Fungi,bacteria and soil pH: the oxalate–carbonate pathway as a model for metabolic interaction

The oxalate–carbonate pathway involves the oxidation of calcium oxalate to low‐magnesium calcite and represents a potential long‐term terrestrial sink for atmospheric CO ₂. In this pathway, bacterial oxalate degradation is associated with a strong local alkalinization and subsequent carbonate precipitation. In order to test whether this process occurs in soil, the role of bacteria, fungi and calcium oxalate amendments was studied using microcosms. In a model system with sterile soil amended with laboratory cultures of oxalotrophic bacteria and fungi, the addition of calcium oxalate induced a distinct pH shift and led to the final precipitation of calcite. However, the simultaneous presence of bacteria and fungi was essential to drive this pH shift. Growth of both oxalotrophic bacteria and fungi was confirmed by qPCR on the frc (oxalotrophic bacteria) and 16S rRNA genes, and the quantification of ergosterol (active fungal biomass) respectively. The experiment was replicated in microcosms with non‐sterilized soil. In this case, the bacterial and fungal contribution to oxalate degradation was evaluated by treatments with specific biocides (cycloheximide and bronopol). Results showed that the autochthonous microflora oxidized calcium oxalate and induced a significant soil alkalinization. Moreover, data confirmed the results from the model soil showing that bacteria are essentially responsible for the pH shift, but require the presence of fungi for their oxalotrophic activity. The combined results highlight that the interaction between bacteria and fungi is essential to drive metabolic processes in complex environments such as soil.     

Use of the frc gene as a molecular marker to characterize oxalate-oxidizing bacterial abundance and diversity structure in soil

Oxalate catabolism, which can have both medical and environmental implications, is performed by phylogenetically diverse bacteria. The formyl-CoA-transferase gene was chosen as a molecular marker of the oxalotrophic function. Degenerated primers were deduced from an alignment of frc gene sequences available in databases. The specificity of primers was tested on a variety of frc-containing and frc-lacking bacteria. The frc-primers were then used to develop PCR-DGGE and real-time SybrGreen PCR assays in soils containing various amounts of oxalate. Some PCR products from pure cultures and from soil samples were cloned and sequenced. Data were used to generate a phylogenetic tree showing that environmental PCR products belonged to the target physiological group. The extent of diversity visualised on DGGE pattern was higher for soil samples containing carbonate resulting from oxalate catabolism. Moreover, the amount of frc gene copies in the investigated soils was detected in the range of 1.64x10(7) to 1.75x10(8)/g of dry soil under oxalogenic tree (representing 0.5 to 1.2% of total 16S rRNA gene copies), whereas the number of frc gene copies in the reference soil was 6.4x10(6) (or 0.2% of 16S rRNA gene copies). This indicates that oxalotrophic bacteria are numerous and widespread in soils and that a relationship exists between the presence of the oxalogenic trees Milicia excelsa and Afzelia africana and the relative abundance of oxalotrophic guilds in the total bacterial communities. This is obviously related to the accomplishment of the oxalate-carbonate pathway, which explains the alkalinization and calcium carbonate accumulation occurring below these trees in an otherwise acidic soil. The molecular tools developed in this study will allow in-depth understanding of the functional implication of these bacteria on carbonate accumulation as a way of atmospheric CO(2) sequestration.     

Oxalate utilisation is widespread in the actinobacterial genus Kribbella

The type strains of all 33 species in the genus Kribbella were tested for growth on oxalate (^?OOC-COO^?) as sole carbon source. Media were initially formulated to contain sodium oxalate, but even a concentration as low as 7.5 mM oxalate prevented growth. A modified medium based on calcium oxalate was very successful in characterising oxalate utilisation by Kribbella strains (metabolism of oxalate by oxalotrophic bacteria results in visible zones of clearing around the growth streaks on the opaque plates). To assess the variability of oxalate utilisation in Kribbella species, we also tested eight non-type strains for their ability to use oxalate. Thirty of 33 type strains (90.9%) and six of eight non-type strains (75%) were able to use oxalate as a sole carbon source. Based on these results, we propose that oxalate would be an excellent carbon source for the selective isolation of Kribbella strains. Based on the oxalate-utilisation phenotype and analyses of the 19 publicly available Kribbella type-strain genome sequences, we propose a pathway for oxalate metabolism in Kribbella. This pathway is significantly different from those previously proposed for oxalate metabolism in other bacteria, involving the indirect catabolism of oxalate to formate. Formate production is proposed to be involved in energy generation and to be crucial for oxalate import via an oxalate:formate antiporter. To our knowledge, this is the first report of an oxalate:formate antiporter in an aerobic, Gram-positive bacterium.     

Thermodynamic efficiency of bacterial growth calculated from growth yield of Pseudomonas oxalaticus OX1 in the chemostat

In order to determine the thermodynamic efficiency of bacterial growth, Pseudomonas oxalaticus OX1 was grown in carbon-limited continuous cultures. 11 different carbon sources, ranging from oxalate (most oxidised component) to ethanol (most reduced component), were used as limiting substrate in these experiments. From the experimental yield values (expressed as C-mol dry weight produced per C-mol carbon substrate consumed) the thermodynamic efficiencies were calculated. On substrates more reduced than biomass (such as ethanol and glycerol) the thermodynamic efficiency of growth of P. oxalaticus was negative but it reached a maximum of 23 +/- 3% with substrates with a degree of reduction of 3 (citrate) and lower. The actual concentrations of the components involved were incorporated into the calculations but this affected the overall thermodynamic efficiency only to a small extent. This result strengthens the conclusion of Westerhoff et al. (Westerhoff, H.V., Hellingwerf, K.J. and Van Dam, K. (1983) Proc. Natl. Acad. Sci. 80, 305-309) that bacteria have been optimised towards a theoretical thermodynamic efficiency of 24%, corresponding with maximisation of growth rate at optimal efficiency, with highly oxidised substrates.     

Carbonate Accumulation in the Bark of Terminalia bellirica: A New Habitat for the Oxalate-Carbonate Pathway

Oxalate oxidation and carbonate precipitation associated with the oxalogenic tree Terminalia bellirica were investigated. Calcium oxalate crystals, oxalotrophic bacteria (dominated by genera Methylobacterium and Burkholderia), and carbonate accumulation (82% dry weight), were detected in the bark. In contrast, only a slight accumulation of carbonate was observed in soil (1.5%). The combined geochemical and microbiological analyses suggest that bark of an Indian living tree represents a novel habitat for the accumulation of carbonate associated with bacterial oxalate oxidation. The importance of these types of habitats needs to be quantified when considering the biogeochemical cycling of carbon and calcium in tropical ecosystems.     

Monitoring the Size and Metabolic Activity of the Bacterial Community during Biostimulation of Fuel-Contaminated Soil using Competitive PCR and RT-PCR

Efforts to understand and improve soil bioremediation are limited by our ability to determine how treatment variables affect microbial communities. A method was developed to monitor the density and metabolic activity of the total bacterial community in soil. This method was used to monitor the bacterial community in microcosms of Arctic soil after addition of N plus P to stimulate biodegradation of hydrocarbon contaminants. During 29 days of incubation, the total petroleum hydrocarbon level in the soil was reduced from 850 to 360 mg/g of soil. DNA and RNA were extracted from soil using a bead beating method, purified by ammonium acetate precipitation, and assayed by competitive PCR and RT-PCR assays with universal bacterial primers. The copy number of 16S rDNA in the soil microbial community was relatively stable and ranged from 1.7 × 109 to 4.5 × 109/g of soil throughout the incubation. The copy number of 16S rRNA changed substantially and ranged from 5.6 × 1010 to 1.0 × 1012/g of soil. The rRNA:rDNA ratio was highest during the phase of fastest hydrocarbon biodegradation. These results suggest that the treatment to stimulate hydrocarbon biodegradation did not substantially change the density of the bacterial community but did transiently increase its overall metabolic activity.     

Effect of nitrogen and/or oxygen concentration on poly(3-hydroxybutyrate) accumulation by <Emphasis Type="Italic">Halomonas boliviensis</Emphasis>

The behaviour of Halomonas boliviensis during growth in fed-batch culture under different kind of nutrient restrictions was examined. The metabolic switch between growth and accumulation phase is determined by the limitation in one or more essential nutrient for bacterial growth. The aim of this study was to test the effect of applying limitations of a essential nutrient, such as nitrogen, and the influence of different O₂ concentrations on poly(3-hydroxybutyrate) (PHB) production during the accumulation phase. Single limitations of nitrogen and oxygen provoke PHB accumulations of 45 and 37 % (g g^?1), respectively, while N limitation with low O₂ supply causes the highest PHB accumulation of 73 %. The characterization of the PHB production with the strain H. boliviensis would allow a better optimization of the process and enrich the knowledge about the PHB production from strains different than Cupriavidus necator.     

Conjugal transfer of hydrogen-oxidizing ability of Alcaligenes hydrogenophilus to Pseudomonas oxalaticus

Conjugal transfer of hydrogen-oxidizing ability (Hox) of the hydrogen bacterium Alcaligenes hydrogenophilus was examined. Intraspecific cross of plasmid pHG21-a that encodes hydrogenases that mediate hydrogen oxidation was most frequent at 25 C; the optimal temperature for growth was 30 C. The plasmid could be transferred from A. hydrogenophilus to Pseudomonas oxalaticus OX1 and OX4, and the resulting strains gained the capacity for autotrophic growth with H2 and CO2. Plasmid pHG21-a was maintained in P. oxalaticus OX1 and OX4 as stably as in A. hydrogenophilus.     

暗褐网柄牛肝菌子实体内生细菌群落结构研究

暗褐网柄牛肝菌（Phlebopus portentosus）是第一个栽培成功的牛肝菌目（Boletales）真菌,一般采用覆土栽培模式,覆土微生物对子实体的发育具有重要作用,目前的研究主要集中在覆土微生物上,但缺少对子实体内生菌的研究。为揭开暗褐网柄牛肝菌子实体内生菌的组成,本研究基于Illumina MiSeq平台,利用16S rRNA V3~V4区高通量测序进行细菌群落结构分析。结果表明子实体（fruiting body,FB）内部存在很高丰度的细菌,主要以变形菌门（Proteobacteria）为主,在属水平上以贪铜菌属（Cupriavidus）（64.98%）、不动杆菌属（Acinetobacter）（7.62%）、苍白杆菌属（Ochrobactrum）（6.02%）为主。与覆土（casing soil,CS）基质固有的细菌种群进行比较,VENN分析显示子实体内有468个OTUs为独有,占总序列的26.65%,分类上隶属于贪铜菌属、不动杆菌属和Delftia。FB和CS差异性分析以及Lefse标志性微生物分析均显示贪铜菌属、苍白杆菌属、不动杆菌属、嗜糖假单胞菌属（Pelomonas）等为FB特异性、标志性类群。利用Picrust（Phylogenetic investigation of communities by reconstruction of unobserved states）进行Kyoto Encyclopedia of Genes and Genomes（KEGG）通路预测发现,子实体内生菌群的主要代谢途径为膜传输、氨基酸代谢、碳水化合物代谢、外源生物降解与代谢,与覆土菌群有20个代谢通路的差异,并且外源生物降解与代谢、信号转导、其他氨基酸代谢、脂质代谢、细胞过程和信号5个通路的比例要高于覆土,微生物类群的差异可能是导致产生这些功能差异的原因。     

Isolation of Oxalic acid tolerating fungi and decipherization of its potential to control Sclerotinia sclerotiorum through oxalate oxidase like protein

Oxalic acid plays major role in the pathogenesis by Sclerotinia sclerotiorum; it lowers the pH of nearby environment and creates the favorable condition for the infection. In this study we examined the degradation of oxalic acid through oxalate oxidase and biocontrol of Sclerotinia sclerotiorum. A survey was conducted to collect the rhizospheric soil samples from Indo-Gangetic Plains of India to isolate the efficient fungal strains able to tolerate oxalic acid. A total of 120 fungal strains were isolated from root adhering soils of different vegetable crops. Out of 120 strains a total of 80 isolates were able to grow at 10?mM of oxalic acid whereas only 15 isolates were grow at 50?mM of oxalic acid concentration. Then we examined the antagonistic activity of the 15 isolates against Sclerotinia sclerotiorum. These strains potentially inhibit the growth of the test pathogen. A total of three potential strains and two standard cultures of fungi were tested for the oxalate oxidase activity. Strains S7 showed the maximum degradation of oxalic acid (23?%) after 60?min of incubation with fungal extract having oxalate oxidase activity. Microscopic observation and ITS (internally transcribed spacers) sequencing categorized the potential fungal strains into the Aspergillus, Fusarium and Trichoderma. Trichoderma sp. are well studied biocontrol agent and interestingly we also found the oxalate oxidase type activity in these strains which further strengthens the potentiality of these biocontrol agents.     

Coexistence of Burkholderia, Cupriavidus, and Rhizobium sp. nodule bacteria on two Mimosa spp. in Costa Rica

rRNA gene sequencing and PCR assays indicated that 215 isolates of root nodule bacteria from two Mimosa species at three sites in Costa Rica belonged to the genera Burkholderia, Cupriavidus, and Rhizobium. This is the first report of Cupriavidus sp. nodule symbionts for Mimosa populations within their native geographic range in the neotropics. Burkholderia spp. predominated among samples from Mimosa pigra (86% of isolates), while there was a more even distribution of Cupriavidus, Burkholderia, and Rhizobium spp. on Mimosa pudica (38, 37, and 25% of isolates, respectively). All Cupriavidus and Burkholderia genotypes tested formed root nodules and fixed nitrogen on both M. pigra and M. pudica, and sequencing of rRNA genes in strains reisolated from nodules verified identity with inoculant strains. Inoculation tests further indicated that both Cupriavidus and Burkholderia spp. resulted in significantly higher plant growth and nodule nitrogenase activity (as measured by acetylene reduction assays) relative to plant performance with strains of Rhizobium. Given the prevalence of Burkholderia and Cupriavidus spp. on these Mimosa legumes and the widespread distribution of these plants both within and outside the neotropics, it is likely that both beta-proteobacterial genera are more ubiquitous as root nodule symbionts than previously believed.     

Effect of the insecticide actellic on soil microorganisms and their activity

The insecticide Actellic or its active component pirimiphosmethyl affected the growth and reproduction of some soil bacteria and fungi, fixation of atmospheric nitrogen, nitrification and overall soil metabolic activity. Both bacterial growth and the activities under study were negatively influenced by high concentrations of the agent (as compared with actual pirimiphosmethyl concentrations routinely used in agriculture) whereas lower concentrations stimulated the overall metabolic soil activity.     

Simultaneous Monitoring of Cell Number and Metabolic Activity of Specific Bacterial Populations with a Dual gfp-luxAB Marker System

A dual marker system was developed for simultaneous quantification of bacterial cell numbers and their activity with the luxAB and gfp genes, encoding bacterial luciferase and green fluorescent protein (GFP), respectively. The bioluminescence phenotype of the luxAB biomarker is dependent on cellular energy status. Since cellular metabolism requires energy, bioluminescence output is directly related to the metabolic activity of the cells. By contrast, GFP fluorescence has no energy requirement. Therefore, by combining these two biomarkers, total cell number and metabolic activity of a specific marked cell population could be monitored simultaneously. Two different bacterial strains, Escherichia coli DH5α and Pseudomonas fluorescens SBW25, were chromosomally tagged with the dual marker cassette, and the cells were monitored under different conditions by flow cytometry, plate counting, and luminometry. During log-phase growth, the luciferase activity was proportional to the number of GFP-fluorescent cells and culturable cells. Upon entrance into stationary phase or during starvation, luciferase activity decreased due to a decrease in cellular metabolic activity of the population, but the number of GFP-fluorescing cells and culturable cells remained relatively stable. In addition, we optimized a procedure for extraction of bacterial cells from soil, allowing GFP-tagged bacteria in soil samples to be quantitated by flow cytometry. After 30 days of incubation of P. fluorescens SBW25::gfp/lux in soil, the cells were still maintained at high population densities, as determined by GFP fluorescence, but there was a slow decline in luciferase activity, implicating nutrient limitation. In conclusion, the dual marker system allowed simultaneous monitoring of the metabolic activity and cell number of a specific bacterial population and is a promising tool for monitoring of specific bacteria in situ in environmental samples.     

Isolation and selection of an L-aminoacylase-producing bacterium, Pseudomonas sp. BA2

The enrichment culture method was used to detect and isolate L-aminoacylase-producing bacteria from soil, using N -acetyl-L-alanine as inducer and substrate. Isolated bacterial strains were screened for growth and enzyme activity. Strain BA2 displayed both the highest intracellular L-aminoacylase activity and the most profuse growth. Furthermore, BA2 cells did not show any D-aminoacylase activity. This strain was an obligately aerobic rod-shaped bacterium and stained Gram-negative, and was therefore identified as Pseudomonas. Its morphological and biochemical characteristics corresponded to those of Pseudomonas fluorescens biovar I.     

Impact of rhizobial inoculation on Acacia senegal (L.) Willd. growth in greenhouse and soil functioning in relation to seed provenance and soil origin

Rhizobial inoculation has a positive impact on plants growth; however, there is little information about its effect on soil microbial communities and their activity in the rhizosphere. It was therefore necessary to test the effect of inoculation of Acacia senegal (L.) Willd. seedlings with selected rhizobia on plant growth, structure and diversity of soil bacterial communities and soil functioning in relation to plant provenance and soil origin. In order to carry out this experiment, three A. senegal seeds provenance from Kenya, Niger, and Senegal were inoculated with selected rhizobial strains. They have been further grown during 4 months in greenhouse conditions in two non-disinfected soils, Dahra and Goudiry coming respectively from arid and semi-arid areas. The principal component analysis (ACP) showed an inoculation effect on plant growth, rhizospheric bacterial diversity and soil functioning. However, the performances of the rhizobial strains varied in relation to the seed provenance and the soil origin. The selected rhizobial strains, the A. senegal provenance and the soil origin have modified the structure and the diversity of soil bacterial communities as measured by principal component analysis/denaturing gradient gel electrophoresis analyses. It is interesting to note that bacterial communities of Dahra soil were highly structured according to A. senegal provenance, whereas they were structured in relation to rhizobial inoculation in Goudiry soil. Besides, the impact of inoculation on soil microbial activities measured by fluorescein diacetate analyses varied in relation to plant provenance and soil origin. Nevertheless, total microbial activity was about two times higher in Goudiry, arid soil than in Dahra, semi-arid soil. Our results suggest that the rhizobial inoculation is a suitable tool for improving plants growth and soil fertility. Yet, the impact is dependent on inoculants, plant provenance and soil origin. It will, therefore, be crucial to identify the appropriate rhizobial strains and plant provenance or species in relation to the soil type.     

Burkholderia spp. are the most competitive symbionts of Mimosa, particularly under N-limited conditions

Bacteria isolated from Mimosa nodules in Taiwan, Papua New Guinea, Mexico and Puerto Rico were identified as belonging to either the α- or β-proteobacteria. The β-proteobacterial Burkholderia and Cupriavidus strains formed effective symbioses with the common invasive species Mimosa diplotricha , M. pigra and M. pudica , but the α-proteobacterial Rhizobium etli and R. tropici strains produced a range of symbiotic phenotypes from no nodulation through ineffective to effective nodulation, depending on Mimosa species. Competition studies were performed between three of the α-proteobacteria ( R. etli TJ167, R. tropici NGR181 and UPRM8021) and two of the β-rhizobial symbionts ( Burkholderia mimosarum PAS44 and Cupriavidus taiwanensis LMG19424) for nodulation of these invasive Mimosa species. Under flooded conditions, B. mimosarum PAS44 out-competed LMG19424 and all three α-proteobacteria to the point of exclusion. This advantage was not explained by initial inoculum levels, rates of bacterial growth, rhizobia-rhizobia growth inhibition or individual nodulation rate. However, the competitive domination of PAS44 over LMG19424 was reduced in the presence of nitrate for all three plant hosts. The largest significant effect was for M. pudica , in which LMG19424 formed 57% of the nodules in the presence of 0.5 mM potassium nitrate. In this host, ammonium also had a similar, but lesser, effect. Comparable results were also found using an N-containing soil mixture, and environmental N levels are therefore suggested as a factor in the competitive success of the bacterial symbiont in vivo .     

Isolation and Characterization of New Cyclohexylacetic Acid-Degrading Bacteria

Six cyclohexylacetic acid-degrading strains were isolated from soil samples in Japan and identified as members of the genera Cupriavidus (strain KUA-1), Rhodococcus, and Dietzia by 16S rRNA gene sequence analysis. For the first time members of these genera were shown to be capable of degrading cyclohexylacetic acid. A selected strain, KUA-1, which is the first reported Gram-negative organism capable of growth on cyclohexylacetic acid, was identified as a Cupriavidus metallidurans, based on morphologic and physiologic characteristics and its 16S rRNA gene sequence. Metabolite analysis by HPLC-MS indicated that 1-cyclohexenylacetic acid is an intermediate of cyclohexaneacetic acid metabolism in strain KUA-1.     

Development of antioxidant activity in milk whey during fermentation with lactic acid bacteria

AIMS: To investigate the production of antioxidant activity during fermentation with commonly used dairy starter cultures. Moreover, to study the development of antioxidant activity during fermentation, and the connection to proteolysis and bacterial growth. METHODS AND RESULTS: Antioxidant activity was measured by analysing the radical scavenging activity using a spectrophotometric decolorization assay and lipid peroxidation inhibition was assayed using liposomal model system with a fluorescence method. Milk was fermented with 25 lactic acid bacterial (LAB) strains, and from these six strains, exhibiting the highest radical scavenging activity was selected for further investigation. Leuconostoc mesenteroides ssp. cremoris strains, Lactobacillus jensenii (ATCC 25258) and Lactobacillus acidophilus (ATCC 4356) showed the highest activity with both the methods used. However, the radical scavenging activity was stronger than lipid peroxidation inhibition activity. The development of radical scavenging activity was connected to proteolysis with four strains. Molecular distribution profiles showed that fermentates with high scavenging activity also possessed a higher proportion of peptides in the molecular mass range of 4-20 kDa, while others had mostly large polypeptides and compounds below 4 kDa. In addition, the amount of hydrophobic amino acids was higher in these fermentates. CONCLUSIONS: The development of antioxidant activity was strain-specific characteristic. The development of radical scavengers was more connected to the simultaneous development of proteolysis whereas, lipid peroxidation inhibitory activity was related to bacterial growth. However, high radical scavenging activity was not directly connected to the high degree of proteolysis. SIGNIFICANCE AND IMPACT OF THE STUDY: To the best of our knowledge, this seems to be the first report, which screens possible antioxidant activity among most common dairy LAB strains. Use of such strains improve nutritional value of fermented dairy products.