Marcela Hernández

Isolation of culturable phosphobacteria with both phytate-mineralization and phosphate-solubilization activity from the rhizosphere of plants grown in a volcanic soil

Chilean volcanic soils contain large amounts of total and organic phosphorus, but P availability is low. Phosphobacteria [phytate-mineralizing bacteria (PMB) and phosphate-solubilizing bacteria (PSB)] were isolated from the rhizosphere of perennial ryegrass (Lolium perenne), white clover (Trifolium repens), wheat (Triticum aestivum), oat (Avena sativa), and yellow lupin (Lupinus luteus) growing in volcanic soil. Six phosphobacteria were selected, based on their capacity to utilize both Na-phytate and Ca-phosphate on agar media (denoted as PMPSB), and characterized. The capacity of selected PMPSB to release inorganic P (Pi) from Na-phytate in broth was also assayed. The results showed that from 300 colonies randomly chosen on Luria–Bertani agar, phosphobacteria represented from 44% to 54% in perennial ryegrass, white clover, oat, and wheat rhizospheres. In contrast, phosphobacteria represented only 17% of colonies chosen from yellow lupin rhizosphere. This study also revealed that pasture plants (perennial ryegrass and white clover) have predominantly PMB in their rhizosphere, whereas PSB dominated in the rhizosphere of crops (oat and wheat). Selected PMPSB were genetically characterized as Pseudomonas, Enterobacter, and Pantoea; all showed the production of phosphoric hydrolases (alkaline phosphatase, acid phosphatase, and naphthol phosphohydrolase). Assays with PMPSB resulted in a higher Pi liberation compared with uninoculated controls and revealed also that the addition of glucose influenced the Pi-liberation capacity of some of the PMPSB assayed.

Different bacterial populations associated with the roots and rhizosphere of rice incorporate plant-derived carbon

ABSTRACT Microorganisms associated with the roots of plants have an important function in plant growth and in soil carbon sequestration. Rice cultivation is the second largest anthropogenic source of atmospheric CH4, which is a significant greenhouse gas. Up to 60% of fixed carbon formed by photosynthesis in plants is transported below ground, much of it as root exudates that are consumed by microorganisms. A stable isotope probing (SIP) approach was used to identify microorganisms using plant carbon in association with the roots and rhizosphere of rice plants. Rice plants grown in Italian paddy soil were labeled with 13CO2 for 10 days. RNA was extracted from root material and rhizosphere soil and subjected to cesium gradient centrifugation followed by 16S rRNA amplicon pyrosequencing to identify microorganisms enriched with 13C. Thirty operational taxonomic units (OTUs) were labeled and mostly corresponded to Proteobacteria (13 OTUs) and Verrucomicrobia (8 OTUs). These OTUs were affiliated with the Alphaproteobacteria, Betaproteobacteria, and Deltaproteobacteria classes of Proteobacteria and the “Spartobacteria” and Opitutae classes of Verrucomicrobia. In general, different bacterial groups were labeled in the root and rhizosphere, reflecting different physicochemical characteristics of these locations. The labeled OTUs in the root compartment corresponded to a greater proportion of the 16S rRNA sequences (∼20%) than did those in the rhizosphere (∼4%), indicating that a proportion of the active microbial community on the roots greater than that in the rhizosphere incorporated plant-derived carbon within the time frame of the experiment.

Autotrophic Growth of Bacterial and Archaeal Ammonia Oxidizers in Freshwater Sediment Microcosms Incubated at Different Temperatures

ABSTRACT Both bacteria and archaea potentially contribute to ammonia oxidation, but their roles in freshwater sediments are still poorly understood. Seasonal differences in the relative activities of these groups might exist, since cultivated archaeal ammonia oxidizers have higher temperature optima than their bacterial counterparts. In this study, sediment collected from eutrophic freshwater Lake Taihu (China) was incubated at different temperatures (4°C, 15°C, 25°C, and 37°C) for up to 8 weeks. We examined the active bacterial and archaeal ammonia oxidizers in these sediment microcosms by using combined stable isotope probing (SIP) and molecular community analysis. The results showed that accumulation of nitrate in microcosms correlated negatively with temperature, although ammonium depletion was the same, which might have been related to enhanced activity of other nitrogen transformation processes. Incubation at different temperatures significantly changed the microbial community composition, as revealed by 454 pyrosequencing targeting bacterial 16S rRNA genes. After 8 weeks of incubation, [13C]bicarbonate labeling of bacterial amoA genes, which encode the ammonia monooxygenase subunit A, and an observed increase in copy numbers indicated the activity of ammonia-oxidizing bacteria in all microcosms. Nitrosomonas sp. strain Is79A3 and Nitrosomonas communis lineages dominated the heavy fraction of CsCl gradients at low and high temperatures, respectively, indicating a niche differentiation of active bacterial ammonia oxidizers along the temperature gradient. The 13C labeling of ammonia-oxidizing archaea in microcosms incubated at 4 to 25°C was minor. In contrast, significant 13C labeling of Nitrososphaera-like archaea and changes in the abundance and composition of archaeal amoA genes were observed at 37°C, implicating autotrophic growth of ammonia-oxidizing archaea under warmer conditions.

Microbial Diversity in Hummock and Hollow Soils of Three Wetlands on the Qinghai-Tibetan Plateau Revealed by 16S rRNA Pyrosequencing

The wetlands of the Qinghai-Tibetan Plateau are believed to play an important role in global nutrient cycling, but the composition and diversity of microorganisms in this ecosystem are poorly characterized. An understanding of the effects of geography and microtopography on microbial populations will provide clues to the underlying mechanisms that structure microbial communities. In this study, we used pyrosequencing-based analysis of 16S rRNA gene sequences to assess and compare the composition of soil microbial communities present in hummock and hollow soils from three wetlands (Dangxiong, Hongyuan and Maduo) on the Qinghai-Tibetan Plateau, the world’s highest plateau. A total of 36 bacterial phyla were detected. Proteobacteria (34.5% average relative abundance), Actinobacteria (17.3%) and Bacteroidetes (11%) had the highest relative abundances across all sites. Chloroflexi, Acidobacteria, Verrucomicrobia, Firmicutes, and Planctomycetes were also relatively abundant (1–10%). In addition, archaeal sequences belonging to Euryarchaea, Crenarchaea and Thaumarchaea were detected. Alphaproteobacteria sequences, especially of the order Rhodospirillales, were significantly more abundant in Maduo than Hongyuan and Dangxiong wetlands. Compared with Hongyuan soils, Dangxiong and Maduo had significantly higher relative abundances of Gammaproteobacteria sequences (mainly order Xanthomonadales). Hongyuan wetland had a relatively high abundance of methanogens (mainly genera Methanobacterium, Methanosarcina and Methanosaeta) and methanotrophs (mainly Methylocystis) compared with the other two wetlands. Principal coordinate analysis (PCoA) indicated that the microbial community structure differed between locations and microtopographies and canonical correspondence analysis indicated an association between microbial community structure and soil properties or geography. These insights into the microbial community structure and the main controlling factors in wetlands of the Qinghai-Tibetan Plateau provide a valuable background for further studies on biogeochemical processes in this distinct ecosystem.

Ammonia oxidizers are pioneer microorganisms in the colonization of new acidic volcanic soils from South of Chile

Ammonia oxidation, performed by specialized microorganisms belonging to the Bacteria and Archaea, is the first and most limiting step of soil nitrification. Nitrification has not yet been examined in young volcanic soils. The aim of the present work was to evaluate the abundance and diversity of ammonia-oxidizing bacteria (AOB) and archaea (AOA) in acidic volcanic soils (andisols) of different defined ages to determine their relative contribution to nitrification and soil colonization. Soil was collected from three vegetated sites on Llaima Volcano (Chile) recolonized after lava eruptions in 1640, 1751 and 1957. Quantitative polymerase chain reaction, terminal restriction fragment length polymorphism and clone sequence analyses of the amoA gene were performed for the AOA and AOB communities. All soils showed high nitrification potentials, but they were highest in the younger soils. Archaeal amoA genes outnumbered bacterial amoA genes at all sites, and AOA abundances were found to be proportional to the nitrification potentials. Sequencing indicated the presence of AOA related to Nitrososphaera and Nitrosotalea, and AOB related primarily to Nitrosospira and sporadically to Nitrosomonas. The study showed that both AOA and AOB are early colonizers of andisols, but that AOA outnumber AOB and play an important role in nitrification.

Response of methanogenic microbial communities to desiccation stress in flooded and Rain-fed paddy soil from Thailand

Rice paddies in central Thailand are flooded either by irrigation (irrigated rice) or by rain (rain-fed rice). The paddy soils and their microbial communities thus experience permanent or arbitrary submergence, respectively. Since methane production depends on anaerobic conditions, we hypothesized that structure and function of the methanogenic microbial communities are different in irrigated and rain-fed paddies and react differently upon desiccation stress. We determined rates and relative proportions of hydrogenotrophic and aceticlastic methanogenesis before and after short-term drying of soil samples from replicate fields. The methanogenic pathway was determined by analyzing concentrations and δ13C of organic carbon and of CH4 and CO2 produced in the presence and absence of methyl fluoride, an inhibitor of aceticlastic methanogenesis. We also determined the abundance (qPCR) of genes and transcripts of bacterial 16S rRNA, archaeal 16S rRNA and methanogenic mcrA (coding for a subunit of the methyl coenzyme M reductase) and the composition of these microbial communities by T-RFLP fingerprinting and/or Illumina deep sequencing. The abundances of genes and transcripts were similar in irrigated and rain-fed paddy soil. They also did not change much upon desiccation and rewetting, except the transcripts of mcrA, which increased by more than two orders of magnitude. In parallel, rates of CH4 production also increased, in rain-fed soil more than in irrigated soil. The contribution of hydrogenotrophic methanogenesis increased in rain-fed soil and became similar to that in irrigated soil. However, the relative microbial community composition on higher taxonomic levels was similar between irrigated and rain-fed soil. On the other hand, desiccation and subsequent anaerobic reincubation resulted in systematic changes in the composition of microbial communities for both Archaea and Bacteria. It is noteworthy that differences in the community composition were mostly detected on the level of operational taxonomic units (OTUs; 97% sequence similarity). The treatments resulted in change of the relative abundance of several archaeal OTUs. Some OTUs of Methanobacterium, Methanosaeta, Methanosarcina, Methanocella and Methanomassiliicoccus increased, while some of Methanolinea and Methanosaeta decreased. Bacterial OTUs within Firmicutes, Cyanobacteria, Planctomycetes and Deltaproteobacteria increased, while OTUs within other proteobacterial classes decreased.

Enhancing functional metagenomics of complex microbial communities using stable isotopes

Exploring the function of genes encoded by uncultivated microorganisms is one of the major challenges facing microbiologists. Functions can be predicted by sequence comparisons to known genes and proteins, but proof of function requires the analysis of gene products by in vitro or in vivo expression, which is referred to as functional metagenomics. Using this approach, genetic material is retrieved from the environment, cloned, and expressed under laboratory conditions in order to screen for specific biochemical activities. Stable-isotope probing (SIP) is an approach for capturing genetic material of active microorganisms in environmental samples. This method facilitates functional metagenomics by directing the search toward microorganisms that are likely to possess genes of relevance to a specific research objective. In this chapter, we discuss how combined DNA-SIP and metagenomic research has been used for enhancing functional screening efforts. In addition, we highlight emerging methods, such as mRNA-SIP and Raman microspectroscopy, that can help retrieve genetic material from targeted microbial groups for the discovery of novel functions.