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At least 19 records

Modest functional diversity decline and pronounced composition shifts of microbial communities in a mixed waste-contaminated aquifer

Background: Microbial taxonomic diversity declines with increased environmental stress. Yet, few studies have explored whether phylogenetic and functional diversities track taxonomic diversity along the stress gradient. Here, we investigated microbial communities within an aquifer in Oak Ridge, Tennessee, USA, which is characterized by a broad spectrum of stressors, including extremely high levels of nitrate, heavy metals like cadmium and chromium, radionuclides such as uranium, and extremely low pH (< 3). Results: Both taxonomic and phylogenetic α-diversities were reduced in the most impacted wells, while the decline in functional α-diversity was modest and statistically insignificant, indicating a more robust buffering capacity to environmental stress. Differences in functional gene composition (i.e., functional β-diversity) were pronounced in highly contaminated wells, while convergent functional gene composition was observed in uncontaminated wells. The relative abundances of most carbon degradation genes were decreased in contaminated wells, but genes associated with denitrification, adenylylsulfate reduction, and sulfite reduction were increased. Compared to taxonomic and phylogenetic compositions, environmental variables played a more significant role in shaping functional gene composition, suggesting that niche selection could be more closely related to microbial functionality than taxonomy. Conclusions: Overall, we demonstrated that despite a reduced taxonomic α-diversity, microbial communities under stress maintained functionality underpinned by environmental selection.

59 BASIC BIOLOGICAL SCIENCES↗

METABOLIC: high-throughput profiling of microbial genomes for functional traits, metabolism, biogeochemistry, and community-scale functional networks

Background Advances in microbiome science are being driven in large part due to our ability to study and infer microbial ecology from genomes reconstructed from mixed microbial communities using metagenomics and single-cell genomics. Such omics-based techniques allow us to read genomic blueprints of microorganisms, decipher their functional capacities and activities, and reconstruct their roles in biogeochemical processes. Currently available tools for analyses of genomic data can annotate and depict metabolic functions to some extent; however, no standardized approaches are currently available for the comprehensive characterization of metabolic predictions, metabolite exchanges, microbial interactions, and microbial contributions to biogeochemical cycling. Results We present METABOLIC (METabolic And BiogeOchemistry anaLyses In miCrobes), a scalable software to advance microbial ecology and biogeochemistry studies using genomes at the resolution of individual organisms and/or microbial communities. The genome-scale workflow includes annotation of microbial genomes, motif validation of biochemically validated conserved protein residues, metabolic pathway analyses, and calculation of contributions to individual biogeochemical transformations and cycles. The community-scale workflow supplements genome-scale analyses with determination of genome abundance in the microbiome, potential microbial metabolic handoffs and metabolite exchange, reconstruction of functional networks, and determination of microbial contributions to biogeochemical cycles. METABOLIC can take input genomes from isolates, metagenome-assembled genomes, or single-cell genomes. Results are presented in the form of tables for metabolism and a variety of visualizations including biogeochemical cycling potential, representation of sequential metabolic transformations, community-scale microbial functional networks using a newly defined metric “MW-score” (metabolic weight score), and metabolic Sankey diagrams. METABOLIC takes ~ 3 h with 40 CPU threads to process ~ 100 genomes and corresponding metagenomic reads within which the most compute-demanding part of hmmsearch takes ~ 45 min, while it takes ~ 5 h to complete hmmsearch for ~ 3600 genomes. Tests of accuracy, robustness, and consistency suggest METABOLIC provides better performance compared to other software and online servers. To highlight the utility and versatility of METABOLIC, we demonstrate its capabilities on diverse metagenomic datasets from the marine subsurface, terrestrial subsurface, meadow soil, deep sea, freshwater lakes, wastewater, and the human gut. Conclusion METABOLIC enables the consistent and reproducible study of microbial community ecology and biogeochemistry using a foundation of genome-informed microbial metabolism, and will advance the integration of uncultivated organisms into metabolic and biogeochemical models. METABOLIC is written in Perl and R and is freely available under GPLv3 at https://github.com/AnantharamanLab/METABOLIC.

59 BASIC BIOLOGICAL SCIENCES↗

Biodegradation of Fresh vs. Oven-Dried Inedible Crop Residue in a Continuously Stirred Tank Reactor

The degradation of soluble organics and mineral recovery from fresh and oven-dried biomass were compared in an Intermediate-Scale Aerobic Bioreactor (8 L working volume) to determine if drying crop residue improves performance in a continuously stirred tank reactor (CSTR). The study was conducted in an Intermediate-Scale Aerobic Bioreactor (ISAB) CSTR with dimensions of 390 mm height x 204 mm diameter. The pH in the bioreactor was controlled at 6.0, temperature at 30 C, and aeration at 7.0 L/min. Gases monitored were CO2 evolution and dissolved oxygen. Homogeneously mixed wheat cultures, used either fresh or oven-dried biomass and were leached, then placed in the ISAB for a 4-day degradation period. Studies found that mineral recovery was greater for leached oven-dried crop residue. However, after activity by the mixed microbial communities in the ISAB CSTR, there were little notable differences in the measured mineral recovery and degradation of soluble organic compounds. Degradation of soluble organic compounds was also shown to improve for leached oven-dried crop residue, but after mixing in the CSTR the degradation of the fresh biomass seemed to be slightly greater. Time for the biomass to turn in the CSTR appeared to be one factor for the experimental differences between the fresh and oven-dried biomass. Other factors, although not as defined, were the differing physical structures in the cell walls and varying microbial components of the fresh and oven-dried treatments due to changes in chemical composition after drying of the biomass.

Crawford, Kamau↗

Development of an Intermediate-Scale Aerobic Bioreactor to Regenerate Nutrients from Inedible Crop Residues

Three Intermediate-Scale Aerobic Bioreactors were designed, fabricated, and operated. They utilized mixed microbial communities to bio-degrade plant residues. The continuously stirred tank reactors operated at a working volume of 8 L, and the average oxygen mass transfer coefficient, k(sub L)a, was 0.01 s(exp -1). Mixing time was 35 s. An experiment using inedible wheat residues, a replenishment rate of 0.125/day, and a solids loading rate of 20 gdw/day yielded a 48% reduction in biomass. Bioreactor effluent was successfully used to regenerate a wheat hydroponic nutrient solution. Over 80% of available potassium, calcium, and other minerals were recovered and recycled in the 76-day wheat growth experiment.

Finger, Barry W.↗

Diagnosing and Predicting Mixed-Culture Fermentations with Unicellular and Guild-Based Metabolic Models

Microbiomes are vital to human health, agriculture, and protecting the environment. Predicting behavior of self-assembled or synthetic microbiomes, however, remains a challenge. In this work, we used unicellular and guild-based metabolic models to investigate production of medium-chain fatty acids by a mixed microbial community that is fed multiple organic substrates. Modeling results provided insights into metabolic pathways of three medium-chain fatty acid-producing guilds and identified potential strategies to increase production of medium-chain fatty acids. This work demonstrates the role of metabolic models in augmenting multi-omic studies to gain greater insights into microbiome behavior.

59 BASIC BIOLOGICAL SCIENCES↗

Eukaryotic genomes from a global metagenomic data set illuminate trophic modes and biogeography of ocean plankton

ABSTRACT Metagenomics is a powerful method for interpreting the ecological roles and physiological capabilities of mixed microbial communities. Yet, many tools for processing metagenomic data are neither designed to consider eukaryotes nor are they built for an increasing amount of sequence data. EukHeist is an automated pipeline to retrieve eukaryotic and prokaryotic metagenome-assembled genomes (MAGs) from large-scale metagenomic sequence data sets. We developed the EukHeist workflow to specifically process large amounts of both metagenomic and/or metatranscriptomic sequence data in an automated and reproducible fashion. Here, we applied EukHeist to the large-size fraction data (0.8–2,000 µm) from Tara Oceans to recover both eukaryotic and prokaryotic MAGs, which we refer to as TOPAZ (Tara Oceans Particle-Associated MAGs). The TOPAZ MAGs consisted of >900 environmentally relevant eukaryotic MAGs and >4,000 bacterial and archaeal MAGs. The bacterial and archaeal TOPAZ MAGs expand upon the phylogenetic diversity of likely particle- and host-associated taxa. We use these MAGs to demonstrate an approach to infer the putative trophic mode of the recovered eukaryotic MAGs. We also identify ecological cohorts of co-occurring MAGs, which are driven by specific environmental factors and putative host-microbe associations. These data together add to a number of growing resources of environmentally relevant eukaryotic genomic information. Complementary and expanded databases of MAGs, such as those provided through scalable pipelines like EukHeist, stand to advance our understanding of eukaryotic diversity through increased coverage of genomic representatives across the tree of life. IMPORTANCE Single-celled eukaryotes play ecologically significant roles in the marine environment, yet fundamental questions about their biodiversity, ecological function, and interactions remain. Environmental sequencing enables researchers to document naturally occurring protistan communities, without culturing bias, yet metagenomic and metatranscriptomic sequencing approaches cannot separate individual species from communities. To more completely capture the genomic content of mixed protistan populations, we can create bins of sequences that represent the same organism (metagenome-assembled genomes [MAGs]). We developed the EukHeist pipeline, which automates the binning of population-level eukaryotic and prokaryotic genomes from metagenomic reads. We show exciting insight into what protistan communities are present and their trophic roles in the ocean. Scalable computational tools, like EukHeist, may accelerate the identification of meaningful genetic signatures from large data sets and complement researchers’ efforts to leverage MAG databases for addressing ecological questions, resolving evolutionary relationships, and discovering potentially novel biodiversity.

59 BASIC BIOLOGICAL SCIENCES↗

Chain elongators, friends, and foes

Bioproduction of medium chain carboxylic acids has recently emerged as an alternative strategy to valorize low-value organic waste and side-streams. Key to this route is chain elongation, an anaerobic microbial process driven by ethanol, lactic acid, or carbohydrates. Additionally, because these technologies use wastes as feedstocks, mixed microbial communities are often considered as biocatalysts. Understanding and steering these microbiomes is key to optimize bioprocess performance. From a meta-analysis of publicly available sequencing data, we (i) explore how the current collection of isolated chain elongators compares to microbiome members, (ii) discuss the main beneficial and antagonistic interactions with community partners, and (iii) identify the key research gaps and needs to help understand chain elongation microbiomes, and design/steer these novel bioproduction processes.

59 BASIC BIOLOGICAL SCIENCES↗

Stochasticity in microbiology: managing unpredictability to reach the Sustainable Development Goals

Pure (single) cultures of microorganisms and mixed microbial communities (microbiomes) have been important for centuries in providing renewable energy, clean water and food products to human society and will continue to play a crucial role to pursue the Sustainable Development Goals. To use microorganisms effectively, microbial engineered processes require adequate control. Microbial communities are shaped by manageable deterministic processes, but also by stochastic processes, which can promote unforeseeable variations and adaptations. Here, we highlight the impact of stochasticity in single culture and microbiome engineering. First, we discuss the concepts and mechanisms of stochasticity in relation to microbial ecology of single cultures and microbiomes. Second, we discuss the consequences of stochasticity in relation to process performance and human health, which are reflected in key disadvantages and important opportunities. Third, we propose a suitable decision tool to deal with stochasticity in which monitoring of stochasticity and setting the boundaries of stochasticity by regulators are central aspects. Stochasticity may give rise to some risks, such as the presence of pathogens in microbiomes. We argue here that by taking the necessary precautions and through clever monitoring and interpretation, these risks can be mitigated.

59 BASIC BIOLOGICAL SCIENCES↗

Utilization of the water soluable fraction of wheat straw as a plant nutrient source

Recovery of water soluble, inorganic nutrients from the inedible portion of wheat was found to be an effective means of recycling nutrients within hydroponic systems. Through aqueous extraction (leaching), 60 percent of the total inorganic nutrient weight was removed from wheat straw and roots, although the recovery of individual nutrients varied. Leaching also removed about 20 percent of the total organic carbon from the biomass. In terms of dry weight, the leachate was comprised of approximately 60 percent organic and 40 percent inorganic compounds. Direct use of wheat straw leachate in static hydroponic systems had an inhibitory effect on wheat growth, both in the presence and absence of microorganisms. Biological treatment of leachate either with a mixed microbial community or the oyster mushroom Pleurotus ostreatus L., prior to use in hydroponic solutions, significantly reduced both the organic content and the inhibitory effects of the leachate. The inhibitory effects of unprocessed leachate appear to be a result of rapidly acting phytotoxic compounds that are detoxified by microbial activity. Leaching holds considerable promise as a method for nutrient recycling in a Controlled Ecological Life Support System (CELSS).

Mackowiak, C. L.↗

Controlling Microbial Byproducts using Model-Based Substrate Monitoring and Control Strategies

We have developed a computer-controlled bioreactor system to study various aspects of microbially-mediated nitrogen cycling. The system has been used to investigate methods for controlling microbial denitrification (the dissimilatory reduction of nitrate to N2O and N2) in hydroponic plant growth chambers. Such chambers are key elements of advanced life support systems being designed for use on long duration space missions, but nitrogen use efficiency in them is reduced by denitrification. Control software architecture was designed which permits the heterogeneous control of system hardware using traditional feedback control, and quantitative and qualitative models of various system features. Model-based feed forward control entails prediction of future systems in states and automated regulation of system parameters to achieve desired and avoid undesirable system states. A bacterial growth rate model based on the classic Monod model of saturation kinetics was used to evaluate the response of several individual denitrifying species to varying environmental conditions. The system and models are now being applied to mixed microbial communities harvested from the root zone of a hydroponic growth chamber. The use of a modified Monod organism interaction model was evaluated as a means of achieving more accurate description of the dynamic behavior of the communities. A minimum variance parameter estimation routine was also' used to calibrate the constant parameters in the model by iterative evaluation of substrate (nitrate) uptake and growth kinetics. This representation of processes and interactions aids in the formulation of control laws. The feed forward control strategy being developed will increase system autonomy, reduce crew intervention and limit the accumulation of undesirable waste products (NOx).

Smernoff, David T.↗

Ecological Dichotomies Arise in Microbial Communities Due to Mixing of Deep Hydrothermal Waters and Atmospheric Gas in a Circumneutral Hot Spring

Understanding the source and availability of energy capable of supporting life in hydrothermal environments is central to predicting the ecology of microbial life on early Earth when volcanic activity was more widespread. Little is known of the substrates supporting microbial life in circumneutral to alkaline springs, despite their relevance to early Earth habitats.

59 BASIC BIOLOGICAL SCIENCES↗

Development and Validation of a Process Model and Open-Source Process Simulator for Microalgae-Based Tertiary Phosphorus Recovery

Microalgae-based tertiary wastewater treatment has the potential to meet stringent effluent phosphorus limits, with the added benefit of producing a marketable feedstock. However, the lack of validated mechanistic models and their implementation in process simulators have limited the adoption of this technology. In this study, an updated lumped pathway metabolic model (Phototrophic-Mixotrophic Process Model, PM 2 ), including both photoautotrophic and heterotrophic metabolisms of microalgae, was developed to predict effluent phosphorus concentration and biomass yield in response to dynamic influent and varying environmental conditions. The model was implemented in QSDsan – an open-source, Python-based design and simulation platform – for robust simulation under uncertainty. A global sensitivity analysis was performed to prioritize model parameters for calibration. The model was then calibrated and validated using batch experimental data and 45 days of continuous online monitoring data from a full-scale (568 m 3 ·d -1 ) microalgae-based tertiary wastewater treatment plant (EcoRecover process). In particular, along with dynamic influent composition, temperature and light intensity data with diel variation were provided as model inputs to reflect the microalgal behavior under day-night cycling. Overall, the QSDsan-based microalgae process simulator was able to predict effluent phosphorus within 0.02–0.04 mg-P·L -1 , while also capturing the general trends of state variables according to nutrient availability.

Lumped pathway metabolic model↗

Mixed and membrane-separated culturing of synthetic cyanobacteria-yeast consortia reveals metabolic cross-talk mimicking natural cyanolichens

Metabolite exchange mediates crucial interactions in microbial communities, significantly impacting global carbon and nitrogen cycling. Understanding these chemically-mediated interactions is essential for elucidating natural community functions and developing engineered synthetic communities. This study investigated membrane-separated bioreactors (mBRs) as a novel tool to identify transient metabolites and their producers/consumers in mixed microbial communities. We compared three co-culture methods (direct mixed, 2-chamber mBR, and 3-chamber mBR) to grow a synthetic binary community of the cyanobacterium Synechococcus elongatus PCC 7942 and the fungus Rhodotorula toruloides NBRC 0880, as well as axenic S. elongatus. Despite not being natural lichen constituents, these organisms exhibited interactions resembling those in cyanolichens. S. elongatus fixed CO 2 into sugars as the primary shared metabolite, while R. toruloides secreted various biochemicals, predominantly sugar alcohols, mirroring the metabolite exchange observed in natural lichens. The mBR systems successfully captured metabolite gradients and revealed rapidly consumed compounds, including TCA cycle intermediates and amino acids. Our approach demonstrated that the 2-chamber mBR optimally balanced metabolite exchange and growth dynamics. This study provides insights into cross-species metabolic interactions and presents a valuable tool for investigating and engineering synthetic microbial communities with potential applications in biotechnology and environmental science.

59 BASIC BIOLOGICAL SCIENCES↗

Isoflavone supplementation, via red clover hay, alters the rumen microbial community and promotes weight gain of steers grazing mixed grass pastures

Biochanin A, an isoflavone present in the pasture legume red clover (Trifloium pratense L.), alters fermentation in the rumen of cattle and other ruminants. Biochanin A inhibits hyperammonia-producing bacteria and promotes cellulolytic bacteria and fiber catalysis in vitro and ex vivo. Consequently, biochanin A supplementation improves weight gain in grazing steers. Red clover contains biologically active isoflavones that may act synergistically. Therefore, the objective was to evaluate the effect of two levels of red clover hay on growth performance and the microbial community in growing steers grazing mixed grass pastures. A grazing experiment was conducted over 2 early growing seasons (2016 and 2017) with 36 cross-bred steers and twelve rumen-fistulated, growing Holstein steers for evaluation of average daily gain and rumen microbiota, respectively. Steers were blocked by body weight and assigned to pastures with one of four treatments: 1) pasture only, 2) pasture + dry distillers’ grains (DDG), 3) pasture + DDG + low level of red clover hay (~15% red clover diet), or 4) pasture + DDG + high level of red clover hay (~30% red clover diet). DDG were added to treatments to meet protein requirements and to balance total protein supplementation between treatments. All supplementation strategies (DDG ± red clover hay) increased average daily gains in comparison to pasture-only controls (P < 0.05), with a low level of red clover supplementation being the most effective (+0.17 kg d-1 > DDG only controls; P < 0.05). Similarly, hyper-ammonia-producing bacteria inhibition (10–100-fold; P < 0.05), fiber catalysis (+10–25%; P < 0.05) and short chain fatty acid concentrations were greatest with the low red clover supplement (+~25%; P < 0.05). These results provide evidence that lower levels or red clover supplementation may be optimal for maximizing overall microbial community function and animal performance in grazing steers.

54 ENVIRONMENTAL SCIENCES↗

Impact of prescribed fire on soil microbial communities in a Southern Appalachian Forest clear-cut

Escalating wildfire frequency and severity, exacerbated by shifting climate patterns, pose significant ecological and economic challenges. Prescribed burns, a common forest management tool, aim to mitigate wildfire risks and protect biodiversity. Nevertheless, understanding the impact of prescribed burns on soil and microbial communities in temperate mixed forests, considering temporal dynamics and slash fuel types, remains crucial. Our study, conducted at the University of Tennessee Forest Resources AgResearch and Education Center in Oak Ridge, TN, employed controlled burns across various treatments, and the findings indicate that low-intensity prescribed burns have none or minimal short-term effects on soil parameters but may alter soil nutrient concentrations, as evidenced by significant changes in porewater acetate, formate, and nitrate concentrations. These burns also induce shifts in microbial community structure and diversity, with Proteobacteria and Acidobacteria increasing significantly post-fire, possibly aiding soil recovery. In contrast, Verrucomicrobia showed a notable decrease over time, and other specific microbial taxa correlated with soil pH, porewater nitrate, ammonium , and phosphate concentrations. Our research contributes to understanding the intricate relationships between prescribed fire, soil dynamics, and microbial responses in temperate mixed forests in the Southern Appalachian Region, which is valuable for informed land management practices in the face of evolving environmental challenges.

Microbiology↗

Biotic degradation of acetonitrile. Limitations, controls and conversion rates

This document has been prepared to support Washington River Protection Solutions (WRPS) in their evaluation of technologies to remediate organic compounds in the Liquid Effluent Retention Facility (LERF)/Effluent Treatment Facility (ETF) during direct feed low activity waste (DFLAW) operations at Hanford. This report summarizes the results of a review of the available literature describing aerobic and anaerobic biological treatment of acetonitrile and proposes a test matrix to evaluate microbial degradation rates of acetonitrile. Numerous studies describe acetonitrile degradation by pure culture strains of bacteria, fungi, and yeast; however, the purpose of this review was to provide estimations of degradation under conditions that more closely approximate the Liquid Effluent Retention Facility (LERF) basins at the Hanford Site. To this end, documented limitations, controls, and conversion rates are summarized for mixed and natural microbial communities being maintained or operated as a biological treatment for wastewater processing. Based on the literature evaluation, the inherent capacity and rates of microbial degradation of acetonitrile are sufficiently high that if applied in the LERF basins, acetonitrile removal would be achieved. To be clear, though, published studies have not been conducted under conditions that approximate or that could be directly related to the LERF basins. Thus, a focused test matrix the encompasses LERF relevant conditions to specifically measure microbial degradation of acetonitrile is proposed and the results would provide the strong technical justification for use of this low-cost efficient biological process at LERF during DFLAW operations and beyond.

12 MANAGEMENT OF RADIOACTIVE AND NON-RADIOACTIVE W↗

Complex subsurface hydrothermal fluid mixing at a submarine arc volcano supports distinct and highly diverse microbial communities

Hydrothermally active submarine volcanoes are mineral-rich biological oases contributing significantly to chemical fluxes in the deep sea, yet little is known about the microbial communities inhabiting these systems. Here we investigate the diversity of microbial life in hydrothermal deposits and their metagenomics-inferred physiology in light of the geological history and resulting hydrothermal fluid paths in the subsurface of Brothers submarine volcano north of New Zealand on the southern Kermadec arc. From metagenome-assembled genomes we identified over 90 putative bacterial and archaeal genomic families and nearly 300 previously unknown genera, many potentially endemic to this submarine volcanic environment. While magmatically influenced hydrothermal systems on the volcanic resurgent cones of Brothers volcano harbor communities of thermoacidophiles and diverse members of the superphylum “DPANN,” two distinct communities are associated with the caldera wall, likely shaped by two different types of hydrothermal circulation. The communities whose phylogenetic diversity primarily aligns with that of the cone sites and magmatically influenced hydrothermal systems elsewhere are characterized predominately by anaerobic metabolisms. These populations are probably maintained by fluids with greater magmatic inputs that have interacted with different (deeper) previously altered mineral assemblages. However, proximal (a few meters distant) communities with gene-inferred aerobic, microaerophilic, and anaerobic metabolisms are likely supported by shallower seawater-dominated circulation. Furthermore, mixing of fluids from these two distinct hydrothermal circulation systems may have an underlying imprint on the high microbial phylogenomic diversity. Collectively our results highlight the importance of considering geologic evolution and history of subsurface processes in studying microbial colonization and community dynamics in volcanic environments.

59 BASIC BIOLOGICAL SCIENCES↗

Effects of artificial defoliation of pines on the structure and physiology of the soil fungal community of a mixed pine-spruce forest

Loss of photosynthetic area can affect soil microbial communities by altering the availability of fixed carbon. We used denaturing gradient gel electrophoresis (DGGE) and Biolog filamentous-fungus plates to determine the effects of artificial defoliation of pines in a mixed pine-spruce forest on the composition of the fungal community in a forest soil. As measured by DGGE, two fungal species were affected significantly by the defoliation of pines (P < 0.001); the frequency of members of the ectomycorrhizal fungus genus Cenococcum decreased significantly, while the frequency of organisms of an unidentified soil fungus increased. The decrease in the amount of Cenococcum organisms may have occurred because of the formation of extensive hyphal networks by species of this genus, which require more of the carbon fixed by their host, or because this fungus is dependent upon quantitative differences in spruce root exudates. The defoliation of pines did not affect the overall composition of the soil fungal community or fungal-species richness (number of species per core). Biolog filamentous-fungus plate assays indicated a significant increase (P < 0.001) in the number of carbon substrates utilized by the soil fungi and the rate at which these substrates were used, which could indicate an increase in fungal-species richness. Thus, either small changes in the soil fungal community give rise to significant increases in physiological capabilities or PCR bias limits the reliability of the DGGE results. These data indicate that combined genetic and physiological assessments of the soil fungal community are needed to accurately assess the effect of disturbance on indigenous microbial systems.

Pinus/growth & development/physiology↗