Effect of acetate on molecular and physiological aspects of Clostridium beijerinckii NCIMB 8052 solvent production and strain degeneration

Abstract

The addition of sodium acetate to chemically defined MP2 medium was found to increase and stabilize solvent production and also increase glucose utilization by Clostridium beijerinckii NCIMB 8052. RNA and enzyme analyses indicated that coenzyme A (CoA) transferase was highly expressed and has higher activity in C. beijerinckii NCIMB 8052 grown in MP2 medium containing added sodium acetate than in the microorganism grown without sodium acetate. RNA analysis suggested the existence of a sol operon and confirmed the presence of a ptb-buk operon in C. beijerinckii NCIMB 8052. In addition to CoA transferase, C. beijerinckii NCIMB 8052 grown in MP2 medium containing added acetate demonstrated higher acetate kinase- and butyrate kinase-specific activity than when the culture was grown in MP2 medium containing no added acetate. Southern blot analysis with chromosomal DNA isolated from solventogenic and degenerated C. beijerinckii NCIMB 8052 indicated that C. beijerinckii NCIMB 8052 strain degeneration does not involve loss of the CoA transferase genes. The addition of acetate to MP2 medium may induce the expression of the sol operon, which ensures solvent production and prevents strain degeneration in C. beijerinckii NCIMB 8052.

FIG. 1

Solvent production by C. beijerinckii NCIMB 8052 grown in 50 ml of MP2 medium containing 100 mM MES buffer and 0 to 100 mM sodium acetate. The samples were examined following 72 h of incubation at 35°C. The values represent the means of triplicate samples, and the error bars represent standard deviations. Conc., concentration.

FIG. 2

Solvent and acid profiles during 1-liter batch fermentation by C. beijerinckii NCIMB 8052 grown in MP2 medium containing 0 (A), 20 (B), and 60 (C) mM sodium acetate. The data are the averages of duplicate experiments.

FIG. 3

Putative arrangement of sol operon from C. beijerinckii NRRL 593 (28) and Northern blot analysis of RNA isolated from C. beijerinckii NCIMB 8052 following growth in 1 liter of MP2 medium containing 0, 20, or 60 mM sodium acetate and probed with a DNA probe containing entire ctfB and partial ctfA genes from C. beijerinckii NRRL B593 (dotted line). The bars and arrows indicate the sizes of the bands and their corresponding genes in the operon, respectively. Samples were collected following growth for 6, 24, and 48 h as indicated. Lane M represents biotin-labeled RNA size markers (Ambion), and their corresponding sizes are given on the right. The Northern blot is representative of duplicate experiments. conc., concentration.

FIG. 4

Arrangement of ptb-buk operon from C. beijerinckii NCIMB 8052 and Northern blot analysis of RNA isolated from C. beijerinckii NCIMB 8052 following growth in 1 liter of MP2 medium containing 0, 20, or 60 mM sodium acetate and probed with a DNA probe complementary to ptb from C. beijerinckii NCIMB 8052, as indicated by the dotted line. The bars and arrows indicate the sizes of the bands and their corresponding genes in the operon, respectively. Samples were collected following growth for 6, 24, and 48 h, as indicated. Lane M represents biotin-labeled RNA size markers (Ambion), and their corresponding sizes are given on the right. The Northern blot is representative of duplicate experiments. conc., concentration.

FIG. 5

Specific activity profiles of CoA transferase (A), acetate kinase (B), butyrate kinase (C), and phosphotransbutyrylase (D) of C. beijerinckii NCIMB 8052 following growth on MP2 medium containing 0 or 20 mM sodium acetate. Symbols: □, 0 mM sodium acetate; ⧫, 20 mM sodium acetate. The data represent the averages of duplicate experiments, in which each sample was assayed three times; standard deviations are within 5%.

FIG. 6

Effect of added sodium acetate on the stability of C. beijerinckii NCIMB 8052. Cultures were grown on MP2 medium containing 100 mM MES buffer and 0 (−Ac) or 20 (+Ac) mM sodium acetate. Optical density (O.D.) and total solvent concentration (conc.) were measured for 48-h cultures. Subculturing took place every 24 h for 19 days. The data represent the averages of duplicate experiments.

FIG. 7

Southern hybridization of chromosomal DNA from C. beijerinckii NCIMB 8052 and SA2. Chromosomal DNA was digested with HindIII and probed with ctfAB probe and ptb probe. Two X-ray films were overlapped when taking the picture. Lane 1, solvent-producing C. beijerinckii NCIMB 8052 culture; lane 2, degenerated C. beijerinckii NCIMB 8052 culture; lane 3, C. beijerinckii SA2 culture. SA2 is a butanol-tolerant degenerated isolate derived from C. beijerinckii NCIMB 8052 (3).