Clostridium thermopapyrolyticum sp. nov., a Cellulolytic Thermophile

Abstract

A new thermophilic, cellulolytic species of the genus Clostridium was isolated from riverside mud. The guanine-plus-cytosine content of its DNA is 34 mol%. The isolate ferments a wide range of carbohydrates. Its major fermentation products are ethanol, butanol, acetate, butyrate, lactate, carbon dioxide, and hydrogen. Hydrogen sulfide is also produced. The name Clostridium thermupapyrulyticum is proposed. The type strain has been deposited in the South American Biotechnology and Applied Microbiology microbiological resource center (United Nations Educational, Scientific, and Cultural Organization) culture collection, Tucuman, Argentina, as UBA 305.The genus Clostridium includes four cellulolytic and thermophilic species: Clostridium thermocellum (lo), C. stercorarium (8), C. thermolacticum (7), and C. thermocopriae (5).Thermophilic bacteria are of particular interest for the industrial conversion of cellulose to liquid fuels. For this reason, we searched for cellulolytic thermophiles in different anaerobic environments.One isolate from riverside mud digested cellulose and fermented a wide range of carbohydrates. This isolate was chosen for further studies. In this article, we present its taxonomic features and propose it as a new species named Clostridiurn thermopapyrolyticum . MATERIALS AND METHODSMedia and culture conditions. CM4 (2) was the basal medium used throughout this study. This medium was supplemented with carbon sources as indicated below. Cellulose powder (standard grade; Whatman, Maidstone, England) and strips of Whatman no. 1 filter paper were used as cellulosic substrates. Solid medium contained 1.5% (wthol) agar (Merck, Darmstadt, Germany). The Hungate method (4) was used for liquid cultures. The headspace gas was oxygen-free nitrogen. The cultures were plated either in an anaerobic hood (Coy Laboratory Products, Ann Arbor, Mich.) in an atmosphere of 80% N,-10% H,-10% CO, or on a laboratory bench. In the latter case, the plates were incubated in anaerobic jars at 60°C with anaerobiosis generators (Anaerocult; Merck).Isolation procedure and stock culture maintenance. Onehalf-gram samples of mud were added to 5 ml of liquid medium containing a filter paper strip (1 by 5 cm) as the sole carbon source and incubated at 60°C until the strip was degraded. The cultures were then vigorously shaken and streaked onto plates supplemented with 0.6% (wthol) cellobiose. After 48 h of incubation, isolated colonies were restreaked and repicked twice on the same medium. The purified colonies were transferred to liquid CM4 containing a filter paper strip of the same size as that mentioned above with no other carbon source. After complete paper degradation, the cultures were plated on cellobiose agar. The whole * Corresponding author. procedure was repeated once more. Stock cultures were kept at 4°C in medium containing a strip of filter paper. This stock was used as an inoculum for the cultures used to perform the physical and biochemical assays described below. All assays were done in triplicate.Temperature studi...

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“…Thermoanaerobacterium thermosaccharolyticum GSU5 was isolated from animal dung collected in a pasture plain in Buenos Aires, Argentina, in 1987. The strain was originally designated as Clostridium thermopapyrolyticum due to its phenotypic characteristics (Mendez et al, 1991). Stock cultures were kept at 4°C in Hungate tubes containing growth medium with a strip of filter paper for several decades.…”

Recent Advances in Second Generation Ethanol Production by Thermophilic Bacteria

“…Their habitats include geothermal areas [16][17][18], deep sea vents [19], river bank sediments [20], and artificial habitats such as self-heated compost piles [21], municipal solid waste or sewage sludge [22], oil wells [23], and thermally treated foods [24]. This suggests that thermophilic anaerobes are somewhat ubiquitous in nature.…”

Butanol production from renewable biomass: Rediscovery of metabolic pathways and metabolic engineering

“…Cellulose needs to be degraded into fermentable sugars before it can be utilized by the cells. Several anaerobic microorganisms, including C. acetobutylicum [25], Clostridium thermupapyrulyticum [49], and some Clostridium sp. [50], naturally possess either the cellulose degrading extracellular enzyme complex called cellulosome or cellulolytic activity.…”

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