Earthworm effects without earthworms: inoculation of raw organic matter with worm-worked substrates alters microbial community functioning

Background

Earthworms are key organisms in organic matter decomposition because of the interactions they establish with soil microorganisms. They enhance decomposition rates through the joint action of direct effects (i.e. effects due to direct earthworm activity such as digestion, burrowing, etc) and indirect effects (i.e. effects derived from earthworm activities such as cast ageing). Here we test whether indirect earthworm effects affect microbial community functioning in the substrate, as when earthworms are present (i. e., direct effects).

Methodology/Principal Findings

To address these questions we inoculated fresh organic matter (pig manure) with worm-worked substrates (vermicompost) produced by three different earthworm species. Two doses of each vermicompost were used (2.5 and 10%). We hypothesized that the presence of worm-worked material in the fresh organic matter will result in an inoculum of different microorganisms and nutrients. This inoculum should interact with microbial communities in fresh organic matter, thus promoting modifications similar to those found when earthworms are present. Inoculation of worm-worked substrates provoked significant increases in microbial biomass and enzyme activities (β-glucosidase, cellulase, phosphatase and protease). These indirect effects were similar to, although lower than, those obtained in pig manure with earthworms (direct and indirect earthworm effects). In general, the effects were not dose-dependent, suggesting the existence of a threshold at which they were triggered.

Conclusion/Significance

Our data reveal that the relationships between earthworms and microorganisms are far from being understood, and suggest the existence of several positive feedbacks during earthworm activity as a result of the interactions between direct and indirect effects, since their combination produces stronger modifications to microbial biomass and enzyme activity.     

Variations in microbial isotopic fractionation during soil organic matter decomposition

The soil microbial biomass (SMB) is known to participate in key soil processes such as the decomposition of soil organic matter (SOM). However, its contribution to the isotopic composition of the SOM is not clear yet. Shifts in the ¹³C and ¹⁵N natural abundances of the SMB and SOM fractions (mineralised, water soluble and non-extractable) were investigated by incubating an unamended arable soil for 6 months. Microbial communities were also studied using Fatty Acid Methyl Ester specific isotope analysis. The SMB was significantly ¹³C and ¹⁵N-enriched relative to other fractions throughout the incubation. However, significant isotopic variations with time were also observed due to the rapid consumption of relatively ¹³C-enriched water soluble compounds. The increase in the difference in SMB and water soluble ¹⁵N compositions as the water soluble C/N ratio decreased, indicated a shift from N assimilation to N dissimilation during the incubation. These changes also induced modifications of the microbial community structure. Once the system reached a steady-state (after 1 month), the isotopic trends appeared to corroborate those obtained in long term experiments in the field in that there was a constant microbial isotopic fractionation leading to a ¹³C and ¹⁵N enrichment of the SOM over the long-term. This work also suggests that caution must be exercised when interpreting short term incubation studies since perturbations associated with experimental set-up can have an important effect on C and N dynamics, microbial fractionation of ¹³C and ¹⁵N and microbial community structure.     

Comparative efficacy of three epigeic earthworms under different deciduous forest litters decomposition

An experiment was conducted during 1998-1999, in a deciduous forest located in the semi-arid tropics of central India, to evaluate the suitability of different forest litters as food material for the tropical epigeic earthworms i.e. Eisenia fetida (Savigny), Perionyx excavatus (Perrier) and Dicogaster bolaui (michaelsen). The aim was to examine the influence of these earthworms on the decomposition processes of three types of forest litters i.e. Tectona grandis (teak), Madhuca indica (mahua) and Butea monosperma (palas), on the maintenance of quality in a vermicomposting system, and to assess the effect of applications of in situ prepared vermicomposts on the growth of forest trees. The results indicated that T. grandis litter was the most suitable food material for the earthworms possibly because it contained high reserves of mineral nutrients. Comparisons of the survival and reproduction rates of the three epigeic earthworm species indicated that a higher reproduction rate was maintained for E. fetida compared to P. excavatus and D. bolaui in the decomposition of these forest litters. The rates of growth and population increases of E. fetida approximately doubled after 12 weeks of litter decomposition. The litter decomposition process was associated strongly with the quality of the materials and their chemical composition. Irrespective of earthworm inoculations, the levels of available nutrient such as NH(4)-N, NO(3)-N, available P and K increased significantly (pM. indica litter compost>B. monosperma litter compost. The mature decomposed litter had lower C/N ratios (11.3-24.8:1), water-soluble carbon (0.30-0.58%), water-soluble carbohydrates (0.35-0.71%) and larger cation exchange capacity/total organic carbon ratios than the values in the parent forest litter. The lignin content increased with maturation with a concomitant decrease in cellulose resulting in higher lignin/cellulose ratios.Application of all three vermicomposts to forest trees significantly improved their heights and diameters over those of control trees, although the increases were lower than those resulting from the chemical fertilizer applications. However, soil biological activities i.e. soil respiration, soil microbial biomass carbon and dehydrogenase activity were greater by application of vermicomposts over that after application of inorganic fertilizer in a new plantation of T. grandis.     

Dynamics of C, N, P and microbial community composition in particulate soil organic matter during residue decomposition

Decomposing residues can be an important source of nutrients for plants, especially of N and P, but the relationship between N and P release and microbial community dynamics have rarely been studied. Two pea (Pisum sativum L.) residues with contrasting chemical composition, shoots from flowering pea (Pea-Y) with 2.9 mg P and 36 mg N kg⁻¹ and from mature pea (Pea-M) with 0.3 mg P and 13 mg N kg⁻¹, were added at a rate of 20 g kg soil⁻¹ to a sandy soil low in nutrients. Particulate organic matter (POM) was isolated on days (d) 0, 5, 15, 28, 42 and 61 after residue addition and analysed for C, N, P and microbial community structure (fatty acid methyl ester analysis). The recovery of POM from residue-amended soils decreased over time to 30–40% of added amounts for both residues. Apart from d 0, the N concentration in POM was lower in residue-amended soil than in the control. Due to a rapid decrease in P concentration during the first 5 days in Pea-Y and a slow increase over the whole experiment in Pea-M, P concentrations in POM on d 61 were similar in all treatments. In Pea-Y, the dynamics of C, N and P were coupled, with amounts of C, N and P decreasing during the first 15 days and remaining stable thereafter. In Pea-M, a steady loss of C from POM was contrasted by a slight increase in P. As a result, the C/P ratio decreased from 1,330 on d 0 to 390 on d 61. The C/N ratio of Pea-M decreased only during the second phase of decomposition. The different nutrient dynamics in Pea-Y and Pea-M led to similar amounts of N and P in POM towards the end of the incubation. Microbial community composition in the POM in Pea-Y and Pea-M remained distinct from the control, even though it changed over time. POM was shown to be an important source of potentially available nutrients after addition of plant residues. In the unamended soil, stable nutrient amounts in POM suggested very low net nutrient release from native POM compared to POM after residue addition.     

Methane fluxes in permafrost habitats of the Lena Delta: effects of microbial community structure and organic matter quality

For the understanding and assessment of recent and future carbon dynamics of arctic permafrost soils the processes of CH(4) production and oxidation, the community structure and the quality of dissolved organic matter (DOM) were studied in two soils of a polygonal tundra. Activities of methanogens and methanotrophs differed significantly in their rates and distribution patterns among the two investigated profiles. Community structure analysis showed similarities between both soils for ester-linked phospholipid fatty acids (PLFAs) and differences in the fraction of unsaponifiable PLFAs and phospholipid ether lipids. Furthermore, a shift of the overall composition of the microbiota with depth at both sites was indicated by an increasing portion of iso- and anteiso-branched fatty acids related to the amount of straight-chain fatty acids. Although permafrost soils represent a large carbon pool, it was shown that the reduced quality of organic matter leads to a substrate limitation of the microbial metabolism. It can be concluded from our and previous findings first that microbial communities in the active layer of an Arctic polygon tundra are composed by members of all three domains of life, with a total biomass comparable to temperate soil ecosystems, and second that these microorganisms are well adapted to the extreme temperature gradient of their environment.     

Nanoscale structure of organic matter could explain litter decomposition

Soil organic matter (SOM) turnover is crucial for soil quality and fertility in biogeochemical carbon cycle dynamics that can influence the fluxes of greenhouse gases. This research was focused to acquire deeper understanding of the mechanisms leading to decomposition of plant tissue and SOM persistence against both aerobic and anaerobic biodegradation. Decomposition rates of a various biomass types were studied conducting experiments in both aerobic and anaerobic environments. Different analytical approaches were applied in order to characterize biomass at chemical and physical levels. Combined statistical approaches were used to examine the relationships between carbon mineralization and chemical/physical characteristics. The obtained results revealed that degradation was significantly and negatively correlated with the micro-porosity surface (surface of pores of 0.3–1.5 nm of diameter). The multiple regressions performed by using partial least squares modelling enabled describing biomass biodegradability under either aerobic and anaerobic condition by using micro-porosity and aromatic-C content (assumed to be representative of lignin) as independent variables (R² = 0.97, R _cv ²  = 0.95 for aerobic condition; R² = 0.99, R _cv ²  = 0.98 for anaerobic condition, respectively). These results corroborate the hypothesis that plant tissues are physically protected from enzymatic attack by a microporous “sheath” that limits enzyme penetration into cell wall, and demonstrate the key role played by aromatic carbon, because of its chemical protection of the other cell wall polymers and its contribution to the three-dimensional (3D) cell wall structure.     

Microbial and decomposition efficiencies of monoculture and polyculture vermireactors,based on epigeic and anecic earthworms

Efforts have been made to evaluate the microbial and decomposition efficiency of three different vermireactors: (i) polyculture (introducing equal numbers of anecic and epigeic earthworms), (ii) monoculture (anecic) and (iii) monoculture (epigeic), designed by using earthworms of two different ecological categories i.e. anecic (Lampito mauritii Kinberg) and epigeic (Eisenia fetida (Savigny)). The microbial load of vermireactors was measured through substrate-induced respiration rate (SIR), microbial biomass N content and rate of dehydrogenase activity, while mineralization rate was evaluated measuring some chemical parameters of the substrate. Earthworms caused a decrease (as compared to initial value) in pH (41.9–80.7%), organic C (10.3–14.2%) and C:N ratio (41.9–80.7%) and an increase in total N (29.1–58.8%), NH₄-N (876.1–1485.7%), NO₃-N (29081.8–56792.6%), available P (16–19.4%) and exchangeable K (9.8–13.5%) contents of the substrate. The mineralization efficiency of the reactors was in the order: polyculture (epigeic + anecic) > monoculture (anecic) > monoculture (epigeic). The polyculture reactor showed the maximum rate of SIR (2.91 ± 0.2 mg CO₂ g⁻¹ substrate), microbial biomass N (3108.1 ± 289.2 mg N g⁻¹ substrate), and dehydrogenase activity (2453.3 ± 379.8 μg g⁻¹ substrate 24 h), while in the monoculture (epigeic) the lowest values of the same parameters were observed. It is concluded that the observed differences among reactors were due to different feeding behaviour and niche structures of epigeic and anecic earthworms. Data suggests that burrowing earthworms in waste-decomposing-system not only enhance the microbial efficiencies, but at the same time also accelerate the organic matter mineralization in a vermireactor. However, most of the previous studies were based on monoculture reactors (using epigeic earthworms) which have been recommended for waste decomposition operations, but this study revealed that polyculture vermicomposting (adding of burrowing worms with epigeic earthworms in vermicomposting system) might be beneficial for rapid decomposition of organic wastes.     

Interactive effects of solar radiation and dissolved organic matter on bacterial activity and community structure

We studied the interactive effects of dissolved organic matter (DOM) and solar radiation on the activity and community structure of bacteria from an alpine lake. Activity was assessed both at the community level as leucine incorporation rates and at the single-cell level by microautoradiography. Fluorescent in situ hybridization and signal amplification by catalysed reporter deposition (CARD-FISH) was used to track changes in the bacterial community composition. Bacteria-free filtrates of different DOM sources (lake, algae or soil) were incubated either in the dark or exposed to solar radiation. Afterwards, the natural bacterial assemblage was inoculated and the cultures incubated in the dark for 24-48 h. Bacterial activity was enhanced in the first 24 h in the soil and algal DOM amendments kept in the dark. After 48 h, the enhancement effect was greatly reduced. The initial bacterial community was dominated by Betaproteobacteria followed by Actinobacteria. The relative abundance (expressed as a percentage of DAPI-stained cells) of Betaproteobacteria increased first in dark incubated DOM amendments, but after 48 h no significant differences were detected among treatments. In contrast, the relative abundance of Actinobacteria increased in pre-irradiated DOM treatments. Although Betaproteobacteria dominated at the end of the experiment, the relative abundance of their R-BT subgroup differed among treatments. Changes in bacterial community activity were significantly correlated with those of the relative abundance and activity of Betaproteobacteria, whereas the contribution of Actinobacteria to the bulk activity was very modest. Our results indicate a negative effect of DOM photoalteration on the bulk bacterial activity. The magnitude of this effect was time-dependent and related to rapid changes in the bacterial assemblage composition.     

Soil and plant effects on microbial community structure

We investigated the effects of two different plant species (corn and soybean) and three different soil types on microbial community structure in the rhizosphere. Our working hypothesis was that the rhizosphere effect would be strongest on fast-growing aerobic heterotrophs, while there would be little or no rhizosphere effect on oligotrophic and other slow-growing microorganisms. Culturable bacteria and fungi had larger population densities in the rhizosphere than in bulk soil. Communities were characterized by soil fatty acid analysis and by substrate utilization assays for bacteria and fungi. Fatty acid analysis revealed a very strong soil effect but little plant effect on the microbial community, indicating that the overall microbial community structure was not affected by the rhizosphere. There was a strong rhizosphere effect detected by the substrate utilization assay for fast-growing aerobic heterotrophic bacterial community structure, with soil controls and rhizosphere samples clearly distinguished from each other. There was a much weaker rhizosphere effect on fungal communities than on bacterial communities as measured by the substrate utilization assays. At this coarse level of community analysis, the rhizosphere microbial community was impacted most by soil effects, and the rhizosphere only affected a small portion of the total bacteria.     

Nitrate addition stimulates microbial decomposition of organic matter in salt marsh sediments

Salt marshes sequester carbon at rates more than an order of magnitude greater than their terrestrial counterparts, helping to mitigate climate change. As nitrogen loading to coastal waters continues, primarily in the form of nitrate, it is unclear what effect it will have on carbon storage capacity of these highly productive systems. This uncertainty is largely driven by the dual role nitrate can play in biological processes, where it can serve as a nutrient‐stimulating primary production or a thermodynamically favorable electron acceptor fueling heterotrophic metabolism. Here, we used a controlled flow‐through reactor experiment to test the role of nitrate as an electron acceptor, and its effect on organic matter decomposition and the associated microbial community in salt marsh sediments. Organic matter decomposition significantly increased in response to nitrate, even at sediment depths typically considered resistant to decomposition. The use of isotope tracers suggests that this pattern was largely driven by stimulated denitrification. Nitrate addition also significantly altered the microbial community and decreased alpha diversity, selecting for taxa belonging to groups known to reduce nitrate and oxidize more complex forms of organic matter. Fourier Transform‐Infrared Spectroscopy further supported these results, suggesting that nitrate facilitated decomposition of complex organic matter compounds into more bioavailable forms. Taken together, these results suggest the existence of organic matter pools that only become accessible with nitrate and would otherwise remain stabilized in the sediment. The existence of such pools could have important implications for carbon storage, since greater decomposition rates as N loading increases may result in less overall burial of organic‐rich sediment. Given the extent of nitrogen loading along our coastlines, it is imperative that we better understand the resilience of salt marsh systems to nutrient enrichment, especially if we hope to rely on salt marshes, and other blue carbon systems, for long‐term carbon storage.     

Analysis of the microbial community structure in a membrane bioreactor during initial stages of filtration

Membrane biofouling was investigated during the early stages of filtration in a laboratory-scale membrane bioreactor operated on molasses wastewater. The bacterial diversity and composition of the membrane biofilm and activated sludge were analyzed using terminal restriction fragment length polymorphism coupled with 16S rRNA clone library construction and sequencing. The amount of extracellular polymeric substances produced by bacteria was investigated using spectroscopic methods. The results reveal that the bacterial community of activated sludge differs significantly from that of the membrane biofilm, especially at the initial phase. Phylogenetic analysis based on 16S rRNA gene sequences identified 25 pioneer OTUs responsible for membrane surface colonization. Also, the relationship between the identified bacterial strains and the system specifications was explored.     

Linking temperature sensitivity of soil organic matter decomposition to its molecular structure,accessibility, and microbial physiology

Temperature sensitivity of soil organic matter (SOM) decomposition may have a significant impact on global warming. Enzyme‐kinetic hypothesis suggests that decomposition of low‐quality substrate (recalcitrant molecular structure) requires higher activation energy and thus has greater temperature sensitivity than that of high‐quality, labile substrate. Supporting evidence, however, relies largely on indirect indices of substrate quality. Furthermore, the enzyme‐substrate reactions that drive decomposition may be regulated by microbial physiology and/or constrained by protective effects of soil mineral matrix. We thus tested the kinetic hypothesis by directly assessing the carbon molecular structure of low‐density fraction (LF) which represents readily accessible, mineral‐free SOM pool. Using five mineral soil samples of contrasting SOM concentrations, we conducted 30‐days incubations (15, 25, and 35 °C) to measure microbial respiration and quantified easily soluble C as well as microbial biomass C pools before and after the incubations. Carbon structure of LFs (<1.6 and 1.6–1.8 g cm^?3) and bulk soil was measured by solid‐state ¹³C‐NMR. Decomposition Q₁₀ was significantly correlated with the abundance of aromatic plus alkyl‐C relative to O‐alkyl‐C groups in LFs but not in bulk soil fraction or with the indirect C quality indices based on microbial respiration or biomass. The warming did not significantly change the concentration of biomass C or the three types of soluble C despite two‐ to three‐fold increase in respiration. Thus, enhanced microbial maintenance respiration (reduced C‐use efficiency) especially in the soils rich in recalcitrant LF might lead to the apparent equilibrium between SOM solubilization and microbial C uptake. Our results showed physical fractionation coupled with direct assessment of molecular structure as an effective approach and supported the enzyme‐kinetic interpretation of widely observed C quality‐temperature relationship for short‐term decomposition. Factors controlling long‐term decomposition Q₁₀ are more complex due to protective effect of mineral matrix and thus remain as a central question.     

Illuminating microbial species-specific effects on organic matter remineralization in marine sediments

Marine microorganisms play a fundamental role in the global carbon cycle by mediating the sequestration of organic matter in ocean waters and sediments. A better understanding of how biological factors, such as microbial community composition, influence the lability and fate of organic matter is needed. Here, we explored the extent to which organic matter remineralization is influenced by species-specific metabolic capabilities. We carried out aerobic time-series incubations of Guaymas Basin sediments to quantify the dynamics of carbon utilization by two different heterotrophic marine isolates (Vibrio splendidus 1A01; Pseudoalteromonas sp. 3D05). Continuous measurement of respiratory CO₂ production and its carbon isotopic compositions (¹³C and ¹⁴C) shows species-specific differences in the rate, quantity and type of organic matter remineralized. Each species was incubated with hydrothermally-influenced versus unimpacted sediments, resulting in a ~2-fold difference in respiratory CO₂ yield across the experiments. Genomic analysis indicated that the observed carbon utilization patterns may be attributed in part to the number of gene copies encoding for extracellular hydrolytic enzymes. Our results demonstrate that the lability and remineralization of organic matter in marine environments is not only a function of chemical composition and/or environmental conditions, but also a function of the microorganisms that are present and active.     

Rhizodeposition shapes rhizosphere microbial community structure in organic soil

The aims of the study were to determine group specificity in microbial utilization of root-exudate compounds and whole rhizodeposition; quantify the proportions of carbon acquired by microbial groups from soil organic matter and rhizodeposition, respectively; and assess the importance of root-derived C as a driver of soil microbial community structure. Additions of 13C-labelled root-exudate compounds to organic soil and steady-state labelling of Lolium perenne, coupled to compound-specific isotope ratio mass spectrometry, were used to quantify group-specific microbial utilization of rhizodeposition. Microbial utilization of glucose and fumaric acid was widespread through the microbial community, but glycine was utilized by a narrower range of populations, as indicated by the enrichment of phospholipid fatty acid (PLFA) analysis fractions. In L. perenne rhizospheres, high rates of rhizodeposit utilization by microbial groups showed good correspondence with increased abundance of these groups in the rhizosphere. Although rhizodeposition was not the quantitatively dominant C source for microbes in L. perenne rhizospheres, relative utilization of this C source was an important driver of microbial group abundance in organic soil.     

Growth and turnover of microbial biomass during the decomposition of organic matter(Polygonum cuspidatum) in vitro

Air-dried fresh and dead specimens ofPolygonum cuspidatum were incubated for 250 days in the laboratory, and the growth and turnover of microbial biomass-C in the organic matter were studied. The biomass-C in the fresh leaf and fresh stem attained maximum levels on day 14 and day 7, respectively, and then settled down to stable levels. In the dead leaf and dead stem, increase in biomass-C ceased by day 4 and the biomass-C levels did not change thereafter. The turnover time of the biomass-C was estimated from the amount of biomass-C and the release rate of CO₂-C. The turnover was rapid in the early period of incubation. Then the turnover time became longer and after incubation for 70 days the values approached those in natural soils (longer than 16 days). During the incubation period, nitrogen was not mineralized in any organic matter. In the dead leaf and dead stem, asymbiotic nitrogen fixation activity increased after incubation for about 40 days and disappeared by the end of the incubation period, whereas nitrogen fixation was hardly detected in the fresh leaf and fresh stem.     

Soil microbial diversity affects soil organic matter decomposition in a silty grassland soil

Soil microorganisms play a pivotal role in soil organic matter (SOM) turn-over and their diversity is discussed as a key to the function of soil ecosystems. However, the extent to which SOM dynamics may be linked to changes in soil microbial diversity remains largely unknown. We characterized SOM degradation along a microbial diversity gradient in a two month incubation experiment under controlled laboratory conditions. A microbial diversity gradient was created by diluting soil suspension of a silty grassland soil. Microcosms containing the same sterilized soil were re-inoculated with one of the created microbial diversities, and were amended with ¹³C labeled wheat in order to assess whether SOM decomposition is linked to soil microbial diversity or not. Structural composition of wheat was assessed by solid-state ¹³C nuclear magnetic resonance, sugar and lignin content was quantified and labeled wheat contribution was determined by ¹³C compound specific analyses. Results showed decreased wheat O-alkyl-C with increasing microbial diversity. Total non-cellulosic sugar-C derived from wheat was not significantly influenced by microbial diversity. Carbon from wheat sugars (arabinose-C and xylose-C), however, was highest when microbial diversity was low, indicating reduced wheat sugar decomposition at low microbial diversity. Xylose-C was significantly correlated with the Shannon diversity index of the bacterial community. Soil lignin-C decreased irrespective of microbial diversity. At low microbial diversity the oxidation state of vanillyl–lignin units was significantly reduced. We conclude that microbial diversity alters bulk chemical structure, the decomposition of plant litter sugars and influences the microbial oxidation of total vanillyl–lignins, thus changing SOM composition.