Final Report Summary - DYNOMIWAS (DYNAMICS OF THE ORGANIC MATTER AND THE MICROBIAL COMMUNITY RELATED TO ITS CYCLING IN ARID AREAS. INVOLVEMENT OF AMENDMENTS BASED ON ORGANIC WASTES)
INTRODUCTION
The constant loss of organic matter from Mediterranean soils is largely due to the scarce plant cover which does not provide enough organic carbon inputs into soil. Under these conditions, soils are unable to efficiently buffer anthropic activities that could lead to processes of desertification.
Organic matter in soils has several roles: it promotes the soil physical structure, acts as a buffer for pollutants, increases the water-holding capacity, and promotes vegetal development, etc. In this sense, the application of exogenous organic materials from wastes (i.e. sludges or compost) increases the total organic carbon content in degraded soils. However, there are no evidences on the fate, persistence and microbial assimilation of individual molecules which compound organic matter.
The general objective of this proposal was to study the dynamics of organic matter and the function of the microbial community associated with its turnover in semiarid areas. The specific objectives were: i) to analyze the effect of the addition of new organic wastes with different degrees of stability (for example, charcoal, sludges, composts, etc.) on the microbiological quality of soil; ii) identify groups of microorganisms related to C sequestration in desert areas; iii) evaluate the major chemical and physical sinks for carbon sequestration by isotopic studies, and iv) identify enzymatic processes related to organic matter turnover.
EXPERIMENTAL SET-UP. ACTIVITIES
Three types of activities were conducted with the aim to answer the proposed objectives. The first group of experiments (A) evaluated the response of soil microbial community and soil quality to the addition of exogenous sources of organic matter, such composts and sewage sludges. The second group of experiments aimed to evaluate the persistence, carbon sequestration and assimilation of specific organic molecules in soil. The third group experiments (C) is devoted to the standardization of protein extraction methodologies in soil and proteome analysis as a tool for understanding microbial functionality in soil.
A) Effects of organic amendments in soil
These experiments have been conducted either under controlled laboratory incubations and field conditions with the aim to test the effectiveness of organic amendments in different conditions.
Under laboratory conditions, two doses (5g and 10g) of two organic amendments (sewage sludge from wastewater plant and compost from this sludge) were applied to a semiarid soil (500g) and incubated during 360 days. Several indicators of microbial activity and soil quality were evaluated such basal respiration, microbial biomass, enzyme activities and humic-enzyme complexes.
Under field conditions, the effects of different doses of organic materials (from municipal solid wastes) were evaluated at long-term after the addition of organic amendments 25 years ago. In this case, microbial community was evaluated in the bases of their microbial activity and community structure by genomic tools (pyrosequencing).
B) Persistence, carbon sequestration and microbial assimilation of organic molecules in semiarid soils
Tracer experiments with isotopic-enriched compounds (13C-glucose, 13C-cellulose and 13C-lignin, 99 atom%) were used in two semiarid soils. Organic molecules were incubated in soils during 4 months. The amount of 13C that persisted in bulk soil, water-soluble fractions or humic-substances fractions was evaluated by mass spectrometry methodologies at the University of California, Davis. The assimilation of 13C by the microbial community was evaluated after extraction, fractionation and analysis of the isotopic composition of phospholipid fatty acids (PLFA-SIP) in collaboration with UFZ-Leipzig (Dpt. Isotope Biogeochemistry, Dr. H. Richnow) by gas-chromatography-combustion-isotope-ratio mass spectrometer (GC-C-IRMS). The amount of organic carbon mineralized from exogenous substrates or from soil organic matter was also evaluated by the analysis of the isotopic signature of CO2 by GC-C-IRMS.
C) Soil proteomics
Soil proteins were extracted using different approaches. Methods based on a previous cell-separation or direct protein-extraction strategies were used. Several semiarid soils were used.
RESEARCH RESULTS
A) Effects of organic amendments in soil
The different carbon fractions, such as the total organic carbon (TOC), water-soluble carbon (WSC), and microbial biomass carbon (MBC), increased in amended soils compared to the control soil without amendment, as well as dehydrogenase and hydrolytic enzymes (β-glucosidase and urease) activities. For instance, after 360 days the total organic carbon reached 1.41 % in soil amended with a high dose of sewage sludge and water-soluble carbon content reached 56 mg kg-1 in soil amended with high dose of composted municipal solid waste. The immobilized enzymes in the soil humic extracts exhibited different behaviors compared to total activity, depending on the origin of the organic material which suggests neo-formation of humic-enzyme complexes. Addition of composted municipal solid waste or sewage sludge increased the activity of β-glucosidase linked to humic substances.
The long-term effects of organic amendment in the microbial community were evaluated in a soil that received singly application of two doses of organic domestic waste at 65 Mg ha-1 (LD plots) and 195 Mg ha-1 (HD plots) 25 years ago. A control soil without organic amendment was also evaluated. Pyrosequencing of 16S- and 18S-rRNA genes did not reveal significant differences in phylogenetic diversity between restored and control soils. However, principal coordinates analysis of unweighted Unifrac distances showed variation in the structure of bacterial and fungal communities of HD plots. The number of Alpha-proteobacteria sequences was higher in HD plots than in LD and control plots, while Actinobacteria abundance diminished in HD plots. In contrast to Basidiomycota, the number of Ascomycota sequences responded positively to restoration. Changes in microbial phylogenetic structure were related to changes in functional structure established by multivariate analysis of community-level-physiological profiles. Interestingly, despite the absence of phylogenetic diversity, restoration decreased the catabolic diversity in HD plots. This effect is likely due to the aboveground plant influences in restored plots. Overall, in the long-term, soil restoration under semiarid conditions did not increase microbial diversity but influenced microbial community structure and functionality.
B) Persistence, carbon sequestration and microbial assimilation of organic molecules in semiarid soils
We studied priming effects and microbial utilization of glucose as an example of bioavailable labile molecule in the carbon cycle of a semiarid soil. The soil, which has low content of total organic carbon (5.0 g kg-1), was amended with U13C-glucose (99 atom %) at concentration of 75 µg C g-1 soil (LD) or 300 µg C g-1 soil (HD). Glucose-derived carbon remained in soil after two months of incubation. The percentage of residual carbon stabilized was greater in LD with 40% of the initial 13C added compared to 30% of the initial 13C added in the HD. Glucose was subjected to an intense mineralization in the first 17-days of 22.8% and 40.94% for the LD and HD, respectively. The stable isotope probing (SIP) of phospholipid fatty acids (PLFAs) by gas-chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) showed that bacteria dominated glucose metabolism in comparison to fungi. Gram-negative populations were initially more involved in glucose assimilation than Gram-positive bacteria. In the fatty acids fraction, up to 95% of the 13C was predominantly found in fatty acids typical for Gram-negative bacteria. However, after 4 and 17 days the 13C-enrichment in Gram-positive biomarkers increased. The mineralization of soil organic matter triggered by glucose additions was more intense in HD (3.6% of soil TOC) than LD (1.0% of soil TOC). Our data indicated that the intense metabolism of SOM due to priming phenomena compromises the potential carbon sequestration in this semiarid soil amended with glucose.
In a similar experiment, 13C-labeled cellulose and 13C-labeled lignin were added to two semiarid soils (75 µg 13C g-1 soil) with different properties and degradation level. Abanilla soil is a bare, highly degraded soil without plant cover growing on it and a total organic C content of 5.0 g kg-1; Santomera soil is covered by a plant cover (20%) based on xerophytic shrubs and has a total organic C content of 12.0 g kg-1. Both the persistence and availability of added carbon was evaluated by analysis of the carbon isotope signature of bulk soil-derived carbon and extractable carbon fractions. Up to 25% and 78% of the added 13C lasted in bulk soil after 4 months of incubation with cellulose and lignin, respectively. The substrate mineralization was dependent on the soil properties and degradation level. The amount of 13C-cellulose mineralized was 39.7% and 9.3% in Santomera and Abanilla soil, respectively, and 34.9% and 8.1% for 13C-lignin. Stable isotope probing (SIP) of phospholipid fatty acids (PLFA-SIP) analysis revealed that only a small fraction of the microbial community assimilated the carbon derived from these compounds. Noteworthy, the assimilation of cellulose- and lignin-derived carbon was higher in Santomera soil. In both soils, the assimilation of cellulose was mediated by fungi and Gram-negative bacteria. For instance, the fatty acid 18:2ω6,9 indicative of fungi reached the maximum incorporation of cellulose-derived carbon with 4 atom % at 20 days of incubation in Abanilla soil, while its enrichment in Santomera was faster and reached 8 atom % during the first day. We conclude that soil properties and molecule complexity play an important role in the carbon cycling of soils under semiarid conditions.
C) Soil proteomics
A methodological approach was applied in order to retrieve functional information by proteomics of soils subjected to glucose addition. This approach revealed a great importance of glucose in microbial development and the further feasibility of proteome procedures after cell-density separation. The yield of protein extraction was dependent on the soil type and texture.
Moreover, different protocols aiming the extraction of proteins from bulk soils are being tested. The proteome analysis is being conducted by LTQ-Orbitrap MS/MS at the ETH-Zurich. Results have revealed that methods based on boiling with an SDS-buffer and DTT provide the highest amount of proteins and with higher ecological information than other methods.