Effect of Temperature on Structure and Function of the Methanogenic Archaeal Community in an Anoxic Rice Field Soil

Soil temperatures in Italian rice fields typically range between about 15 and 30°C. A change in the incubation temperature of anoxic methanogenic soil slurry from 30°C to 15°C typically resulted in a decrease in the CH₄ production rate, a decrease in the steady-state H₂ partial pressure, and a transient accumulation of acetate. Previous experiments have shown that these changes were due to an alteration of the carbon and electron flow in the methanogenic degradation pathway of organic matter caused by the temperature shift (K. J. Chin and R. Conrad, FEMS Microbiol. Ecol. 18:85–102, 1995). To investigate how temperature affects the structure of the methanogenic archaeal community, total DNA was extracted from soil slurries incubated at 30 and 15°C. The archaeal small-subunit (SSU) rRNA-encoding genes (rDNA) of these environmental DNA samples were amplified by PCR with an archaeal-specific primer system and used for the generation of clone libraries. Representative rDNA clones (n = 90) were characterized by terminal restriction fragment length polymorphism (T-RFLP) and sequence analysis. T-RFLP analysis produced for the clones terminally labeled fragments with a characteristic length of mostly 185, 284, or 392 bp. Sequence analysis allowed determination of the phylogenetic affiliation of the individual clones with their characteristic T-RFLP fragment lengths and showed that the archaeal community of the anoxic rice soil slurry was dominated by members of the families Methanosarcinaceae (185 bp) and Methanosaetaceae (284 bp), the kingdom Crenarchaeota (185 or 284 bp), and a novel, deeply branching lineage of the (probably methanogenic) kingdom Euryarchaeota (392 bp) that has recently been detected on rice roots (R. Großkopf, S. Stubner, and W. Liesack, Appl. Environ. Microbiol. 64:4983–4989, 1998). The structure of the archaeal community changed when the temperature was shifted from 30°C to 15°C. Before the temperature shift, the clones (n = 30) retrieved from the community were dominated by Crenarchaeota (70%), “novel Euryarchaeota” (23%), and Methanosarcinacaeae (7%). Further incubation at 30°C (n = 30 clones) resulted in a relative increase in members of the Methanosarcinaceae (77%), whereas further incubation at 15°C (n = 30 clones) resulted in a much more diverse community consisting of 33% Methanosarcinaceae, 23% Crenarchaeota, 20% Methanosaetaceae, and 17% novel Euryarchaeota. The appearance of Methanosaetaceae at 15°C was conspicuous. These results demonstrate that the structure of the archaeal community in anoxic rice field soil changed with time and incubation temperature.     

Bacterial Populations Colonizing and Degrading Rice Straw in Anoxic Paddy Soil

Rice straw is a major substrate for the production of methane, a greenhouse gas, in flooded rice fields. The bacterial community degrading rice straw under anoxic conditions was investigated with molecular methods. Rice straw was incubated in paddy soil anaerobically for 71 days. Denaturing gradient gel electrophoresis (DGGE) of the amplified bacterial 16S rRNA genes showed that the composition of the bacterial community changed during the first 15 days but then was stable until the end of incubation. Fifteen DGGE bands with different signal intensities were excised, cloned, and sequenced. In addition, DNA was extracted from straw incubated for 1 and 29 days and the bacterial 16S rRNA genes were amplified and cloned. From these clone libraries 16 clones with different electrophoretic mobilities on a DGGE gel were sequenced. From a total of 31 clones, 20 belonged to different phylogenetic clusters of the clostridia, i.e., clostridial clusters I (14 clones), III (1 clone), IV (1 clone), and XIVa (4 clones). One clone fell also within the clostridia but could not be affiliated to one of the clostridial clusters. Ten clones grouped closely with the genera Bacillus (3 clones), Nitrosospira (1 clone), Fluoribacter (1 clones), and Acidobacterium (2 clones) and with clone sequences previously obtained from rice field soil (3 clones). The relative abundances of various phylogenetic groups in the rice straw-colonizing community were determined by fluorescence in situ hybridization (FISH). Bacteria were detached from the incubated rice straw with an efficiency of about 80 to 90%, as determined by dot blot hybridization of 16S rRNA in extract and residue. The number of active (i.e., a sufficient number of ribosomes) Bacteria detected with a general eubacterial probe (Eub338) after 8 days of incubation was 61% of the total cell counts. This percentage decreased to 17% after 29 days of incubation. Most (55%) of the active cells on day 8 belonged to the genus Clostridium, mainly to clostridial clusters I (24%), III (6%), and XIVa (24%). An additional 5% belonged to the Cytophaga-Flavobacterium cluster of the Cytophaga-Flavobacterium-Bacteroides phylum, 4% belonged to the α, β, and γ Proteobacteria, and 1.3% belonged to the Bacillus subbranch of the gram-positive bacteria with a low G+C content. The results show that the bacterial community colonizing and decomposing rice straw developed during the first 15 days of incubation and was dominated by members of different clostridial clusters, especially clusters I, III, and XIVa.     

Novel Euryarchaeotal Lineages Detected on Rice Roots and in the Anoxic Bulk Soil of Flooded Rice Microcosms

Because excised, washed roots of rice (Oryza sativa) immediately produce CH₄ when they are incubated under anoxic conditions (P. Frenzel and U. Bosse, FEMS Microbiol. Ecol. 21:25–36, 1996), we employed a culture-independent molecular approach to identify the methanogenic microbial community present on roots of rice plants. Archaeal small-subunit rRNA-encoding genes were amplified directly from total root DNA by PCR and then cloned. Thirty-two archaeal rice root (ARR) gene clones were randomly selected, and the amplified primary structures of ca. 750 nucleotide sequence positions were compared. Only 10 of the environmental sequences were affiliated with known methanogens; 5 were affiliated with Methanosarcina spp., and 5 were affiliated with Methanobacterium spp. The remaining 22 ARR gene clones formed four distinct lineages (rice clusters I through IV) which were not closely related to any known cultured member of the Archaea. Rice clusters I and II formed distinct clades within the phylogenetic radiation of the orders “Methanosarcinales” and Methanomicrobiales. Rice cluster I was novel, and rice cluster II was closely affiliated with environmental sequences obtained from bog peat in northern England. Rice cluster III occurred on the same branch as Thermoplasma acidophilum and marine group II but was only distantly related to these taxa. Rice cluster IV was a deep-branching crenarchaeotal assemblage that was closely related to clone pGrfC26, an environmental sequence recovered from a temperate marsh environment. The use of a domain-specific oligonucleotide probe in a fluorescent in situ hybridization analysis revealed that viable members of the Archaea were present on the surfaces of rice roots. In addition, we describe a novel euryarchaeotal main line of descent, designated rice cluster V, which was detected in anoxic rice paddy soil. These results indicate that there is an astonishing richness of archaeal diversity present on rice roots and in the surrounding paddy soil.     

Diauxic Growth in Rice Suspension Cells Grown on Mixed Carbon Sources of Acetate and Glucose

Diauxic growth was observed in rice (Oryza sativa L.) suspension cells growing on acetate (10 mM) and glucose (10 mM). Cells used acetate during the first growth phase and the acetate level in the medium was rapidly decreased, whereas the level of glucose remained essentially unchanged. After acetate was depleted from the medium, cells started to use glucose, forming the second growth phase. It appears that uptake of [14C]glucose was repressed during the first growth phase and became active during the second growth phase. In contrast, uptake of [14C]acetate occurred actively throughout the diauxic growth. By further demonstrating the specific induction of isocitrate lyase (EC 4.1.3.1), a glyoxylate cycle enzyme, and hexokinase (EC 2.7.1.1), a glycolysis enzyme, during the first and second growth phases, respectively, it was clearly shown that rice cells use acetate first and do not use both carbon sources simultaneously. This kind of diauxic growth pattern has been observed in bacteria. To our knowledege, this study is the first report demonstrating the presence of diauxic growth in plant cells.     

Phototrophic N2 Fixation Suppressed by Activated Sulfate Reduction in Anoxic Rice Soil Slurries

Interactions between sulfate reduction (SR) and phototrophic nitrogenase activities were investigated in rice soil slurries mixed with rice straw. Activation of SR by adding exogenous sulfate suppressed acetylene-reducing activity (ARA) of the slurries, which was associated with phototrophic purple bacteria (PB) enumerated to 108-109 MPN g-1 dry weight (dw) soil. Adding 5 mm sodium molybdate, an inhibitor of SR, markedly increased ARA. However, in the slurries receiving both molybdate and exogenous sulfate, the effects declined simultaneously with partial recovery of SR. These results indicate outcompetition of sulfate-reducing bacteria (SRB) with PB in rice soil, when sulfate concentrations are high enough to support SR. The increasing effects of molybdate on ARA continued during the incubation in the sulfate-depleted condition, probably because of absence of SR and toxicity of molybdate to methanogenesis. Accordingly, stopping activities of the competitive microorganisms may be efficient to increase N2 fixation in rice soil.     

The Metabolism of Glucose and Acetate in Aspergillus niger

1. The metabolism of a citric-acid-forming strain of A. nigerwhengrown on a glucose-acetate medium has been investigated.
2. Acetate greatly accelerated the rate of utilization of glucose.
3. Citric acid production from glucose was much increased bythepresence of acetate.
4. The formation of oxalate from glucose-acetatecultures was muchless than from acetate alone.
5. In some cultureslarge amounts of glucose and acetate were consumedbut no acidicproducts were formed.

     

Anoxic Responses of Seedling Roots of Rice Cultivars Varying in Tolerance to Submergence

Differences in tolerance to submergence and anoxia exhibited by cultivar-specific rice (Oryza sativa L.) extend to the primary root tips and axes of 3-day-old seedlings. This paper considers the physiological mechanisms which might account for rice root intolerance to anoxia, particularly those implicated in pH regulation and sugar metabolism in relation to hypoxic acclimation. Hypoxic treatment and the presence of glucose during anoxia did not permit root tips and axes of intolerant cultivars to survive 24-h anoxia. The absence of typical glycolytic and fermentative enzyme induction together with no improvement of ethanol production and energy status during anoxia suggest that intolerant cultivars are not capable of hypoxic acclimation at the level of energy and sugar metabolism. However, root tip survival was enhanced in buffered medium after hypoxic treatment, suggesting a relationship between hypoxic treatment and improved pH regulation.     

Culturable Populations of Sporomusa spp. and Desulfovibrio spp. in the Anoxic Bulk Soil of Flooded Rice Microcosms

Most-probable-number (MPN) counts were made of homoacetogenic and other bacteria present in the anoxic flooded bulk soil of laboratory microcosms containing 90- to 95-day-old rice plants. MPN counts with substrates known to be useful for the selective enrichment or the cultivation of homoacetogenic bacteria (betaine, ethylene glycol, 2,3-butanediol, and 3,4,5-trimethoxybenzoate) gave counts of 2.3 × 10³ to 2.8 × 10⁵ cells per g of dry soil. Homoacetogens isolated from the terminal positive steps of these dilution cultures belonged to the genus Sporomusa. Counts with succinate, ethanol, and lactate gave much higher MPNs of 5.9 × 10⁵ to 3.4 × 10⁷ cells per g of dry soil and led to the isolation of Desulfovibrio spp. Counting experiments on lactate and ethanol which included Methanospirillum hungatei in the medium gave MPNs of 2.3 × 10⁶ to 7.5 × 10⁸ cells per g of dry soil and led to the isolation of Sporomusa spp. The latter strains could grow with betaine, ethylene glycol, 2,3-butanediol, and/or 3,4,5-trimethoxybenzoate, but apparently most cells of Sporomusa spp. did not initiate growth in counting experiments with those substrates. Spores apparently accounted for 2.2% or less of the culturable bacteria. It appears that culturable Desulfovibrio spp. and Sporomusa spp. were present in approximately equal numbers in the bulk soil. Multiple, phylogenetically-distinct, phenotypically-different, strains of each genus were found in the same soil system.     

醋酸纤维素膜为基础的葡萄糖生物传感器的研制

用共价法将酶固定在醋酸纤维素膜上,方法简便易行,制造的酶膜稳定,比活力高。同时采用该方法制备了葡萄糖氧化酶酶膜,与氧电极组装成测定葡萄糖的生物传感器,线性范围为50~800mg／dl,仪器工作的最适pH为6．0,最适温度为40℃。将该膜与过氧化氢电极组装得到的传感器具有以下特性：线性范围为10~200mg／dl,最适pH为6．0,测定结果与酶试制盒有良好相关性。     

Ferredoxin-Dependent Conversion of Acetaldehyde to Acetate and H2 in Extracts of S Organism

S organism, an anaerobic gram-negative rod, which is one of two bacterial species isolated from the culture known as "Methanobacillus omelianskii," ferments ethanol to acetate and H(2). The present study shows that extracts of this organism contain ferredoxin and produce acetate from acetaldehyde via aldehyde: ferredoxin oxidoreductase activity. Electrons generated in the reaction are given off as H(2) by a previously demonstrated ferredoxin-linked hydrogenase system. Extracts were shown to contain good phosphotransacetylase and acetokinase activities, but no mechanism of adenosine triphosphate generation during acetaldehyde conversion to acetate could be detected. No evidence could be obtained for coenzyme A (CoASH) or phosphate requirement or for formation of acetyl CoA or acetyl phosphate.     

Succession of Bacterial Populations during Plant Residue Decomposition in Rice Field Soil

The incorporation of rice residues into paddy fields strongly enhances methane production and emissions. Although the decomposition processes of plant residues in rice field soil has been documented, the structure and dynamics of the microbial communities involved are poorly understood. The purpose of the present study was to determine the dynamics of short-chain fatty acids and the structure of bacterial communities during residue decomposition in a rice field soil. The soil was anaerobically incubated with the incorporation of rice root or straw residues for 90 days at three temperatures (15, 30, and 45°C). The dynamics of fatty acid intermediates showed an initial cumulative phase followed by a rapid consumption phase and a low-concentration quasi-steady state. Correspondingly, the bacterial populations displayed distinct successions during residue decomposition. Temperature showed a strong effect on the dynamics of bacterial populations. Members of Clostridium (clusters I and III) were most dominant in the incubations, particularly in the early successions. Bacteroidetes and Chlorobi were abundant in the later successions at 15 and 30°C, while Acidobacteria were selected at 45°C. We suggest that the early successional groups are responsible for the decomposition of the easily degradable fraction of residues, while the late successional groups become more important in decomposing the less-degradable or resistant fraction of plant residues. The bacterial succession probably is related to resource availability during residue decomposition. The fast-growing organisms are favored at the beginning, while the slow-growing bacteria are better adapted in the later stages, when substrate availability is limiting.Rice residues, including root and straw residues, serve as the major carbon source in paddy fields. It has been estimated that the amounts of organic matter supplied annually to paddy fields range from 1,700 to 3,470 kg ha⁻¹, and more than 65% of them were derived from plant residues (19, 23). The incorporation of rice residues into paddy fields helps sustain soil organic matter, improve physical and chemical properties, and increase nutrient availability (15, 39). However, it also strongly enhances methane production and emissions (6, 45, 46).Numerous studies have been carried out on residue decomposition and CH₄ production in rice field soils (11-13, 22). The rate of decomposition usually is separated into a fast phase and a slowdown phase (24). According to the dynamics of the intermediates H₂ and fatty acids and the activities of polysaccharolytic enzymes, Glissmann and Conrad (13) proposed five stages for residue decomposition, including the production and consumption of reducing sugars, the production and consumption of H₂ and fatty acids, and the production of CH₄. The rate of decomposition was affected by the composition of residues (11, 18). Root residues generally are decomposed slower than straw residues (24). The fermentation pathway also could differ depending on the residue material, resulting in different fatty acid intermediates (13).A complex microbial assemblage consisting of hydrolytic, fermenting, homoacetogenic, syntrophic, and methanogenic microorganisms are involved in the anaerobic decomposition of organic residues (5, 41, 48). Plant residues are composed of complex components. With the decomposition process, the proportion of labile component decreases while the resistant components relatively accumulate. Changes in the activity and structure of the microbial community thus are anticipated during the processes of residue decomposition. However, little has been known about the dynamics of microbial populations during residue decomposition in anoxic rice soil. Using culture-independent methods, Weber et al. (47) showed that the structure of the bacterial community shifted between early and late stages. Microscopic observation revealed the differential colonizations of bacterial populations on different parts of straw residue, indicating the effects of residue quality and niche condition (18). The microbial community appears to form a spatially well organized architecture, with the fermenting bacteria colonizing the residue particles, while the syntrophic bacteria and methanogens mainly inhabit the adjacent soil during the decomposition process (12).Air temperature exhibits a large seasonal variation in southeastern Asia, where rice is widely cultivated. The lowest and highest records in our research site, for instance, were −5 and 40°C, respectively, in 2006, when we collected the soil samples. It has been demonstrated that temperature has a strong effect on residue decomposition and CH₄ production (8, 22, 33). However, it is uncertain whether the effect also is reflected in the structure and function of degrading microbial communities in the soil. Therefore, the purpose of our project was to determine the effect of temperature on microbial communities during the processes of plant residue decomposition in a Chinese rice field soil. The dynamics of methanogenesis and methanogenic archaea have been reported previously (33). Here, we show the results on fatty acid intermediates and the responsible bacterial communities.     

Archaeal Population Dynamics during Sequential Reduction Processes in Rice Field Soil

The population dynamics of Archaea after flooding of an Italian rice field soil were studied over 17 days. Anoxically incubated rice field soil slurries exhibited a typical sequence of reduction processes characterized by reduction of nitrate, Fe³⁺, and sulfate prior to the initiation of methane production. Archaeal population dynamics were followed using a dual approach involving molecular sequence retrieval and fingerprinting of small-subunit (SSU) rRNA genes. We retrieved archaeal sequences from four clone libraries (30 each) constructed for different time points (days 0, 1, 8, and 17) after flooding of the soil. The clones could be assigned to known methanogens (i.e., Methanosarcinaceae, Methanosaetaceae, Methanomicrobiaceae, and Methanobacteriaceae) and to novel euryarchaeotal (rice clusters I, II, and III) and crenarchaeotal (rice clusters IV and VI) lineages previously detected in anoxic rice field soil and on rice roots (R. Grosskopf, S. Stubner, and W. Liesack, Appl. Environ. Microbiol. 64:4983–4989, 1998). During the initiation of methanogenesis (days 0 to 17), we detected significant changes in the frequency of individual clones, especially of those affiliated with the Methanosaetaceae and Methanobacteriaceae. However, these findings could not be confirmed by terminal restriction fragment length polymorphism (T-RFLP) analysis of SSU rDNA amplicons. Most likely, the fluctuations in sequence composition of clone libraries resulted from cloning bias. Clonal SSU rRNA gene sequences were used to define operational taxonomic units (OTUs) for T-RFLP analysis, which were distinguished by group-specific TaqI restriction sites. Sequence analysis showed a high degree of conservation of TaqI restriction sites within the different archaeal lineages present in Italian rice field soil. Direct T-RFLP analysis of archaeal populations in rice field soil slurries revealed the presence of all archaeal lineages detected by cloning with a predominance of terminal restriction fragments characteristic of rice cluster I (389 bp), Methanosaetaceae (280 bp), and Methanosarcinaceae/rice cluster VI (182 bp). In general, the relative gene frequency of most detected OTUs remained rather constant over time during the first 17 days after flooding of the soil. Most minor OTUs (e.g., Methanomicrobiaceae and rice cluster III) and Methanosaetaceae did not change in relative frequency. Rice cluster I (37 to 30%) and to a lesser extent rice cluster IV as well as Methanobacteriaceae decreased over time. Only the relative abundance of Methanosarcinaceae (182 bp) increased, roughly doubling from 15 to 29% of total archaeal gene frequency within the first 11 days, which was positively correlated to the dynamics of acetate and formate concentrations. Our results indicate that a functionally dynamic ecosystem, a rice field soil after flooding, was linked to a relatively stable archaeal community structure.     

Influence of Glucose on the Transmembrane Action Potential of Anoxic Papillary Muscle

The response of the cat papillary muscle to anoxia has been found to alter depending on the glucose concentration in the medium. At a glucose concentration of 5 mM anoxia caused a marked reduction in force of contraction and action potential duration within 20 minutes. At a glucose concentration of 50 mM anoxia induced similar changes in the force of contraction but little or no change in action potential duration. Elevation of glucose concentration during an anoxic interval reversed the anoxia-induced changes in action potential but had little effect on force of contraction. This effect of glucose could be partially duplicated by xylose and 2-deoxyglucose and in addition, 2-deoxyglucose has been found to prevent the effect of subsequently added glucose. These sugars appear to be transported by a system responsible for glucose transport but are not metabolized to any extent. It would appear therefore that transport of glucose is in some way related to transport of potassium as increased potassium permeability is thought by many to be responsible for anoxia-induced changes in action potential duration.     

Microscale Biosensor for Measurement of Volatile Fatty Acids in Anoxic Environments

A microscale biosensor for acetate, propionate, isobutyrate, and lactate is described. The sensor is based on the bacterial respiration of low-molecular-weight, negatively charged species with a concomitant reduction of NO₃⁻ to N₂O. A culture of denitrifying bacteria deficient in N₂O reductase was immobilized in front of the tip of an electrochemical N₂O microsensor. The bacteria were separated from the outside environment by an ion-permeable membrane and supplied with nutrients (except for electron donors) from a medium reservoir behind the N₂O sensor. The signal of the sensor, which corresponded to the rate of N₂O production, was proportional to the supply of the electron donor to the bacterial mass. The selectivity for volatile fatty acids compared to other organic compounds was increased by selectively enhancing the transport of negatively charged compounds into the sensor by electrophoretic migration (electrophoretic sensitivity control). The sensor was susceptible to interference from O₂, N₂O, NO₂⁻, H₂S, and NO₃⁻. Interference from NO₃⁻ was low and could be quantified and accounted for. The detection limit was equivalent to about 1 μM acetate, and the 90% response time was 30 to 90 s. The response of the sensor was not affected by changes in pH between 5.5 and 9 and was also unaffected by changes in salinity in the range of 2 to 32‰. The functioning of the sensor over a temperature span of 7 to 30°C was investigated. The concentration range for a linear response was increased five times by increasing the temperature from 7 to 19.5°C. The life span of the biosensor varied between 1 and 3 weeks after manufacturing.     

Dependency of Glucose Transport on d-Glucoside 3-Dehydrogenase as a Conversion System of Glucose to 3-Ketoglucose in Agrobacterium tumefaciens

Alzheimer’s disease (AD) is characterized by progressive cognitive impairment and the formation of senile plaques. Silymarin, an extract of milk thistle, has long been used as a medicinal herb for liver diseases. Here we report marked suppression of amyloid β-protein (Aβ) fibril formation and neurotoxicity in PC12 cells after silymarin treatment in vitro. In vivo studies had indicated a significant reduction in brain Aβ deposition and improvement in behavioral abnormalities in amyloid precursor protein (APP) transgenic mice that had been preventively treated with a powdered diet containing 0.1% silymarin for 6 months. The silymarin-treated APP mice also showed less anxiety than the vehicle-treated APP mice. These behavioral changes were associated with a decline in Aβ oligomer production induced by silymarin intake. These results suggest that silymarin is a promising agent for the prevention of AD.