Determination of microbial biomass and fungal and bacterial distribution in cattle faeces

As an important component of organic fertilizers, animal faeces require methods for determining diet effects on their microbial quality to improve nutrient use efficiency in soil and to decrease gaseous greenhouse emissions to the environment. The objectives of the present study were (i) to apply the chloroform fumigation extraction (CFE) method for determining microbial biomass in cattle faeces, (ii) to determine the fungal cell-membrane component ergosterol, and (iii) to measure the cell-wall components fungal glucosamine and bacterial muramic acid as indices for the microbial community structure. Additionally, ergosterol and amino sugar data provide independent control values for the reliability of the microbial biomass range obtained by the CFE method. A variety of extractant solutions were tested for the CFE method to obtain stable extracts and reproducible microbial biomass C and N values, leading to the replacement of the original 0.5 M K₂SO₄ extractant for 0.05 M CuSO₄. The plausibility of the data was assessed in a 28-day incubation study at 25 °C with cattle faeces of one heifer, where microbial biomass C and N were repeatedly measured together with ergosterol. Here, the microbial biomass indices showed dynamic characteristics and possible shifts in the microbial community. In faeces of five different heifers, the mean microbial biomass C/N ratio was 5.6, the mean microbial biomass to organic C ratio was 2.2%, and the mean ergosterol to microbial biomass C ratio was 1.1‰. Ergosterol and amino sugar analysis revealed a significant contribution of fungi, with a percentage of more than 40% to the microbial community. All three methods are expected to be suitable tools for analysing the quality of cattle faeces.     

Above- and belowground carbon inputs affect seasonal variations of soil microbial biomass in a subtropical monsoon forest of southwest China

Soil microbial activity drives carbon and nutrient cycling in terrestrial ecosystems. Soil microbial biomass is commonly limited by environmental factors and soil carbon availability. We employed plant litter removal, root trenching and stem-girdling treatments to examine the effects of environmental factors, above- and belowground carbon inputs on soil microbial C in a subtropical monsoon forest in southwest China. During the experimental period from July 2006 through April 2007, 2 years after initiation of the treatments, microbial biomass C in the humus layer did not vary with seasonal changes in soil temperature or water content. Mineral soil microbial C decreased throughout the experimental period and varied with soil temperature and water content. Litter removal reduced mineral soil microbial C by 19.0% in the ungirdled plots, but only 4.0% in girdled plots. Root trenching, stem girdling and their interactions influenced microbial C in humus layer. Neither root trenching nor girdling significantly influenced mineral soil microbial C. Mineral soil microbial C correlated with following-month plant litterfall in control plots, but these correlations were not observed in root-trenching plots or girdling plots. Our results suggest that belowground carbon retranslocated from shoots and present in soil organic matter, rather than aboveground fresh plant litter inputs, determines seasonal fluctuation of mineral soil microbial biomass.     

Low importance for a fungal based food web in arable soils under mineral and organic fertilization indicated by Collembola grazers

We investigated the Collembola community at an arable field where mineral and organic fertilizers have been applied at low and high rates for 27 years. As food resources for Collembola, the soil microbial community was analyzed using phospholipid fatty acids (PLFAs). A special focus was put on AM fungi, which were estimated by the marker 16:1ω5 in PLFA (viable hyphae) and neutral lipid fatty acid (NLFA – storage fat in spores) fractions. Additionally, whole cellular lipids in crop plant tissues and manure were assessed. Greater Collembola species richness occurred in plots where mineral fertilizer was added. In contrast, soil microbial biomass including AM fungal hyphae increased with addition of organic fertilizer, while the amount of AM fungal spores and biomass of saprotrophic fungi were not affected by fertilizer type. The lipid pattern in wheat roots was altered by fertilizer type, application rate and their interaction, indicating different rhizosphere communities. In sum, the availability and composition of food resources for Collembola changed considerably due to farm management practice. The major diet of three dominant Collembola species, Isotoma viridis, Willemia anophthalma and Polyacanthella schäffer was determined by lipid profiling. Multivariate analysis demonstrated species specific lipid patterns, suggesting greater importance of species than management practice on the diet choice. Nevertheless, feeding strategy was affected by fertilizer type and availability of resources, as trophic biomarker fatty acids indicated feeding on wheat roots (and to some extent saprotrophic fungi) with mineral and a shift to soil organic matter (litter, detritus) with organic fertilization. Although AM fungi dominated the soil fungal community, the AMF marker 16:1ω5 was not detected in Collembola lipids, indicating that these were not consumed. The very low amount of saprotrophic fungi in the soil and the fact that Collembola as major fungal grazers did not feed on AM fungi indicates that the fungal energy channel in the investigated arable field is of little importance to the faunal food web.     

Effect of land management and soil texture on seasonal variations in soil microbial biomass in dry tropical agroecosystems in Tanzania

Soil microbes are an essential component of most terrestrial ecosystems; as decomposers they are responsible for regulating nutrient dynamics, and they also serve as a highly labile nutrient pool. Here, we evaluated seasonal variations in microbial biomass carbon (MBC) and nitrogen (MBN) as well as microbial activity (as qCO₂) for 16 months with respect to several factors relating to soil moisture and nutrients under different land management practices (plant residue application, fertilizer application) in both clayey (38% clay) and sandy (4% clay) croplands in Tanzania. We observed that MBC and MBN tended to decrease during the rainy season whereas they tended to increase and remain at high levels during the dry season in all treatment plots at both of our test sites, although soil moisture did not correlate with MBC or MBN. qCO₂ correlated with soil moisture in all treatment plots at both sites, and hence soil microbes act as decomposers mainly during the rainy season. Although the effect of seasonal variation of soil moisture on the dynamics of MBC, MBN, and qCO₂ was certainly greater than that attributable to plant residue application, fertilizer application, or soil texture, plant residue application early in the rainy season clearly increased MBC and MBN in both clayey and sandy soils. This suggests that plant residue application can help to not only counter the N loss caused by leaching but also synchronize crop N uptake and N release from soil microbes by utilizing these microbes as an ephemeral nutrient pool during the early crop growth period. We also found substantially large seasonal variations in MBC and MBN, continuously high qCO₂, and rapid turnover of soil microbes in sandy soil compared to clayey soil. Taken together, our results indicate that soil microbes, acting as both a nutrient pool and decomposers, have a more substantial impact on tropical sandy soil than on clayey soil.     

The microbial transformation of fungal mycelium as the predecessor of humic acids

This work includes investigation of microbial decomposition of fungal mycelium by pure and mixed bacteria cultures. The decomposition of mycelium is estimated according to CO₂ emission activity by gas chromatography and the change in the quantity of bacterial cells by luminescence microscopy. A comparative stability of fungal melanin to microbial destruction is found. It is shown that fungal mycelium was decomposed first mainly by a native complex of microorganisms and later by gram-positive bacteria. In two months of laboratory work, the fungal biomass decomposed by 25–37%. The results of our research verify the possibility of including melanin in humic acids.     

Greater humification of belowground than aboveground biomass carbon into particulate soil organic matter in no-till corn and soybean crops

Quantifying the amount of carbon (C) incorporated from decomposing residues into soil organic carbon (C_S) requires knowing the rate of C stabilization (humification rate) into different soil organic matter pools. However, the differential humification rates of C derived from belowground and aboveground biomass into C_S pools has been poorly quantified. We estimated the contribution of aboveground and belowground biomass to the formation of C_S in four agricultural treatments by measuring changes in δ¹³C natural abundance in particulate organic matter (C_POM) associated with manipulations of C₃ and C₄ biomass. The treatments were (1) continuous corn cropping (C₄ plant), (2) continuous soybean cropping (C₃), and two stubble exchange treatments (3 and 4) where the aboveground biomass left after the grain harvest was exchanged between corn and soybean plots, allowing the separation of aboveground and belowground C inputs to C_S based on the different δ¹³C signatures. After two growing seasons, C_POM was primarily derived from belowground C inputs, even though they represented only ∼10% of the total plant C inputs as residues. Belowground biomass contributed from 60% to almost 80% of the total new C present in the C_POM in the top 10 cm of soil. The humification rate of belowground C inputs into C_POM was 24% and 10%, while that of aboveground C inputs was only 0.5% and 1.0% for soybean and corn, respectively. Our results indicate that roots can play a disproportionately important role in the C_POM budget in soils. Keywords Particulate organic matter; root carbon inputs; carbon isotopes; humification rate; corn; soybean.     

Soil microbial biomass phosphorus as an indicator of phosphorus availability in a Gleyic Andosol

Abstract

The relationship between plant phosphorus (P) uptake and soil microbial biomass phosphorus (biomass P) or available phosphorus (Truog P) was estimated in a Gleyic Andosol in Sapporo, Hokkaido, in a 4-year field trial (2004–2007). Every year, the soil was treated in duplicate (each plot 36?m²) or triplicate (each plot 24?m²) with chemical fertilizer, cow manure compost or sewage sludge compost, and then kidney beans (Phaseolus vulgaris) were planted. Pooled data of the shoot content of P at harvest over the 4?years was significantly correlated with biomass P determined 1?month after the application of fertilizer (P?<?0.01). A multivariate analysis revealed that the grain yield was significantly positively correlated with the shoot content of P (P?<?0.01) and significantly negatively correlated with the shoot content of calcium (P?<?0.05), but not correlated with the shoot content of either nitrogen or potassium. These results suggest that P is the most limiting element to affect the productivity of kidney bean plants in this trial and that biomass P is an important P source that explains the differences in P availability among soil amendments. Biomass P is a better indicator of P availability for kidney beans grown in Gleyic Andosols compared with Truog P, which is widely used in Japan.     

Carbon,nitrogen and microbial biomass interrelationships during the decomposition of wheat straw: A mechanistic simulation model

The behavior of added carbon as crop residues and nitrogen in agricultural ecosystems is most often quantitatively described by empirically derived first-order rate reactions. A mechanistic approach may be more precise for describing interrelations between C, N and microbial populations during short periods of active decomposition. The effect of N on the disappearance of C from a wheat straw system, and the response of the biomass to N additions, was simulated using microbial growth and maintenance terms derived from the literature. Results of the simulation were compared with microbial growth and wheat straw decomposition measurements made with an electrolytic respirometer. Straw decomposition rate was shown to be strongly dependent on available C and N during initial decomposition. When N is limiting, excess available C apparently is immobilized as polysaccharides.     

Carbon and nitrogen relationships in the microbial biomass of soils in beech (Fagus sylvatica L.) forests

Soils from 38 German forest sites, dominated by beech trees (Fagus sylvatica L.) were sampled to a depth of about 10 cm after careful removal of overlying organic layers. Microbial biomass N and C were measured by fumigation-extraction. The pH of the soils varied between 3.5 and 8.3, covering a wide range of cation exchange capacity, organic C, total N, and soil C:N values. Maximum biomass C and biomass N contents were 2116 g C m^-2 and 347 g N m^-2, while minimum contents were 317 and 30 g m^-2, respectively. Microbial biomass N and C were closely correlated. Large variations in microbial biomass C:N ratios were observed (between 5.4 and 17.3, mean 7.7), indicating that no simple relationship exists between these two parameters. The frequency distribution of the parameters for C and N availability to the microflora divided the soils into two subgroups (with the exception of one soil): (1) microbial: organic C>12 mg g^-1, microbial:total N>28 mg g^-1 (n=23), a group with high C and N availability, and (2) microbial:organic C12 mg g^-1, microbial:total N28 mg g^-1 (n=14), a group with low C and N availability. With the exception of a periodically waterlogged soil, the pH of all soils belonging to subgroup 2 was below 5.0 and the soil C:N ratios were comparatively high. Within these two subgroups no significant correlation between the microbial C:N ratio and soil pH or any other parameter measured was found. The data suggest that above a certain threshold (pH 5.0) microbial C:N values vary within a very small range over a wide range of pH values. Below this threshold, in contrast, the range of microbial C:N values becomes very large.     

Carbon stable isotope fractionation and trophic transfer of fatty acids in fungal based soil food chains

Stable isotope analysis has been used as a powerful tool in food web studies in terrestrial ecosystems. In addition the occurrence and abundance of fatty acids may serve as indicator for feeding strategies of soil animals. Here we combine both approaches and investigate the fatty acid composition, δ¹³C values of bulk tissues and individual fatty acids in soil organisms. The fungi Chaetomium globosum and Cladosporium cladosporioides were isotopically labelled by fructose derived from either C₃ or C₄ plants, and the fungal-feeding nematode Aphelenchoides sp. was reared on C. globosum. Fungi and nematodes were used as diet for the Collembolan Protaphorura fimata. The sugar source was fractionated differently by fungal lipid metabolism in a species-specific manner that points to a sensitivity of physiological processing to the non-random distribution of ¹³C/¹²C isotopes in the molecule. As a general trend stearic acid (18:0) was depleted in ¹³C compared to the precursor palmitic acid (16:0), whereas its desaturation to oleic acid (18:1 ω9) favoured the ¹³C-rich substrate.Fatty acid profiles of P. fimata varied due to food source, indicating incorporation of dietary fatty acids into Collembolan tissue. Individuals feeding on fungi had lower amounts in C20 fatty acids, with monoenoic C20 forms not present. This pattern likely separates primary consumers (fungivores) from predators (nematode feeders). The isotopic discrimination in ¹³C for bulk Collembola ranged between −2.6 and 1.4‰ and was dependent on fungal species and C₃/C₄ system, suggesting differences at metabolic branch points and/or isotope discrimination of enzymes. Comparison of δ¹³C values in individual fatty acids between consumer and diet generally showed depletion (i.e. de novo synthesis) or no changes (i.e. dietary routing), but the fractionation was not uniform and affected by the type of ingested food. Fatty acid carbon isotopes were more variable than those of bulk tissues, likely due to both the distrimination by enzymes and the different lipid origin (i.e. neutral or polar fraction).     

Ecophysiology of the soil microbial biomass and its relation to the soil microbial N pool

Abstract. Microbial osmoregulation as a factor regulating the nitrogen and carbon contents of soil microbial biomass was studied in two experiments. In the first the percentages of the carbon and nitrogen occurring in the cytoplasm of Aspergillus flavus and Pseudomonas sp. were shown to be strongly influenced by osmotic stress. In the second, biomass carbon and nitrogen initially increased with increasing water stress (osmotic and matric) up to −1.0 and −1.5 MPa, respectively, but declined under greater osmotic stress. As the soil microbial carbon and nitrogen pools are affected by these stresses, allowance must be made for them when interpreting biomass measurements in water-stressed soils.     

Surface and subsurface microbial biomass, community structure and metabolic activity as a function of soil depth and season

Microbial biomass, size and community structure along with an estimate of microbial activity and soil chemical parameters were determined at three depths in two soils (e.g. sandy loam Ultic Hapludalf and silt loam Mollic Hapludalf) replicated three times under one winter and summer season. Microbial biomass and community structure were estimated from phospholipid-PO₄ content and fatty acid methyl ester (FAME) measurements. Microbial activity and assimilative capacity were estimated using a ³H-acetate incorporation into phospholipids and by incubating the soil samples at the average winter and summer temperatures, 3 and 20 °C, respectively. We found that the size of the microbial biomass in both the surface and the subsurface soils was not significantly affected by the seasonal variation but activity increased by as much as 83% at the summer temperatures in the surface soil. We demonstrated using FAME analysis that for both soils seasonal changes in the subsurface microbial community occurred. These findings suggest that winter conditions will shift the population activity level in both the surface and subsurface systems and the biochemical structure of the community in the subsurface. In all cases, the inorganic chemical properties of the soil, as a function of season, remained constant. The greatly increased activity of microbial population at the higher temperature will favor the capacity of the system to utilize nutrients or organic materials that may enter soil. During low temperature seasons the capacity of either surface or subsurface soils to assimilate materials is generally diminished but the reduction reflects changes in metabolism and not a reduced biomass size.     

A comparison of methods for soil microbial population and biomass studies

A comparison was made of 15 different techniques which are used in assessing soil microbial populations and/or biomasses. These include direct observations (fungal standing crop, fluorescein diacetate active mycelia, acridine orange stained bacteria), cultural methods (bacterial plate counts), physiological methods (total microbial, bacterial and fungal biomasses, O₂-uptake), soil enzyme analyses (dehydrogenase, catalase, alkaline and acid phosphatase, protease, amylase), and ATP-analyses. The various techniques were applied to six soils known to have different microbial characteristics. The results are discussed with respect to the convertability of counts and measurements into microbial biomasses, the variability of the techniques, the correlations within comparable groups of methods, and the practical limitations in application of individual methods to different soils.     

Limitations of soil microbial biomass carbon as an indicator of soil pollution in the field

The size of the soil microbial biomass carbon (SMBC) has been proposed as a sensitive indicator for measuring the adverse effects of contaminants on the soil microbial community. In this study of Australian agricultural systems, we demonstrated that field variability of SMBC measured using the fumigation-extraction procedure limited its use as a robust ecotoxicological endpoint. The SMBC varied up to 4-fold across control samples collected from a single field site, due to small-scale spatial heterogeneity in the soil physicochemical environment. Power analysis revealed that large numbers of replicates (3-93) were required to identify 20% or 50% decreases in the size of the SMBC of contaminated soil samples relative to their uncontaminated control samples at the 0.05% level of statistical significance. We question the value of the routine measurement of SMBC as an ecotoxicological endpoint at the field scale, and suggest more robust and predictive microbiological indicators.     

C and N natural abundance of the soil microbial biomass

Stable isotope analysis is a powerful tool in the study of soil organic matter formation. It is often observed that more decomposed soil organic matter is ¹³C, and especially ¹⁵N-enriched relative to fresh litter and recent organic matter. We investigated whether this shift in isotope composition relates to the isotope composition of the microbial biomass, an important source for soil organic matter. We developed a new approach to determine the natural abundance C and N isotope composition of the microbial biomass across a broad range of soil types, vegetation, and climates. We found consistently that the soil microbial biomass was ¹⁵N-enriched relative to the total (3.2 ‰) and extractable N pools (3.7 ‰), and ¹³C-enriched relative to the extractable C pool (2.5 ‰). The microbial biomass was also ¹³C-enriched relative to total C for soils that exhibited a C3-plant signature (1.6 ‰), but ¹³C-depleted for soils with a C4 signature (−1.1 ‰). The latter was probably associated with an increase of annual C3 forbs in C4 grasslands after an extreme drought. These findings are in agreement with the proposed contribution of microbial products to the stabilized soil organic matter and may help explain the shift in isotope composition during soil organic matter formation.     

Effect of long-term fertilizers and manure application on microbial biomass and microbial activity of a tropical agricultural soil

We investigated some aspects of soil quality and community-level physiological profiles (CLPP) of bacteria in soil under a long-term (37 years) trial with either exclusive inorganic fertilizers or fertilizers combined with farmyard manure cultivated with jute–rice–wheat system. The treatments consisted of 100% recommended dose (RD) of NPK, 150% RD of NPK, 100% RD of N, 100% RD of NPK + FYM (10 t ha⁻¹ year⁻¹), and untreated control. Long-term application of 150% RD of NPK lowered the soil pH considerably while the soils in the other treatments remained near neutral. The 100% RD of NPK + FYM treated plot showed significantly highest accumulation of organic carbon, total nitrogen, microbial biomass carbon, basal soil respiration, and fluorescein diacetate hydrolyzing activity among the treatments. CLPP analysis in Biolog Ecoplates revealed that utilization of carbohydrates was enhanced in all input treated regimes, while the same for polymers, carboxylic acids, amino acids, and amines/amides were similar or less than the untreated control. However, within these groups of carbon sources, heterogeneity of individual substrate utilization between treatments was also noted. Taken together, addition of organic supplements showed significantly increased microbial biomass carbon and microbial activity, but input of nutrient supplements, both inorganic and organic, only marginally affected the overall substrate utilization pattern of soil microorganisms.     

Feasibility of a cell separation-proteomic based method for soils with different edaphic properties and microbial biomass

The suitability of a soil proteome analysis based on previous cell extraction by gradient centrifugation was tested in semiarid soils with distinct edaphic properties and microbial biomass after enrichment with carbon and nitrogen. A sandy loam soil with low organic carbon content reached higher microbial biomass (estimated by PLFAs) after stimulation with nutrient sources (glucose and proline) than a naturally rich soil. However, the extractability of soil microbial cells was higher in a poor soil with high electrical conductivity probably due to the high saline content. The number of identified proteins in the poor soils reached 71 with proteins related to energy processes, transport and nucleic acid metabolism representing the highest percentage. High organic carbon content negatively influences cell extraction and protein separation and analysis. Soil texture and/or salinity might be related to the expression of proteins involved in the removal of reactive oxygen species (ROS) such catalase and superoxide dismutase (SOD) under active metabolism and microbial biomass development