Felipe Bastida

Biochar influences the microbial community structure during manure composting with agricultural wastes.

The influence of biochar derived from a hardwood tree (Quercus serrate Murray) on the dynamics of the microbial community during the composting of poultry manure (PM) and cow manure (CM) was evaluated by phospholipid fatty acid analysis (PLFAs). Changes in the PLFA patterns were related to key composting properties (C/N ratio, temperature, and bulk density) as the major drivers of the dynamics of the microbial community. At the beginning of the process, the fungal biomass was significantly greater in PM and CM than in the respective co-composted materials with biochar (PMB and CMB); this difference declined gradually during the process. In contrast, the Gram+ to Gram- ratio was increased by the presence of biochar. After 12 weeks of composting, factor analysis based on the relative abundances of single PLFAs revealed changes in the microbial community structure which depended on the original organic wastes (CM vs PM).

Protein-based stable isotope probing

We describe a stable isotope probing (SIP) technique that was developed to link microbe-specific metabolic function to phylogenetic information. Carbon (13C)- or nitrogen (15N)-labeled substrates (typically with >98% heavy label) were used in cultivation experiments and the heavy isotope incorporation into proteins (protein-SIP) on growth was determined. The amount of incorporation provides a measure for assimilation of a substrate, and the sequence information from peptide analysis obtained by mass spectrometry delivers phylogenetic information about the microorganisms responsible for the metabolism of the particular substrate. In this article, we provide guidelines for incubating microbial cultures with labeled substrates and a protocol for protein-SIP. The protocol guides readers through the proteomics pipeline, including protein extraction, gel-free and gel-based protein separation, the subsequent mass spectrometric analysis of peptides and the calculation of the incorporation of stable isotopes into peptides. Extraction of proteins and the mass fingerprint measurements of unlabeled and labeled fractions can be performed in 2–3 d.

Insights from quantitative metaproteomics and protein-stable isotope probing into microbial ecology

The recent development of metaproteomics has enabled the direct identification and quantification of expressed proteins from microbial communities in situ, without the need for microbial enrichment. This became possible by (1) significant increases in quality and quantity of metagenome data and by improvements of (2) accuracy and (3) sensitivity of modern mass spectrometers (MS). The identification of physiologically relevant enzymes can help to understand the role of specific species within a community or an ecological niche. Beside identification, relative and absolute quantitation is also crucial. We will review label-free and label-based methods of quantitation in MS-based proteome analysis and the contribution of quantitative proteome data to microbial ecology. Additionally, approaches of protein-based stable isotope probing (protein-SIP) for deciphering community structures are reviewed. Information on the species-specific metabolic activity can be obtained when substrates or nutrients are labeled with stable isotopes in a protein-SIP approach. The stable isotopes (13C, 15N, 36S) are incorporated into proteins and the rate of incorporation can be used for assessing the metabolic activity of the corresponding species. We will focus on the relevance of the metabolic and phylogenetic information retrieved with protein-SIP studies and for detecting and quantifying the carbon flux within microbial consortia. Furthermore, the combination of protein-SIP with established tools in microbial ecology such as other stable isotope probing techniques are discussed.

Elucidating MTBE degradation in a mixed consortium using a multidisciplinary approach

The structure and function of a microbial community capable of biodegrading methyl-tert-butyl ether (MTBE) was characterized using compound-specific stable isotope analysis (CSIA), clone libraries and stable isotope probing of proteins (Protein-SIP). The enrichment culture (US3-M), which originated from a gasoline-impacted site in the United States, has been enriched on MTBE as the sole carbon source. The slope of isotopic enrichment factors (epsilon(C) of -2.29+/-0.03 per thousand; epsilon(H) of -58+/-6 per thousand) for carbon and hydrogen discrimination (Deltadelta(2)H/Deltadelta(13)C) was on average equal to Lambda=24+/-2, a value closely related to the reaction mechanism of MTBE degradation in Methylibium petroleiphilum PM1. 16S rRNA gene libraries revealed sequences belonging to M. petroleiphilum PM1, Hydrogenophaga sp., Thiothrix unzii, Rhodobacter sp., Nocardiodes sp. and different Sphingomonadaceae bacteria. Protein-SIP analysis of the culture grown on (13)C-MTBE as the only carbon source revealed that proteins related to members of the Comamonadaceae family, such as Delftia acidovorans, Acidovorax sp. or Comamonas sp., were not (13)C-enriched, whereas proteins related to M. petroleiphilum PM1 showed an average incorporation of 94.5 atom%(13)C. These results indicate a key role for this species in the degradation of MTBE within the US3-M consortia. The combination of CSIA, molecular biology and Protein-SIP facilitated the analysis of an MTBE-degrading mixed culture from a functional and phylogenetic point of view.

Metaproteomics of soils from semiarid environment: Functional and phylogenetic information obtained with different protein extraction methods

UNLABELLED Microbial populations fulfil a critical role in the soil sustainability and their functionality can be ascertained by proteomics based on high-performance mass spectrometry (MS) measurements. However, soil proteomics is compromised by methodological issues, among which extraction is a limiting factor, and still has not been adequately applied in semiarid soils, which usually are nutrient limited. We aim to evaluate the functional and phylogenetic information retrieved from three semiarid soils with distinct edaphic properties and degradation levels. Three extraction methods with different physico-chemical bases were tested [1-3]. The HPLC-amino acid quantification of the extracted protein pellets revealed a tremendous inefficiency of the extraction methods, with a maximally 6.8% of the proteinaceous material being extracted in comparison with the protein content in the bulk soil. The composition of the proteomes extracted was analysed after SDS-PAGE and liquid chromatography coupled with electrospray-MS/MS. Choureys method, based on boiling and DTT, yielded a high diversity of bacterial proteins and revealed differences in the community composition at the phylum level among the three soils. The overall metabolic information obtained by both extraction methods was similar, but Choureys method provided additionally valuable bio-geochemical insights which suggest an ecological adaptation of microbial communities from semiarid soils for carbon and nitrogen fixation. BIOLOGICAL SIGNIFICANCE Microbial communities inhabiting the soil perform critical reactions for the sustainability of the planet. At biochemical level, soil proteomics is starting to provide incipient insights into the microbial functionality of soils. However, methodological comparisons are needed to assess which methods are more suitable. Precisely, such information under arid and semiarid environments is missing. By using amino acid quantification of extracted proteomes and LC-MS/MS based proteomics, we provide a novel methodological evaluation of the functional, phylogenetic and bio-geochemical information obtained by three extraction methods in semiarid soils with distinct edaphic properties.

Soil microbial community structure and activity in monospecific and mixed forest stands, under Mediterranean humid conditions

AimsWe investigated the link between tree community composition and soil microbial community biomass and structure in central-eastern Spain.MethodsThe effects of the forest stand composition on the soil organic matter dynamics and on the structure and activity of the soil microbial community have been determined using phospholipid fatty acid profiles and soil enzymatic activities.ResultsThe soil and litter N and C contents were higher in Pinus nigra Arn. ssp. salzmannii and Quercus ilex mixed forest stands (SBHO) and in long-term unmanaged Pinus nigra Arn. ssp. salzmannii forest stands (SBPC) than in pure Pinus nigra Arn. ssp. salzmannii forest stands (SBPA) and Pinus nigra Arn. ssp. salzmannii and Juniperus thurifera mixed forest stands (SBSJ). The bacterial biomass was significantly higher in SBSJ and SBPA than in SBPC and SBHO. The results show an uncoupling of the soil microbial biomass and its activity. pH is related to microbial biomass and its community structure under a Mediterranean humid climate.ConclusionsThe tree species seem to affect the biomass of the soil microbial community and its structure. The pH, but not the C/N ratio, is a factor influencing the microbial dynamics, biomass, and community structure.

Addition of Urban Waste to Semiarid Degraded Soil: Long-term Effect

Abstract The addition of municipal solid wastes (MSW) is considered as a possible strategy for soil rehabilitation in southeast Spain. The objective of this study was to evaluate the long-term (17 years) effect of five doses of MSW addition on the microbiological, biochemical, and physical properties of semiarid soil. Increased values of several parameters that serve as indicators of general microbiological activity, such as, basal respiration, adenosine triphosphate (ATP) or dehydrogenase activity; microbial population size (microbial biomass C), and extracellular hydrolase activity related to macronutrient cycles, such as, urease, β-glucosidase, and N-α-benzoyl-L-argininamide protease, were observed in the amended soils. The highest MSW doses showed the highest values in these hydrolase activities. The incorporation of municipal waste resulted in a more dense development of the plant cover, 50% greater in higher doses than in the control treatment, which generated a substantial increase in several C fractions. Total organic carbon reached 12 g kg −1 soil with the highest MSW doses, compared to 4.30 g kg −1 soil in the control treatment. The physical properties of the soil were also improved, showing greater percentage of stable aggregates and water holding capacity. Positive correlation coefficients between C fractions and parameters related to microbial activity and aggregate stability were observed. Although these improvements were greater in the soils receiving the highest doses of organic amendment, the increases were not proportional to the amount added, demonstrating the existence of a threshold, above which an increase in the amount of organic matter added is not reflected in an increase in the soils physical, biochemical, and microbiological properties. However, the addition of municipal solid wastes proved its suitability for improving soil quality, thereby indicating the potential of such an amendment, to prevent desertification in Mediterranean areas such as those studied.

Soil restoration with organic amendments: linking cellular functionality and ecosystem processes

A hot topic in recent decades, the application of organic amendments to arid-degraded soils has been shown to benefit microbially-mediated processes. However, despite the importance of soils for global sustainability, a gap has not been addressed yet in soil science: is there any connection between ecosystem-community processes, cellular functionality, and microbial lifestyles (i.e. oligotrophy-copiotrophy) in restored soils? Together with classical ecosystem indicators (fatty-acids, extracellular-enzyme activities, basal respiration), state-of-the-art metaproteomics was applied to fill this gap in a model-restoration experiment initiated 10-years ago by the addition of sewage-sludge and compost. Organic amendment strongly impacted ecosystem processes. Furthermore, the type of material used induced differences in the cellular functionalities through variations in the percentages of proteins involved in translation, transcription, energy production and C-fixation. We conclude that the long-term impact of organic restoration goes beyond ecosystem processes and affects cellular functionalities and phyla-lifestyles coupled with differences in microbial-community structures.

The ecological and physiological responses of the microbial community from a semiarid soil to hydrocarbon contamination and its bioremediation using compost amendment

The linkage between phylogenetic and functional processes may provide profound insights into the effects of hydrocarbon contamination and biodegradation processes in high-diversity environments. Here, the impacts of petroleum contamination and the bioremediation potential of compost amendment, as enhancer of the microbial activity in semiarid soils, were evaluated in a model experiment. The analysis of phospholipid fatty-acids (PLFAs) and metaproteomics allowed the study of biomass, phylogenetic and physiological responses of the microbial community in polluted semiarid soils. Petroleum pollution induced an increase of proteobacterial proteins during the contamination, while the relative abundance of Rhizobiales lowered in comparison to the non-contaminated soil. Despite only 0.55% of the metaproteome of the compost-treated soil was involved in biodegradation processes, the addition of compost promoted the removal of polycyclic aromatic hydrocarbons (PAHs) and alkanes up to 88% after 50 days. However, natural biodegradation of hydrocarbons was not significant in soils without compost. Compost-assisted bioremediation was mainly driven by Sphingomonadales and uncultured bacteria that showed an increased abundance of catabolic enzymes such as catechol 2,3-dioxygenases, cis-dihydrodiol dehydrogenase and 2-hydroxymuconic semialdehyde. For the first time, metaproteomics revealed the functional and phylogenetic relationships of petroleum contamination in soil and the microbial key players involved in the compost-assisted bioremediation.

Analysis of structure, function, and activity of a benzene-degrading microbial community.

We identified phylotypes performing distinct functions related to benzene degradation in complex microbial biofilms from an aerated treatment pond containing coconut textile. RNA- and protein-stable isotope probing (SIP) and compound-specific stable isotope analysis were applied to delineate bacteria and predominant pathways involved in the degradation of benzene. In laboratory microcosms, benzene was degraded at rates of ≥ 11 μM per day and per gram coconut textile under oxic conditions. Carbon isotope fractionation with isotopic enrichment factors (ε) of -0.6 to -1‰ and no significant hydrogen isotope fractionation indicated a dihydroxylation reaction for the initial ring attack. The incubation with [(13)C₆]-benzene led to (13)CO₂ formation accompanied by (13)C-labeling of RNA and proteins of the active biomass. Phylogenetic analysis of the (13)C-labeled RNA revealed that phylotypes related to Zoogloea, Ferribacterium, Aquabacterium, and Hydrogenophaga within the Betaproteobacteria predominantly assimilated carbon from benzene. Although the phylogenetic classification of identified (13)C-labeled proteins was biased by the incomplete metagenome information of public databases, it matched with RNA-SIP results at genus level. The detection of (13)C-labeled proteins related to toluene dioxygenase and catechol 2,3-dioxygenase suggests benzene degradation by a dihydroxylation pathway with subsequent meta-cleavage of formed catechol.