Clostridium thermocellum JW20 (ATCC 31549) is a coculture with Thermoanaerobacter ethanolicus

Abstract

A PCR assay based on 16S rRNA sequence differences among four thermophilic anaerobic bacterial strains was used to demonstrate contamination of Clostridium thermocellum JW20 (ATCC 31549) with a Thermoanaerobacter ethanolicus strain. Therefore, we suggest that interpretation of experimental results with C. thermocellum JW20 be viewed with caution. In the search for alternative methods of producing alcohol fuels, biodegradation of fibrous biomass by thermophilic anaerobic bacteria has been studied for the past 20 years (9, 13). Clostridium thermocellum has been the most extensively examined thermophile (6), but it does have limitations with regard to ethanol yield and its ability to use certain carbohydrate fractions (9). Therefore, some work has focused on the use of cocultures of thermophilic bacteria (4, 10). Although from the biotechnological standpoint it may be desirable to use cocultures, it is essential that pure cultures are available for study in order to elucidate the molecular regulation of genes encoding saccharolytic enzymes. However, culture purity can be a problem since cultures of cellulolytic thermophiles are prone to contamination by glycolytic, noncellulolytic thermophilic bacteria, including Clostridium thermosaccharolyticum-like bacteria (7). Several biochemical and physiological assays have been suggested to distinguish among closely related saccharolytic thermophiles and to verify the purity of C. thermocellum cultures (7). However, these assays (e.g., monitoring butyrate formation and growth on glucose at 72°C or 30°C and testing for proteolytic activity) are time consuming and may not be reliable when low levels of contaminants are present. Contrary to earlier findings that C. thermocellum JW20 was able to utilize xylose only after 2 to 5 weeks (7), we recently reported that this strain did not require long adaptation periods to grow on xylose (12). In fact, 95% xylose utilization within 12 h has been routinely observed in our laboratory. Based on this observation, we pursued a search for xylose utilization genes in this strain. Screening of a genomic library prepared from chromosomal DNA purified from the C. thermocellum JW20 culture resulted in the isolation of a genomic clone (5) containing a gene with a nucleotide sequence identical, except for four mismatches, to the xylose isomerase gene (xylA) from Thermoanaerobacter ethanolicus 39E (ATCC 33223) (3), formerly Clostridium thermohydrosulfuricum 39E. Since it is highly unlikely that two distinct bacterial species have virtually identical nucleotide sequences of a gene, we suspected that C. thermocellum JW20 was contaminated with a T. ethanolicus 39E-like organism. This observation, as well as the need to routinely assess culture purity, prompted us to design an assay based on DNA sequence specificity. In this report we describe

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“…Medium was prepared as described in Erbeznik et al (11) and supplemented with an additional 2 g/L of yeast extract (T4YE medium). TTC medium was prepared according to the methods described by Jin et al (12) but used a different carbon source concentration (4 g/L).…”

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“…In fact, some of the strains of C. thermocellum including the original culture by McBee () and strain C. thermocellum JW20 (ATCC 31549) were later determined to be co‐cultures with other bacteria. In the case of C. thermocellum JW20 (ATCC 31549), the co‐culture included a new strain of Thermoanaerobacter pseudethanolicus , similar to T. pseudethanolicus 39E (ATCC 33223; Erbeznik et al ., ). This helped resolve the disputed observations of C. thermocellum growing on xylose (Freier et al ., ).…”

Toxicity effects of compressed and supercritical solvents on thermophilic microbial metabolism

“…Clostridium thermocellum ATCC 31549 was obtained from the American Type Culture Collection (Rockville, MD) and grown in a previously described medium (Erbeznik et al, 1997). Nongrowing (resting) cell suspensions were prepared from growing cultures (1 L) that had been initially provided with 11.7 mM cellobiose.…”