Temperature Adaptations in the Terminal Processes of Anaerobic Decomposition of Yellowstone National Park and Icelandic Hot Spring Microbial Mats

The optimum temperatures for methanogenesis in microbial mats of four neutral to alkaline, low-sulfate hot springs in Yellowstone National Park were between 50 and 60°C, which was 13 to 23°C lower than the upper temperature for mat development. Significant methanogenesis at 65°C was only observed in one of the springs. Methane production in samples collected at a 51 or 62°C site in Octopus Spring was increased by incubation at higher temperatures and was maximal at 70°C. Strains of Methanobacterium thermoautotrophicum were isolated from 50, 55, 60, and 65°C sites in Octopus Spring at the temperatures of the collection sites. The optimum temperature for growth and methanogenesis of each isolate was 65°C. Similar results were found for the potential rate of sulfate reduction in an Icelandic hot spring microbial mat in which sulfate reduction dominated methane production as a terminal process in anaerobic decomposition. The potential rate of sulfate reduction along the thermal gradient of the mat was greatest at 50°C, but incubation at 60°C of the samples obtained at 50°C increased the rate. Adaptation to different mat temperatures, common among various microorganisms and processes in the mats, did not appear to occur in the processes and microorganisms which terminate the anaerobic food chain. Other factors must explain why the maximal rates of these processes are restricted to moderate temperatures of the mat ecosystem.     

Influence of Sulfide and Temperature on Species Composition and Community Structure of Hot Spring Microbial Mats

In solfataric fields in southwestern Iceland, neutral and sulfide-rich hot springs are characterized by thick bacterial mats at 60 to 80°C that are white or yellow from precipitated sulfur (sulfur mats). In low-sulfide hot springs in the same area, grey or pink streamers are formed at 80 to 90°C, and a Chloroflexus mat is formed at 65 to 70°C. We have studied the microbial diversity of one sulfur mat (high-sulfide) hot spring and one Chloroflexus mat (low-sulfide) hot spring by cloning and sequencing of small-subunit rRNA genes obtained by PCR amplification from mat DNA. Using 98% sequence identity as a cutoff value, a total of 14 bacterial operational taxonomic units (OTUs) and 5 archaeal OTUs were detected in the sulfur mat; 18 bacterial OTUs were detected in the Chloroflexus mat. Although representatives of novel divisions were found, the majority of the sequences were >95% related to currently known sequences. The molecular diversity analysis showed that Chloroflexus was the dominant mat organism in the low-sulfide spring (1 mg liter⁻¹) below 70°C, whereas Aquificales were dominant in the high-sulfide spring (12 mg liter⁻¹) at the same temperature. Comparison of the present data to published data indicated that there is a relationship between mat type and composition of Aquificales on the one hand and temperature and sulfide concentration on the other hand.     

Archaeal and Bacterial Glycerol Dialkyl Glycerol Tetraether Lipids in Hot Springs of Yellowstone National Park

Glycerol dialkyl glycerol tetraethers (GDGTs) are core membrane lipids originally thought to be produced mainly by (hyper)thermophilic archaea. Environmental screening of low-temperature environments showed, however, the abundant presence of structurally diverse GDGTs from both bacterial and archaeal sources. In this study, we examined the occurrences and distribution of GDGTs in hot spring environments in Yellowstone National Park with high temperatures (47 to 83°C) and mostly neutral to alkaline pHs. GDGTs with 0 to 4 cyclopentane moieties were dominant in all samples and are likely derived from both (hyper)thermophilic Crenarchaeota and Euryarchaeota. GDGTs with 4 to 8 cyclopentane moieties, likely derived from the crenarchaeotal order Sulfolobales and the euryarchaeotal order Thermoplasmatales, are usually present in much lower abundance, consistent with the relatively high pH values of the hot springs. The relative abundances of cyclopentane-containing GDGTs did not correlate with in situ temperature and pH, suggesting that other environmental and possibly genetic factors play a role as well. Crenarchaeol, a biomarker thought to be specific for nonthermophilic group I Crenarchaeota, was also found in most hot springs, though in relatively low concentrations, i.e., <5% of total GDGTs. Its abundance did not correlate with temperature, as has been reported previously. Instead, the cooccurrence of relatively abundant nonisoprenoid GDGTs thought to be derived from soil bacteria suggests a predominantly allochthonous source for crenarchaeol in these hot spring environments. Finally, the distribution of bacterial branched GDGTs suggests that they may be derived from the geothermally heated soils surrounding the hot springs.     

Distribution of Thermus spp. in Icelandic Hot Springs and a Thermal Gradient

The growth range in nature of bacteria belonging to the genus Thermus was investigated by sampling 55 different hot springs in Iceland. The springs ranged in temperature from 32 to 99°C, and in pH from 2.1 to 10.1. Viable counts of Thermus spp. ranging from 10 to 10⁴ CFU/100 ml of spring water were found in 27 of the springs sampled. The temperature range for these bacteria was found to be 55 to 85°C, and the pH range was from about 6.5 to above 10. Thermus spp. were found in springs containing up to 1 mM dissolved sulfide and having conductivity up to 2,000 μS/cm. The distribution of Thermus spp. in a hot spring thermal gradient was also investigated and found to agree well with the overall distribution in individual springs.     

Characterization of an Invertase with pH Tolerance and Truncation of Its N-Terminal to Shift Optimum Activity toward Neutral pH

Most invertases identified to date have optimal activity at acidic pH, and are intolerant to neutral or alkaline environments. Here, an acid invertase named uninv2 is described. Uninv2 contained 586 amino acids, with a 100 amino acids N-terminal domain, a catalytic domain and a C-terminal domain. With sucrose as the substrate, uninv2 activity was optimal at pH 4.5 and at 45°C. Removal of N-terminal domain of uninv2 has shifted the optimum pH to 6.0 while retaining its optimum temperaure at 45°C. Both uninv2 and the truncated enzyme retained highly stable at neutral pH at 37°C, and they were stable at their optimum pH at 4°C for as long as 30 days. These characteristics make them far superior to invertase from Saccharomyces cerevisiae, which is mostly used as industrial enzyme.     

Changes in Quinone Profiles of Hot Spring Microbial Mats with a Thermal Gradient

The respiratory and photosynthetic quinones of microbial mats which occurred in Japanese sulfide-containing neutral-pH hot springs at different temperatures were analyzed by spectrochromatography and mass spectrometry. All of the microbial mats that developed at high temperatures (temperatures above 68°C) were so-called sulfur-turf bacterial mats and produced methionaquinones (MTKs) as the major quinones. A 78°C hot spring sediment had a similar quinone profile. Chloroflexus-mixed mats occurred at temperatures of 61 to 65°C and contained menaquinone 10 (MK-10) as the major component together with significant amounts of either MTKs or plastoquinone 9 (PQ-9). The sunlight-exposed biomats growing at temperatures of 45 to 56°C were all cyanobacterial mats, in which the photosynthetic quinones (PQ-9 and phylloquinone) predominated and MK-10 was the next most abundant component in most cases. Ubiquinones (UQs) were not found or were detected in only small amounts in the biomats growing at temperatures of 50°C and above, whereas the majority of the quinones of a purple photosynthetic mat growing at 34°C were UQs. A numerical analysis of the quinone profiles was performed by using the following three parameters: dissimilarity index (D), microbial divergence index (MD_q), and bioenergetic divergence index (BD_q). A D matrix tree analysis showed that the hot spring mats consisting of the sulfur-turf bacteria, Chloroflexus spp., cyanobacteria, and purple phototrophic bacteria formed distinct clusters. Analyses of MD_q and BD_q values indicated that the microbial diversity of hot spring mats decreased as the temperature of the environment increased. The changes in quinone profiles and physiological types of microbial mats in hot springs with thermal gradients are discussed from evolutionary viewpoints.     

Nanoarchaeota,Their Sulfolobales Host,and Nanoarchaeota Virus Distribution across Yellowstone National Park Hot Springs

Nanoarchaeota are obligate symbionts with reduced genomes first described from marine thermal vent environments. Here, both community metagenomics and single-cell analysis revealed the presence of Nanoarchaeota in high-temperature (∼90°C), acidic (pH ≈ 2.5 to 3.0) hot springs in Yellowstone National Park (YNP) (United States). Single-cell genome analysis of two cells resulted in two nearly identical genomes, with an estimated full length of 650 kbp. Genome comparison showed that these two cells are more closely related to the recently proposed Nanobsidianus stetteri from a more neutral YNP hot spring than to the marine Nanoarchaeum equitans. Single-cell and catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH) analysis of environmental hot spring samples identified the host of the YNP Nanoarchaeota as a Sulfolobales species known to inhabit the hot springs. Furthermore, we demonstrate that Nanoarchaeota are widespread in acidic to near neutral hot springs in YNP. An integrated viral sequence was also found within one Nanoarchaeota single-cell genome and further analysis of the purified viral fraction from environmental samples indicates that this is likely a virus replicating within the YNP Nanoarchaeota.     

A Comprehensive Census of Microbial Diversity in Hot Springs of Tengchong,Yunnan Province China Using 16S rRNA Gene Pyrosequencing

The Rehai and Ruidian geothermal fields, located in Tengchong County, Yunnan Province, China, host a variety of geochemically distinct hot springs. In this study, we report a comprehensive, cultivation-independent census of microbial communities in 37 samples collected from these geothermal fields, encompassing sites ranging in temperature from 55.1 to 93.6°C, in pH from 2.5 to 9.4, and in mineralogy from silicates in Rehai to carbonates in Ruidian. Richness was low in all samples, with 21–123 species-level OTUs detected. The bacterial phylum Aquificae or archaeal phylum Crenarchaeota were dominant in Rehai samples, yet the dominant taxa within those phyla depended on temperature, pH, and geochemistry. Rehai springs with low pH (2.5–2.6), high temperature (85.1–89.1°C), and high sulfur contents favored the crenarchaeal order Sulfolobales, whereas those with low pH (2.6–4.8) and cooler temperature (55.1–64.5°C) favored the Aquificae genus Hydrogenobaculum. Rehai springs with neutral-alkaline pH (7.2–9.4) and high temperature (>80°C) with high concentrations of silica and salt ions (Na, K, and Cl) favored the Aquificae genus Hydrogenobacter and crenarchaeal orders Desulfurococcales and Thermoproteales. Desulfurococcales and Thermoproteales became predominant in springs with pH much higher than the optimum and even the maximum pH known for these orders. Ruidian water samples harbored a single Aquificae genus Hydrogenobacter, whereas microbial communities in Ruidian sediment samples were more diverse at the phylum level and distinctly different from those in Rehai and Ruidian water samples, with a higher abundance of uncultivated lineages, close relatives of the ammonia-oxidizing archaeon “Candidatus Nitrosocaldus yellowstonii”, and candidate division O1aA90 and OP1. These differences between Ruidian sediments and Rehai samples were likely caused by temperature, pH, and sediment mineralogy. The results of this study significantly expand the current understanding of the microbiology in Tengchong hot springs and provide a basis for comparison with other geothermal systems around the world.     

Isolation, Characterization, and Ecology of Sulfur-Respiring Crenarchaea Inhabiting Acid-Sulfate-Chloride-Containing Geothermal Springs in Yellowstone National Park

Elemental sulfur (S⁰) is associated with many geochemically diverse hot springs, yet little is known about the phylogeny, physiology, and ecology of the organisms involved in its cycling. Here we report the isolation, characterization, and ecology of two novel, S⁰-reducing Crenarchaea from an acid geothermal spring referred to as Dragon Spring. Isolate 18U65 grows optimally at 70 to 72°C and at pH 2.5 to 3.0, while isolate 18D70 grows optimally at 81°C and pH 3.0. Both isolates are chemoorganotrophs, dependent on complex peptide-containing carbon sources, S⁰, and anaerobic conditions for respiration-dependent growth. Glycerol dialkyl glycerol tetraethers (GDGTs) containing four to six cyclopentyl rings were present in the lipid fraction of isolates 18U65 and 18D70. Physiological characterization suggests that the isolates are adapted to the physicochemical conditions of Dragon Spring and can utilize the natural organic matter in the spring as a carbon and energy source. Quantitative PCR analysis of 16S rRNA genes associated with the S⁰ flocs recovered from several acid geothermal springs using isolate-specific primers indicates that these two populations together represent 17 to 37% of the floc-associated DNA. The physiological characteristics of isolates 18U65 and 18D70 are consistent with their potential widespread distribution and putative role in the cycling of sulfur in acid geothermal springs throughout the Yellowstone National Park geothermal complex. Based on phenotypic and genetic characterization, the designations Caldisphaera draconis sp. nov. and Acidilobus sulfurireducens sp. nov. are proposed for isolates 18U65 and 18D70, respectively.     

Selective Phylogenetic Analysis Targeted at 16S rRNA Genes of Thermophiles and Hyperthermophiles in Deep-Subsurface Geothermal Environments

Deep-subsurface samples obtained by deep drilling are likely to be contaminated with mesophilic microorganisms in the drilling fluid, and this could affect determination of the community structure of the geothermal microflora using 16S rRNA gene clone library analysis. To eliminate possible contamination by PCR-amplified 16S rRNA genes from mesophiles, a combined thermal denaturation and enzyme digestion method, based on a strong correlation between the G+C content of the 16S rRNA gene and the optimum growth temperatures of most known prokaryotic cultures, was used prior to clone library construction. To validate this technique, hot spring fluid (76°C) and river water (14°C) were used to mimic a deep-subsurface sample contaminated with drilling fluid. After DNA extraction and PCR amplification of the 16S rRNA genes from individual samples separately, the amplified products from river water were observed to be denatured at 82°C and completely digested by exonuclease I (Exo I), while the amplified products from hot spring fluid remained intact after denaturation at 84°C and enzyme digestion with Exo I. DNAs extracted from the two samples were mixed and used as a template for amplification of the 16S rRNA genes. The amplified rRNA genes were denatured at 84°C and digested with Exo I before clone library construction. The results indicated that the 16S rRNA gene sequences from the river water were almost completely eliminated, whereas those from the hot spring fluid remained.     

Temperature-dependent enumeration and characterization of anaerobic, thermophilic xylan-degrading bacteria present in two Icelandic hot springs

Abstract Anaerobic thermophilic xylan-degrading bacteria present in unenriched and enriched 70°C samples from two Icelandic hot springs were enumerated at 68, 78, 90 and 99°C by the use of the Most-Probable-Number method. Xylan was used as substrate. From the samples taken at 70°C and incubated at the temperatures previously described no growth was observed above 78°C. A total of ten strains were isolated and characterized from the positive MPN enrichment cultures from the MPN experiments. A higher number of different strains could be isolated in the enriched samples compared with the unenriched, control samples from the same hot spring. Introduction of xylan, i.e., in situ enrichment, into one of the hot springs changed the bacterial population, as none of the bacteria isolated from the unenriched samples were isolated from the enriched samples. All the isolated bacteria were asporogenous, non-motile and gram-negative rods. One long thin rod had morphological similarities to members of the genus Dictyoglomus and was found in both hot spring samples.     

Storage Temperature Alters the Expression of Differentiation-Related Genes in Cultured Oral Keratinocytes

Purpose

Storage of cultured human oral keratinocytes (HOK) allows for transportation of cultured transplants to eye clinics worldwide. In a previous study, one-week storage of cultured HOK was found to be superior with regard to viability and morphology at 12°C compared to 4°C and 37°C. To understand more of how storage temperature affects cell phenotype, gene expression of HOK before and after storage at 4°C, 12°C, and 37°C was assessed.

Materials and Methods

Cultured HOK were stored in HEPES- and sodium bicarbonate-buffered Minimum Essential Medium at 4°C, 12°C, and 37°C for one week. Total RNA was isolated and the gene expression profile was determined using DNA microarrays and analyzed with Partek Genomics Suite software and Ingenuity Pathway Analysis. Differentially expressed genes (fold change > 1.5 and P < 0.05) were identified by one-way ANOVA. Key genes were validated using qPCR.

Results

Gene expression of cultures stored at 4°C and 12°C clustered close to the unstored control cultures. Cultures stored at 37°C displayed substantial change in gene expression compared to the other groups. In comparison with 12°C, 2,981 genes were differentially expressed at 37°C. In contrast, only 67 genes were differentially expressed between the unstored control and the cells stored at 12°C. The 12°C and 37°C culture groups differed most significantly with regard to the expression of differentiation markers. The Hedgehog signaling pathway was significantly downregulated at 37°C compared to 12°C.

Conclusion

HOK cultures stored at 37°C showed considerably larger changes in gene expression compared to unstored cells than cultured HOK stored at 4°C and 12°C. The changes observed at 37°C consisted of differentiation of the cells towards a squamous epithelium-specific phenotype. Storing cultured ocular surface transplants at 37°C is therefore not recommended. This is particularly interesting as 37°C is the standard incubation temperature used for cell culture.     

Kinetics of Sulfur Oxidation at Suboptimal Temperatures

Chemolithoautotrophic bacteria were enriched from mine water at incubation temperatures ranging from 4 to 46°C, using elemental sulfur as a substrate in acid mineral salts media. Thiobacillus-type bacteria were successfully enriched for at all test temperatures except 46°C. Changes in pH (−dpH/dt) were used to estimate the rate constants for the enrichment cultures. The rate constants yielded a linear Arrhenius plot, an activation energy of 65 kJ/mol, and a temperature coefficient (Q₁₀) of 2.1 for the 4 to 37°C temperature interval.     

Distribution of ether lipids and composition of the archaeal community in terrestrial geothermal springs: impact of environmental variables

Archaea can respond to changes in the environment by altering the composition of their membrane lipids, for example, by modification of the abundance and composition of glycerol dialkyl glycerol tetraethers (GDGTs). Here, we investigated the abundance and proportions of polar GDGTs (P‐GDGTs) and core GDGTs (C‐GDGTs) sampled in different seasons from Tengchong hot springs (Yunnan, China), which encompassed a pH range of 2.5–10.1 and a temperature range of 43.7–93.6°C. The phylogenetic composition of the archaeal community (reanalysed from published work) divided the Archaea in spring sediment samples into three major groups that corresponded with spring pH: acidic, circumneutral and alkaline. Cluster analysis showed correlation between spring pH and the composition of P‐ and C‐GDGTs and archaeal 16S rRNA genes, indicating an intimate link between resident Archaea and the distribution of P‐ and C‐GDGTs in Tengchong hot springs. The distribution of GDGTs in Tengchong springs was also significantly affected by temperature; however, the relationship was weaker than with pH. Analysis of published datasets including samples from Tibet, Yellowstone and the US Great Basin hot springs revealed a similar relationship between pH and GDGT content. Specifically, low pH springs had higher concentrations of GDGTs with high numbers of cyclopentyl rings than neutral and alkaline springs, which is consistent with the predominance of high cyclopentyl ring‐characterized Sulfolobales and Thermoplasmatales present in some of the low pH springs. Our study suggests that the resident Archaea in these hot springs are acclimated if not adapted to low pH by their genetic capacity to effect the packing density of their membranes by increasing cyclopentyl rings in GDGTs at the rank of community.     

Linkage of High Rates of Sulfate Reduction in Yellowstone Hot Springs to Unique Sequence Types in the Dissimilatory Sulfate Respiration Pathway

Diversity, habitat range, and activities of sulfate-reducing prokaryotes within hot springs in Yellowstone National Park were characterized using endogenous activity measurements, molecular characterization, and enrichment. Five major phylogenetic groups were identified using PCR amplification of the dissimilatory sulfite reductase genes (dsrAB) from springs demonstrating significant sulfate reduction rates, including a warm, acidic (pH 2.5) stream and several nearly neutral hot springs with temperatures reaching 89°C. Three of these sequence groups were unrelated to named lineages, suggesting that the diversity and habitat range of sulfate-reducing prokaryotes exceeds that now represented in culture.     

Mycobacterium parascrofulaceum in Acidic Hot Springs in Yellowstone National Park

Mycobacterium parascrofulaceum was found in Norris Geyser Basin, Yellowstone National Park, in a system composed of two acidic (pH 3.0) springs with temperatures between 56°C at the source and 40°C at the confluence of both springs. Growth and survival assays at 56°C for 60 days were performed, confirming the origin of the strain.     

Infectious Cell Entry Mechanism of Influenza Virus

Interaction between influenza virus WSN strain and MDCK cells was studied by using spin-labeled phospholipids and electron microscopy. Envelope fusion was negligibly small at neutral pH but greatly activated in acidic media in a narrow pH range around 5.0. The half-time was less than 1 min at 37°C at pH 5.0. Virus binding was almost independent of the pH. Endocytosis occurred with a half-time of about 7 min at 37°C at neutral pH, and about 50% of the initially bound virus was internalized after 1 h. Electron micrographs showed binding of virus particles in coated pits in the microvillous surface of plasma membrane and endocytosis into coated vesicles. Chloroquine inhibited virus replication. The inhibition occurred when the drug was added not later than 10 min after inoculation. Chloroquine caused an increase in the lysosomal pH 4.9 to 6.1. The drug did not affect virus binding, endocytosis, or envelope fusion at pH 5.0. Electron micrographs showed many virus particles remaining trapped inside vacuoles even after 30 min at 37°C in the presence of drug, in contrast to only a few particles after 10 min in vacuoles and secondary lysosomes in its absence. Virus replication in an artificial condition, i.e., brief exposure of the inoculum to acidic medium followed by incubation in neutral pH in the presence of chloroquine, was also observed. These results are discussed to provide a strong support for the infection mechanism of influenza virus proposed previously: virus uptake by endocytosis, fusion of the endocytosed vesicles with lysosome, and fusion of the virus envelope with the surrounding vesicle membrane in the secondary lysosome because of the low pH. This allows the viral genome to enter the target cell cytoplasm.     

Reconstitution of a Thermostable Xylan-Degrading Enzyme Mixture from the Bacterium Caldicellulosiruptor bescii

Xylose, the major constituent of xylans, as well as the side chain sugars, such as arabinose, can be metabolized by engineered yeasts into ethanol. Therefore, xylan-degrading enzymes that efficiently hydrolyze xylans will add value to cellulases used in hydrolysis of plant cell wall polysaccharides for conversion to biofuels. Heterogeneous xylan is a complex substrate, and it requires multiple enzymes to release its constituent sugars. However, the components of xylan-degrading enzymes are often individually characterized, leading to a dearth of research that analyzes synergistic actions of the components of xylan-degrading enzymes. In the present report, six genes predicted to encode components of the xylan-degrading enzymes of the thermophilic bacterium Caldicellulosiruptor bescii were expressed in Escherichia coli, and the recombinant proteins were investigated as individual enzymes and also as a xylan-degrading enzyme cocktail. Most of the component enzymes of the xylan-degrading enzyme mixture had similar optimal pH (5.5 to ∼6.5) and temperature (75 to ∼90°C), and this facilitated their investigation as an enzyme cocktail for deconstruction of xylans. The core enzymes (two endoxylanases and a β-xylosidase) exhibited high turnover numbers during catalysis, with the two endoxylanases yielding estimated k_cat values of ∼8,000 and ∼4,500 s⁻¹, respectively, on soluble wheat arabinoxylan. Addition of side chain-cleaving enzymes to the core enzymes increased depolymerization of a more complex model substrate, oat spelt xylan. The C. bescii xylan-degrading enzyme mixture effectively hydrolyzes xylan at 65 to 80°C and can serve as a basal mixture for deconstruction of xylans in bioenergy feedstock at high temperatures.     

Control of Lignin Peroxidase Production by Phanerochaete chrysosporium INA-12 by Temperature Shifting

There are two temperature optima connected with lignin peroxidase synthesis by Phanerochaete chrysosporium INA-12. One, at 37°C, is for the mycelium-growing phase; the other, at 30°C, is for the lignin peroxidase-producing phase. One of six extracellular proteins with ligninase activity increased when cultures were grown at 30°C for the entire fermentation period or when cultures were grown at 37°C for the first 2 days of incubation and then shifted to 30°C, compared with the activity of control cultures grown at 37°C for the entire fermentation period. The unsaturation of fatty acid (Δ/mole) of P. chrysosporium INA-12 mycelium decreased from 1.25 to 1.03 when the growth temperature was shifted from 20 to 40°C.     

Fungi from Geothermal Soils in Yellowstone National Park

Geothermal soils near Amphitheater Springs in Yellowstone National Park were characterized by high temperatures (up to 70°C), high heavy metal content, low pH values (down to pH 2.7), sparse vegetation, and limited organic carbon. From these soils we cultured 16 fungal species. Two of these species were thermophilic, and six were thermotolerant. We cultured only three of these species from nearby cool (0 to 22°C) soils. Transect studies revealed that higher numbers of CFUs occurred in and below the root zone of the perennial plant Dichanthelium lanuginosum (hot springs panic grass). The dynamics of fungal CFUs in geothermal soil and nearby nongeothermal soil were investigated for 12 months by examining soil cores and in situ mesocosms. For all of the fungal species studied, the temperature of the soil from which the organisms were cultured corresponded with their optimum axenic growth temperature.