.02～866.74 kg·hm^-2·a^-1,OM处理土壤碳年变化量显著高于MF、RF和CK处理;双季稻田土壤固碳速率为51.5～650.7 kg·hm^-2·a^-1,表土碳密度为55.64～78.42 t·hm^-2,各施肥处理高低顺序均为OM>RF>MF>CK。各施肥处理双季稻田生态系统水稻的碳吸收为4.42～9.32 t C·hm^-2·a^-1,其高低顺序为OM>RF>MF>CK;与MF处理相比,OM和RF处理稻田土壤净碳汇量分别提高了27.6%和13.6%。各施肥处理双季稻田生态系统的碳成本物质投入变化范围为1.49～2.17 t C·hm^-2·a^-1,年经济收益变化范围为1.30×10³～7.83×10³元·hm^-2·a^-1,其高低顺序为RF>OM>MF>CK;OM、RF和MF处理双季稻田生态系统经济效益的净收益均显著高于CK处理。总之,长期施用有机肥、秸秆还田配施化肥措施均有利于增加双季稻田土壤固碳速率、碳汇效应和经济收益,是提高南方双季稻田土壤有机碳贮量的施肥模式。     

水稻砷的吸收机理及阻控对策

水稻积累砷的能力较其他农作物强,稻米是我国人群从食品中摄入无机砷的主要来源,降低稻米砷含量对增进农产品质量安全有重要意义。本文阐述了稻田砷的生物地球化学转化特征及水稻对不同形态砷的吸收、运输与储存机理,提出阻控稻米砷积累的对策,并讨论了需要进一步研究的问题。     

The mechanism of reduction in waterlogged paddy soil

Elucidation of the laws governing the reduction process in flooded rice paddy soil is indispensable for developing the lowland rice cultivation system by which the oxidation-reduction conditions for optimum growth can be maintained throughout the growth of the crop. The authors’ efforts have been focussed on the microbial metabolism of the reduction process in paddy soil, using waterlogged soil incubated in closed syringes as a simplified model of rice paddy field soil under flooded conditions. Such conditions can be assumed to occur throughout the plowed layer of field soil under flooding, except for the uppermost layer to which oxygen is supplied. One may conclude from the results of this investigation that the type of microbial metabolism in waterlogged soil changes successively according to the oxidation-reduction state from aerobic respiration in the presence of molecular oxygen, which is the most efficient energy-yielding reaction, to methane fermentation, which appears to be a less efficient energy-yielding reaction.