Temperature range variants of Bacillus megaterium

Facultatively and obligately thermophilic variants were isolated from 3 out of 12 tested mesophilic Bacillus megaterium strains. The variants occurred at a frequency of 10^-8–10^-9. The ability to grow at elevated temperatures was cured by means of treatment with acridine orange. Stable revertants were isolated from facultatively and obligately thermophilic variants. An unknown type of megacin was produced by the facultative thermophiles. This megacin attacked mesophilic and obligately thermophilic strains. The thermophiles displayed a few divergent taxonomic characteristics but a close relationship between the strains was indicated by the megacin spectrum and sensitivity to phage. Arrhenius plots revealed that the strains could be considered as temperature range variants and that the temperature characteristic increased with growth at a higher temperature range. The case for a plasmid involvement in the phenomenon is discussed.Abbreviations M Mesophilic - Fp facultatively psychrophilic - Ft facultatively thermophilic - Ot obligately thermophilic     

Temperature response of trehalase from a mesophilic (Neurospora crassa) and a thermophilic (Thermomyces lanuginosus) fungus

The purified trehalases of the mesophilic fungus, Neurospora crassa, and the thermophilic fungus, Thermomyces lanuginosus, had similar temperature and pH optima for activity, but differed in molecular weight, electrophoretic mobility and Michaelis constant. At lower concentration, trehalases from both fungi were inactivated to similar extent at 60°C. While purified trehalase of T. lanuginosus was afforded protection against heat-inactivation by proteinaceous protective factor(s) present in mycelial extracts, by bovine serum albumin and by casein, these did not afford protection to N. crassa trehalase against heat inactivation. Both trehalases exhibited discontinuous Arrhenius plots with temperature of discontinuity at 40°C. The activation energy calculated from the slope of the Arrhenius plot was higher for the T. lanuginosus enzyme. The plots of apparent K _m versus 1/T for trehalases of N. crassa and T. lanuginosus were linear from 30° to 60°C.The results show that purified trehalases of the mesophilic and the thermophilic fungus are distinct. Although, these exhibit similar thermostability of their catalytic function at low concentration, distinctive thermal stability characteristics of thermophilic enzyme become apparent at high protein concentration. This could be brought about in the cell by the enzyme itself, or by other proteins.     

Differences in the catalytic mechanisms of mesophilic and thermophilic indole-3-glycerol phosphate synthase enzymes at their adaptive temperatures

Thermophilic enzymes tend to be less catalytically-active at lower temperatures relative to their mesophilic counterparts, despite having very similar crystal structures. An often cited hypothesis for this general observation is that thermostable enzymes have evolved a more rigid tertiary structure in order to cope with their more extreme, natural environment, but they are also less flexible at lower temperatures, leading to their lower catalytic activity under mesophilic conditions. An alternative hypothesis, however, is that complementary thermophilic-mesophilic enzyme pairs simply operate through different evolutionary-optimized catalytic mechanisms. In this communication, we present evidence that while the steps of the catalytic mechanisms for mesophilic and thermophilic indole-3-glycerol phosphate synthase (IGPS) enzymes are fundamentally similar, the identity of the rate-determining step changes as a function of temperature. Our findings indicate that while product release is rate-determining at 25°C for thermophilic IGPS, near its adaptive temperature (75°C), a proton transfer event, involving a general acid, becomes rate-determining. The rate-determining steps for thermophilic and mesophilic IGPS enzymes are also different at their respective, adaptive temperatures with the mesophilic IGPS-catalyzed reaction being rate-limited before irreversible CO2 release, and the thermophilic IGPS-catalyzed reaction being rate limited afterwards.     

Lipid Fatty Acid Composition and Thermophilicity of Cyanobacteria

An analysis of lipid fatty acid composition in several unicellular and filamentous forms of mesophilic and thermophilic cyanobacteria was performed. At 47°C (the temperature of thermophilic cyanobacteria maintenance in the collection), the unicellular thermophilic Synechococcus strains were devoid of polyenoic acids as distinct from the mesophilic forms of this genus at the temperature of 20°C (the temperature of this cyanobacterial maintenance in the collection). In the thermophilic Synechococcus elongatusIPPAS B-267 strain, a decrease in temperature did not result in the occurrence of C18 polyenoic acids, but the quantitative relationship between the saturated and unsaturated fatty acids (S/U ratio) was decreased twofold. In contrast, the culturing of mesophilic strains at 25–32°C resulted in an increase in the S/U ratio due to an increase in the proportion of the 16:0 acid. In the Synechococcus IPPAS B-434 strain, this treatment resulted in a decrease in the relative content of monoenoic, mainly hexadecenoic, acids. The cyanobacterium Gloeobacter violaceus, which lacks thylakoids, and whose photosystems are formed in a cell membrane, contained polyenoic acids. The filamentous thermophilic cyanobacterium Phormidium laminosum, at the maintenance temperature of 47°C, did contain polyenoic acids, but their proportion was considerably lower than that in the filamentous mesophilic forms, such as Tolypothrix sp. and Spirulina platensis. A relative content of hexadecenoic acids in Ph. laminosum was higher than in the mesophilic forms. A possible role of hexadecenoic acids in the processes of adaptation of cyanobacteria to high temperatures is discussed. A relationship between the characteristics of fatty acid composition fixed by evolution and the changes caused by adaptation to a particular environment is considered.     

Actinomycetes and Fungi in Surface Waters and in Potable Water

In Finnish lakes and rivers used as water supplies, mesophilic fungi and actinomycetes were common, whereas thermophilic fungi and actinomycetes were present only in low concentrations. Fungi and actinomycetes were more abundant in eutrophic and mesotrophic lakes than in oligotrophic lakes. River water contained more thermophilic actinomycetes and fungi and mesophilic actinomycetes than did lake water. Runoff from soil seemed to be an important factor contributing to the incidence of these microbes in water. Chemical coagulation removed actinomycetes and fungi efficiently, but sand filtration allowed their passage. Disinfection could not prevent actinomycetes and fungi from reaching the distribution system. During infiltration in the production of recharged groundwater, mesophilic actinomycetes could even multiply appreciably.     

Indigenous and introduced arbuscular mycorrhizal fungi contribute to plant growth in two agricultural soils from south-western Australia

Arbuscular mycorrhizal (AM) fungi occur in all agricultural soils but it is not easy to assess the contribution they make to plant growth under field conditions. Several approaches have been used to investigate this, including the comparison of plant growth in the presence or absence of naturally occurring AM fungi following soil fumigation or application of fungicides. However, treatments such as these may change soil characteristics other than factors directly involving AM fungi and lead to difficulties in identifying the reason for changes in plant growth. In a glasshouse experiment, we assessed the contribution of indigenous AM fungi to growth of subterranean clover in undisturbed cores of soil from two agricultural field sites (a cropped agricultural field at South Carrabin and a low input pasture at Westdale). We used the approach of estimating the benefit of AM fungi by comparing the curvature coefficients ( C) of the Mitscherlich equation for subterranean clover grown in untreated field soil, in field soil into which inoculum of Glomus invermaium was added and in soil fumigated with methyl bromide. It was only possible to estimate the benefit of mycorrhizas using this approach for one soil (Westdale) because it was the only soil for which a Mitscherlich response to the application of a range of P levels was obtained. The mycorrhizal benefit ( C of mycorrhizal vs. non-mycorrhizal plants or C of inoculated vs. uninoculated plants) of the indigenous fungi corresponded with a requirement for phosphate by plants that were colonised by AM fungi already present in the soil equivalent to half that required by non-mycorrhizal plants. This benefit was independent of the plant-available P in the soil. There was no additional benefit of inoculation on plant growth other than that due to increased P uptake. Indigenous AM fungi were present in both soils and colonised a high proportion of roots in both soils. There was a higher diversity of morphotypes of mycorrhizal fungi in roots of plants grown in the Westdale soil than in the South Carrabin soil that had a history of high phosphate fertilizer use in the field. Inoculation with G. invermaium did not increase the level of colonisation of roots by mycorrhizal fungi in either soil, but it replaced approximately 20% of the root length colonised by the indigenous fungi in Westdale soil at all levels of applied P. The proportion of colonised root length replaced by G. invermaium in South Carrabin soil varied with the level of application of P to the soil; it was higher at intermediate levels of recently added soil P.     

Analysis of the Heat-Shock Response Displayed by Two Chaetomium Species Originating from Different Thermal Environments

Three features of the heat shock response, reorganization of protein expression, intracellular accumulation of trehalose, and alteration in unsaturation degree of fatty acids were investigated in the thermophilic fungus Chaetomium thermophile and compared to the response displayed by a closely related mesophilic species, C. brasiliense. Thermophilic heat shock response paralleled the mesophilic response in many respects like (i) the temperature difference observed between normothermia and the upper limit of translational activity, (ii) the transient nature of the heat shock response at the level of protein expression including both the induction of heat shock proteins (HSPs) as well as the repression of housekeeping proteins, (iii) the presence of representatives of high-molecular-weight HSPs families, (iv) intracellular accumulation of trehalose, and finally (v) modifications in fatty acid composition. On the other hand, a great variability between the two organisms was observed for the proteins expressed during stress, in particular a protein of the HSP60 family that was only observed in C. thermophile. This peptide was also present constitutively at normal temperature and may thus fulfil thermophilic functions. It is shown that accumulation of trehalose does not play a part in thermophily but is only a stress response. C. thermophile contains less polyunsaturated fatty acids at normal temperature than C. brasiliense, a fact that can be directly related to thermophily. When subjected to heat stress, both organisms tended to accumulate shorter and less unsaturated fatty acids.     

Thermophilic fungi: their physiology and enzymes.

Thermophilic fungi are a small assemblage in mycota that have a minimum temperature of growth at or above 20 degrees C and a maximum temperature of growth extending up to 60 to 62 degrees C. As the only representatives of eukaryotic organisms that can grow at temperatures above 45 degrees C, the thermophilic fungi are valuable experimental systems for investigations of mechanisms that allow growth at moderately high temperature yet limit their growth beyond 60 to 62 degrees C. Although widespread in terrestrial habitats, they have remained underexplored compared to thermophilic species of eubacteria and archaea. However, thermophilic fungi are potential sources of enzymes with scientific and commercial interests. This review, for the first time, compiles information on the physiology and enzymes of thermophilic fungi. Thermophilic fungi can be grown in minimal media with metabolic rates and growth yields comparable to those of mesophilic fungi. Studies of their growth kinetics, respiration, mixed-substrate utilization, nutrient uptake, and protein breakdown rate have provided some basic information not only on thermophilic fungi but also on filamentous fungi in general. Some species have the ability to grow at ambient temperatures if cultures are initiated with germinated spores or mycelial inoculum or if a nutritionally rich medium is used. Thermophilic fungi have a powerful ability to degrade polysaccharide constituents of biomass. The properties of their enzymes show differences not only among species but also among strains of the same species. Their extracellular enzymes display temperature optima for activity that are close to or above the optimum temperature for the growth of organism and, in general, are more heat stable than those of the mesophilic fungi. Some extracellular enzymes from thermophilic fungi are being produced commercially, and a few others have commercial prospects. Genes of thermophilic fungi encoding lipase, protease, xylanase, and cellulase have been cloned and overexpressed in heterologous fungi, and pure crystalline proteins have been obtained for elucidation of the mechanisms of their intrinsic thermostability and catalysis. By contrast, the thermal stability of the few intracellular enzymes that have been purified is comparable to or, in some cases, lower than that of enzymes from the mesophilic fungi. Although rigorous data are lacking, it appears that eukaryotic thermophily involves several mechanisms of stabilization of enzymes or optimization of their activity, with different mechanisms operating for different enzymes.     

A comparison of the thermostability of cellulases from various thermophilic fungi

Summary The cellulase activities of six thermophilic fungi were compared. Although the thermophilic fungi grew at relatively high temperatures (>45°C) the optimum temperatures for assaying the various cellulase activities were only slightly higher than the optimum temperatures for the mesophilic fungi, Trichoderma harzianum. Over prolonged incubation (> 24 h) the thermophilic strains demonstrated a higher hydrolytic potential as a result of the greater thermostability of the cellulase components. Although the extracellular cellulase activities had similar pH and temperature optima, in some cases the thermostability of the extracellular components were considerably lower.     

Thermophilic Fungi: Their Physiology and Enzymes

Thermophilic fungi are a small assemblage in mycota that have a minimum temperature of growth at or above 20°C and a maximum temperature of growth extending up to 60 to 62°C. As the only representatives of eukaryotic organisms that can grow at temperatures above 45°C, the thermophilic fungi are valuable experimental systems for investigations of mechanisms that allow growth at moderately high temperature yet limit their growth beyond 60 to 62°C. Although widespread in terrestrial habitats, they have remained underexplored compared to thermophilic species of eubacteria and archaea. However, thermophilic fungi are potential sources of enzymes with scientific and commercial interests. This review, for the first time, compiles information on the physiology and enzymes of thermophilic fungi. Thermophilic fungi can be grown in minimal media with metabolic rates and growth yields comparable to those of mesophilic fungi. Studies of their growth kinetics, respiration, mixed-substrate utilization, nutrient uptake, and protein breakdown rate have provided some basic information not only on thermophilic fungi but also on filamentous fungi in general. Some species have the ability to grow at ambient temperatures if cultures are initiated with germinated spores or mycelial inoculum or if a nutritionally rich medium is used. Thermophilic fungi have a powerful ability to degrade polysaccharide constituents of biomass. The properties of their enzymes show differences not only among species but also among strains of the same species. Their extracellular enzymes display temperature optima for activity that are close to or above the optimum temperature for the growth of organism and, in general, are more heat stable than those of the mesophilic fungi. Some extracellular enzymes from thermophilic fungi are being produced commercially, and a few others have commercial prospects. Genes of thermophilic fungi encoding lipase, protease, xylanase, and cellulase have been cloned and overexpressed in heterologous fungi, and pure crystalline proteins have been obtained for elucidation of the mechanisms of their intrinsic thermostability and catalysis. By contrast, the thermal stability of the few intracellular enzymes that have been purified is comparable to or, in some cases, lower than that of enzymes from the mesophilic fungi. Although rigorous data are lacking, it appears that eukaryotic thermophily involves several mechanisms of stabilization of enzymes or optimization of their activity, with different mechanisms operating for different enzymes.     

Mesophilic and thermophilic BTEX substrate interactions for a toluene-acclimatized biofilter

Benzene, toluene, ethylbenzene and xylene (BTEX) substrate interactions for a mesophilic (25°C) and thermophilic (50°C) toluene-acclimatized composted pine bark biofilter were investigated. Toluene, benzene, ethylbenzene, o-xylene, m-xylene and p-xylene removal efficiencies, both individually and in paired mixtures with toluene (1:1 ratio), were determined at a total loading rate of 18.1 g m^–3 h^–1 and retention time ranges of 0.5–3.0 min and 0.6–3.8 min for mesophilic and thermophilic biofilters, respectively. Overall, toluene degradation rates under mesophilic conditions were superior to degradation rates of individual BEX compounds. With the exception of p-xylene, higher removal efficiencies were achieved for individual BEX compounds compared to toluene under thermophilic conditions. Overall BEX compound degradation under mesophilic conditions was ranked as ethylbenzene >benzene >o-xylene >m-xylene >p-xylene. Under thermophilic conditions overall BEX compound degradation was ranked as benzene >o-xylene >ethylbenzene >m-xylene >p-xylene. With the exception of o-xylene, the presence of toluene in paired mixtures with BEX compounds resulted in enhanced removal efficiencies of BEX compounds, under both mesophilic and thermophilic conditions. A substrate interaction index was calculated to compare removal efficiencies at a retention time of 0.8 min (50 s). A reduction in toluene removal efficiencies (negative interaction) in the presence of individual BEX compounds was observed under mesophilic conditions, while enhanced toluene removal efficiency was achieved in the presence of other BEX compounds, with the exception of p-xylene under thermophilic conditions.     

Mesophilic and thermophilic biotreatment of BTEX-polluted air in reactors

This study compares the removal of a mixture of benzene, toluene, ethylbenzene, and all three xylene isomers (BTEX) in mesophilic and thermophilic (50 degrees C) bioreactors. In the mesophilic reactor fungi became dominant after long-term operation, while bacteria dominated in the thermophilic unit. Microbial acclimation was achieved by exposing the biofilters to initial BTEX loads of 2-15 g m(-3) h(-1), at an empty bed residence time of 96 s. After adaptation, the elimination capacities ranged from 3 to 188 g m(-3) h(-1), depending on the inlet load, for the mesophilic biofilter with removal efficiencies reaching 96%. On the other hand, in the thermophilic reactor the average removal efficiency was 83% with a maximum elimination capacity of 218 g m(-3) h(-1). There was a clear positive relationship between temperature gradients as well as CO(2) production and elimination capacities across the biofilters. The gas phase was sampled at different depths along the reactors observing that the percentage pollutant removal in each section was strongly dependant on the load applied. The fate of individual alkylbenzene compounds was checked, showing the unusually high biodegradation rate of benzene at high loads under thermophilic conditions (100%) compared to its very low removal in the mesophilic reactor at such load (<10%). Such difference was less pronounced for the other pollutants. After 210 days of operation, the dry biomass content for the mesophilic and thermophilic reactors were 0.300 and 0.114 g g(-1) (support), respectively, reaching higher removals under thermophilic conditions with a lower biomass accumulation, that is, lower pressure drop.     

Fibrinolytic activity of some fungi isolated from self-heated composted fertilizer

Mesophilic fungi isolated from organic fertilizer compost samples accounted for 70.94% of the total fungal count, while thermophilic and thermotolerant fungi constituted 29.05% of that count. Eight mesophilic fungal species, namelyAspergillus niger, Monilia sitophila, Paecilomyces divaricata, Penicillium chrysogenum, P. fellutanum, Scopulariopsis brevicaulis, S. brumptii andZygorhynchus japonicus; two thermophilic fungiHumicola grisea andOidiodendron flavum and three thermotolerant speciesAspergillus fumigatus, Thermomyces lanuginosus andZygorhynchus vuilleminii were isolated during the study. Most of the tested fungi showed a proteolytic activity and liquified gelatin in the test tube method and in cup plates. The thermophilic fungusO. flavum was the most potent proteolytic fungus. The comparative fibrinolytic assay revealed the following sequence in the ability of the tested fungi to hydrolyse fibrin:O. fiavum>S. brevicaulis>H. grisea>A. fumigatus>T. lanuginosus.     

A comparative molecular dynamics study of thermophilic and mesophilic ribonuclease HI enzymes

We studied a pair of homologous thermophilic and mesophilic ribonuclease HI enzymes by molecular dynamics simulations. Each protein was subjected to three 5 ns simulations in explicit water at both 310 K and 340 K. The thermophilic enzyme showed larger overall positional fluctuations at both temperatures, while only the mesophilic enzyme at the higher temperature showed significant instability. When the temperature is changed, the relative flexibility of different local segments on the two proteins changed differently. Principal component analysis showed that the simulations of the two proteins explored largely overlapping regions in the conformational space. However, at 340 K, the collective structure variations of the thermophilic protein are different from those of the mesophilic protein. Our results, although not in accordance with the view that hyperthermostability of proteins may originate from their conformational rigidity, are consistent with several recent experimental and simulation studies which showed that thermophilic proteins may be conformationally more flexible than their mesophilic counterparts. The decorrelation between conformational rigidity and hyperthermostability may be attributed to the temperature dependence and long range nature of electrostatic interactions that play more important roles in the structural stability of thermophilic proteins.     

A Comparative Molecular Dynamics Study of Thermophilic and Mesophilic Ribonuclease HI Enzymes

Abstract

We studied a pair of homologous thermophilic and mesophilic ribonuclease HI enzymes by molecular dynamics simulations. Each protein was subjected to three 5 ns simulations in explicit water at both 310 K and 340 K. The thermophilic enzyme showed larger overall positional fluctuations at both temperatures, while only the mesophilic enzyme at the higher temperature showed significant instability. When the temperature is changed, the relative flexibility of different local segments on the two proteins changed differently. Principal component analysis showed that the simulations of the two proteins explored largely overlapping regions in the conformational space. However, at 340 K, the collective structure variations of the thermophilic protein are different from those of the mesophilic protein. Our results, although not in accordance with the view that hyperthermostability of proteins may originate from their conformational rigidity, are consistent with several recent experimental and simulation studies which showed that thermophilic proteins may be conformationally more flexible than their mesophilic counterparts. The decorrelation between conformational rigidity and hyperthermostability may be attributed to the temperature dependence and long range nature of electrostatic interactions that play more important roles in the structural stability of thermophilic proteins.     

Thermophillic and thermotolerant fungi isolated from the thermal effluent of nuclear power generating reactors: Dispersal of human opportunistic and veterinary pathogenic fungi

Over a period of a year, samples of water, foam, microbial mat, soil and air were obtained from areas associated with the cooling canal of a nuclear power station. The seventeen sample sites included water in the cooling canal that was thermally enriched and soil and water adjacent to, upstream, downstream and at a distance from the generator. Air samples were taken at the plant and at various disstances from the plant. Fifty-two species of thermotolerant and thermophilic fungi were isolated. Of these, eleven species are grouped as opportunistic Mucorales or opportunistic Aspergillus sp. One veterinary pathogen was also isolated (Dactylaria gallopava):The opportunistic/pathogenic fungi were found primarily in the intake bay, the discharge bay and the cooling canal. Smaller numbers were obtained at both upstream and downstream locations. Soil samples near the cooling canal reflected an enrichment of thermophilous organisms, the previously mentioned opportunistic Mucorales and Aspergillus spp. Their numbers were found to be greater than that usually encountered in a mesophilic environment. However, air and soil samples taken at various distances from the power station indicated no greater abundance of these thermophilous fungi than would be expected from a thermal enriched environment. Our results indicate that there was no significant dissemination of thermophilous fungi from the thermal enriched effluents to the adjacent environment. These findings are consistent with the results of other investigators.     

Long-term effect of inoculum pretreatment on fermentative hydrogen production by repeated batch cultivations: homoacetogenesis and methanogenesis as competitors to hydrogen production

Long-term effects of inoculum pretreatments (heat, acid, loading-shock) on hydrogen production from glucose under different temperatures (37 °C, 55 °C) and initial pH (7 and 5.5) were studied by repeated batch cultivations. Results obtained showed that it was necessary to investigate the long-term effect of inoculum pretreatment on hydrogen production since pretreatments may just temporarily inhibit the hydrogen consuming processes. After long-term cultivation, pretreated inocula did not enhance hydrogen production compared to untreated inocula under mesophilic conditions (initial pH 7 and pH 5.5) and thermophilic conditions (initial pH 7). However, pretreatment could inhibit lactate production and lead to higher hydrogen yield under thermophilic conditions at initial pH 5.5. The results further demonstrated that inoculum pretreatment could not permanently inhibit either methanogenesis or homoacetogenesis, and methanogenesis and homoacetogenesis could only be inhibited by proper control of fermentation pH and temperature. Methanogenic activity could be inhibited at pH lower than 6, both under mesophilic and thermophilic conditions, while homoacetogenic activity could only be inhibited under thermophilic condition at initial pH 5.5. Microbial community analysis showed that pretreatment did not affect the dominant bacteria. The dominant bacteria were Clostridium butyricum related organisms under mesophilic condition (initial pH 7 and 5.5), Thermoanaerobacterium sp. related organisms under thermophilic condition (initial pH 7), and Thermoanaerobacterium thermosaccharolyticum related organisms under thermophilic condition (initial pH 5.5). Results from this study clearly indicated that the long-term effects of inoculum pretreatments on hydrogen production, methanogenesis, homoacetogenesis and dominant bacteria were dependent on fermentation temperature and pH.     

Environmental factors affecting seed germination of gray birch (Betula populifolia) collected from abandoned anthracite coal mine spoils in northeast Pennsylvania

Germination of gray birch (Betula populifolia) seed collected from anthracite mine spoils in northeastern Pennsylvania was studied. Environmental conditions of the spoil banks are such that high mortality may occur at seed and germination stages because of low moisture availability and thermal stress. The mine spoil banks are harsh environments with respect to key seed germination factors: percent soil moisture as low as 1.8% and soil surface temperatures reaching 59°C. In the field, gray birch typically germinated in mid-April prior to severe environmental stress. Trends in germination success were inversely related to rising soil temperature and decreasing soil moisture availability. Although seeds were capable of survival and germination under laboratory conditions of constant temperatures in excess of 55°C, dramatic decline in germination was observed under fluctuating temperature regimes likely to be experienced in the field. No germinations occurred under fluctuating temperatures in excess of 30°C. Germinations in the field were seen to end after mid-June when substrate temperatures exceeded 30°C.     

Start-up of a thermophilic upflow anaerobic sludge bed (UASB) reactor with mesophilic granular sludge

Summary Fast start-up of thermophilic upflow anaerobic sludge bed (UASB) reactors was achieved at process temperatures of 46, 55 and 64° C, using mesophilic granular sludge as inoculum and fatty acid mixtures as feed. The start-up was brought about by increasing the temperature of mesophilic UASB reactors in a single step, which initially led to a sharp drop in the methane production rate. Thereafter, stable thermophilic methanogenesis was achieved within a period of 1 or 2 weeks depending on the temperature of operation. Mesophilic granules functioned initially as effective carrier material for thermophilic organisms. However, long-term operation led to disintegration of the granules, resulting in wash-out of thermophilic biomass. The temperature optima for acetotrophic methanogenic activity of the sludges cultivated at 46, 55 and 64° C, were similar, but differed significantly from the temperature optimum of the mesophilic inoculum. All the sludges examined were dominated by Methanothrix-like rods. These could be distinguished by antigenic fingerprinting into two subpopulations, one predominant at 36° C and the other predominant at 46° C and above. Offprint requests to: J. B. van Lier