Effect of carbon source on growth temperature and fatty-acid composition Thermomonospora curvata F.J. Stutzenberger*
and T.C. Jenkins
The growth-temperature range of the actinomycete, Themlomonosporu curuata, was influenced by the nature of the soluble carbon sources used, which were derived from cellulose, pectin, starch and xylan. This thermophile had the broadest (38 to 65°C) and narrowest (42 to 59°C) temperature range during growth on cellobiose (from cellulose) and 4-deoxy-L-threo-t-hexoseulose uranic acid (from pectin), respectively. This substrate-temperature interaction was accompanied by changes in cellular fatty acids: uranic-acid-grown cells had relatively low amounts of branched chain fatty acids (particularly iso: 0) and high amounts of monounsaturated fatty acids (particularly cis-18 : 1) compared with cells grown on any other substrate. Moreover, uranic-acid-grown cells could not respond to increased growth temperature by altering the ratio of branched chain fatty acids to straight chain fatty acids. Key words: Actinomycete,
carbon source, fatty acids, Tbemamonospora,
Smmmmmporu curvafa is a facultative thermophile which establishes itself as the numerically dominant cellulolytic population during the high-temperature cornposting of a variety of lignocellulosic biomasses (Niese 1959; Fergus 1964; Stutzenberger et al. 1970). This actinomycete produces a variety of thermostable deploymerizing exoenzymes which degrade insoluble polysaccharides in biomass materials (Lupo & Stutzenberger 1988; Stutzenberger 1991; Stutzenberger & Bodine 1992; Collins et al. 1993). The results of the present study show that the nature of the degradation product from these polysaccharides determines the temperature range over which T. curvata can grow and also influences its fatty-acid composition, an important factor in establishing temperature tolerance (Langworthy & Pond 1986).
Conditions T. crtrvatu
(Stutzenberger et al. 1970). Inocula, prepared from lo-fold concentrates of washed late-growth-phase mycelia, were maintained in 50% (w/v) glycerol at - 86°C. Cultures (100 ml final volume) were started with 1% (v/v) inoculum in mineral salts minimal medium (Stutzenberger 1972) with 0.5% (w/v) of the selected carbon source in 250-ml baffled Erlenmeyer flasks inclined at an angle of 30" from vertical in rotary water baths and turned at 140 rev/min. Carbon sources-cellobiose, glucose, maltose and xylosewere from Sigma. The depolymerization product of pectin (4deoxy-r-threo-5-hexoseulose uranic acid) was prepared by digesting purified apple pectin (Sigma) with purified polygalacturonate lyase (EC 220.127.116.11) from 7. curvata (Stutzenberger 1987). Midgrowth-phase cultures were transferred (I%, v/v) into fresh minimal medium containing each carbon source to eliminate glycerol contamination before determinations of growth and fatty-acid composition were made. Growth of 7. curvatu was measured by the increase in absorbance of washed mycelia (I mg dry well wt/ml is equivalent to an A 610 of 1.7 units in a ~-cm light path). Due to the particulate nature of actinomycete mycelial growth and the subsequent large variation in cell mass determinations, (the mean standard deviation was 14.2%), A,,, values were determined in triplicate for each sample from triplicate flasks and then averaged.
waste compost at the US Public Health Service/Tennessee Valley Authority
F.J. Stutzenberger is with the Department of Microbiology, Clemson University, Clemson, SC 29634-1909. USA; fax: 803 656 1127. T.C. Jenkins is with the Department of Animal, Dairy and Veterinary Sciences at the same university. *Corresponding author. @ 1995 Rapid Science
Culture Sample Preparation Cells from Lo-ml, mid-growth-phase culture samples were rated and washed twice with 40 vol. (relative to packed cell of mineral salts minimal medium by centrifugation (12,000 10 min, 22°C). Washed cells were freeze-dried and weighed 15 X 125 mm screw-capped culture tubes with Teflon liners to fatty-acid analysis.
sepavol.) X g, into prior
of Microbiology 6 Biotechnology. Vol Il. 1995
F.]. Stutzenberger and T.C. Jenkins Table growth
1. Effect of carbon source on the temperature of T. curvafa in mlneral salts minimal medium.
Cellobiose Glucose Maltose Uranic acid Xylose
rate (h-‘) 81
at (“C): 83
NG 0.15 NG NG
* Cultures having less than a doubling were recorded as no growth (NG). t Erratic growth (one out of three temperature).
in dry cell cultures
wt after grew
24 h this
Fattyacid Determination The fatty acids of washed cell samples were methylated with 3 ml 5% HCl in absolute methanol by the method of Browse et al. (1986). using heptadecanoic acid (0.1% w/v) in benzene as an internal standard. Methyl esters were analysed by GC, with a 30 m X 0.25 mm (internal diam.) fused silica capillary column, helium carrier gas and a flame ionization detector. Column temperature, initially 150"C, was held for 4 min and then increased at 4”Clmin to a final temperature of 250°C (held for I min). The fatty acids were identified by retention time comparisons with bacterial fatty acids methyl ester standards containing k-and ant&o-branched chain fatty acids (BCFA) in addition to saturated fatty acids (SFA) and mono-unsaturated fatty acids (MUFA). Fatty-acid identify was further verified by comparison of the GC/ MS spectra with those on a Wiley/NBS database (John Wiley & Sons, New York). Data variance was evaluated using the GLM procedure of the Statistical Analysis System (Anon. 1985).
Results The temperature range over which T. curvafa could grow in minimal medium was determined using cellobiose, maltose, 4-deoxy-L-threo-5-hexoseulose uranic acid and xylose, the soluble carbon sources released, by the action of T. curvata depolymerizing exoenzymes, from cellulose, starch, pectin and xylan, respectively. The temperature range for growth on each carbon source was compared with that on glucose (Table 1). Cellobiose supported growth over the widest range (38 to 65°C) whereas the uranic acid product from pectin depolymerization was the most temperaturerestrictive carbon source (42 to 59°C). At a median temperature of 50”C, the specific growth rates ranged from 0.23 h-’ on the uranic acid to 0.30 h-’ on cellobiose. Alteration of fatty-acid composition is one mechanism of microbial adaptation to temperature changes; the growthtemperature effects on overall fatty-acid composition of i? ctrrvah, were therefore measured, and the correlation of those effects with substrate-related effects statistically evaluated. Over the range of carbon sources, an increase in growth temperature resulted in generally increased amounts
of both BCFA and SFA, whereas MUFA content decreased with increasing growth temperature (Table 2). Cellobiose, glucose, maltose and xylose yielded cells with statistically similar BCFA contents (Table 3). BCFA amounts were lowest and MUFA amounts highest in cells grown on uranic acid. This generally lower BCFA content in uronicacid-grown cells was due in part to lower amounts of anteim17:O (down 16%) and iso-Id:0 (down 24%) compared with the corresponding means from cells grown on the other carbon sources. Moreover, the amounts of these fatty acids were less responsive to temperature changes during growth on uranic acid. For example, when the growth temperature was shifted from 42 to WC, anteiso-ITO fell from 29.6% of total fatty acids to 20.0% in cells grown on cellobiose (the most temperature-permissive substrate), but remained constant in the uranic-acid-grown cells (detailed data not shown). This inability of the actinomycete to respond to temperature changes during growth on the uranic acid was most distinctly reflected in the ratio of its major BCFA (is0-16 : 0) to its straight-chain fatty-acid counterpart. Cells grown on cellobiose, glucose, maltose and xylose adapted to increased growth temperature by markedly altering their iso-16: O/16: 0 ratios; however, uronicacid-grown cells could not respond in a similar fashion (Figure 1). MUFA content generally decreased as the growth temperature was increased over the permissive 42 to 59°C range. Although the decrease was- most marked for cells grown on the uranic acid, it was not sufficient to decrease their total MUFA content to that of cells grown on the other substrates (Figure 2). This higher MUFA content in uronicacid-grown cells was attributable largely to G-18: 1. At the median growth temperature of SO”C, the mean cis-lb: 1 40 .-0 3 0 CD 7 0 co F ’ .g
Figure 1. Influence of growth temperature on iso-16:0/16:0 ratios in T. curvata grown on cellobiose (O), glucose (A), maltose (O), pectin-derived uranic acid (0) and xylose (H) as sole carbon source. Points are means of at least three determinations.
of 7. curvata. Temperature
No. of observations Fatty acid composition Branched Saturated Mono-unsaturated
83.6 f 0.5c 5.8 f 0.5b 3.5 f 0.028
85.0 f 0.08b 5.4 f 0.6b 2.9 f 0.3a
88.2 _+ 0.4a 7.3 k 0.48 1.5 f 0.2b
88.7 f 0.5a 8.4 f 0.5a 0.8 k 0.2c
89.7 + 0.6a 8.9 + 0.68 0.4 iz 0.30
averaged 3.4% of dry cell wt. Values with different superscript letters differ
are means significantly
are means with different
(% of total)
(averaged over (p -C 0.05).
of T. curvata. Carbon
No. of observations Fatty acid composition Branched Saturated Mono-unsaturated
(% of total)’
‘Total fatty-acid content of 7. curvata standard deviation. Within rows, values Table
interactions in T. curvata
87.7 f 0.5= 7.8 f 0.5a 1.6 + 0.3b
89.0 f 0.5a 5.7 f 0.5b 1.1 + 0.4b
88.4 f 0.5a 6.3 f 0.5b 0.9 f 0.4b
81.8 + 0.5b 8.8 f 0.5a 4.3 f 0.5a
89.3 f 0.68 5.9 + 0.6b 0.9 f 0.3b
over the temperature range in which letters differ significantly (p < 0.05).
concentration in uranic-acid-grown-cells was 4.3% of total fatty acid, about four times that in cells grown on any of the other substrates (with a mean of 1.1% of total fatty acids).
Discussion Thermo-adaptation mechanisms in thermophilic bacteria include production of temperature-induced proteins (Wu & Welker 1991; Trent et al. 1994). alteration in nutritional requirements (Campbell & Williams 1953) and a decreased proportion of unsaturated fatty acids as the growth temperature increases (Hasegawa et al. 1980). The cytoplasmic membrane, which contains the bulk of fatty acids in Grampositive microbes (O’Leary & Wilkinson 1988), is particularly vulnerable to the hot external environment (Langworthy & Pond 1986). The fatty acids of T. curvata are mostly (approx. 89%) BCFA, with iso-16: 0 and ante&-17: 0 in greatest abundance; this fatty-acid composition is comparable with that of other Gram-positive thermophiles (Langworthy & Pond 1986; O’Leary & Wilkinson 1988). The decreased amounts of BCFA during growth of T. curvata on the uranic acid and the concomitant narrowing of the growth temperature range are similar to effects caused by the presence of the detergent, Tween 80 (Thies et al. 1994). Moreover, the amounts of cis-18: 1 in both Tween-grown and the present uranic-acid-grown cells were elevated. Since T. ctlrvata adapts by decreasing its cis-18: 1 and iso-16:O contents
+ the standard
during temperature increases (and vice versa), inhibition of this adaptive mechanism when pectin is sole carbon source would restrict growth of the thermophile at both extremes of its temperature range. The general decrease in T. curvata unsaturated fatty-acid content with increase in growth temperature is in agreement with previous observations in both Gram-negative and Gram-positive thermophiles (Souza et al. 1974; Miller 1985;
Figure 2. Influence of growth temperature on mono-unsaturated fatty acids (MUFA) in 7. curvafa grown on cellobiose (O), glucose (A), maltose (O), pectin-derived uranic acid (a) and xylose (m) as sole carbon source. Points are means of at least three determinations.
Langworthy & Pond 1986). Although temperature-dependent substrate specificity has been demonstrated in some Gram-negative thermophiles (Heinen 1971; Merkel & Perry 1977), comparable studies have not, to our knowledge, been done to correlate specific changes in fatty-acid composition to substrate-temperature interaction in thermophilic actinomycetes. During growth of T. curvafa on complex substrates such as plant fibre, such substrate-temperature interactions would be masked by the availability of other carbon sources, including those tested here. However, if temperature-induced changes in cell-membrane fatty-acid composition preclude the uptake of a particular carbon source by changes in its transport of catalytic proteins (Russell 1988), the exclusion of that carbon source would block the induction of the extracellular depolymerizing enzymes which generate that carbon source (Priest 1992). This mechanism provides one explanation for an earlier observation (Stutzenberger & Jenkins 1991) on the temperature-dependent patterns of exoenzyme biosynthesis in T. curvafa. When the actinomycete was grown on protein-extracted luceme fibre, a plant biomass having a well-defined biopolymer composition (Vaughn et al. 19&t), its ability to synthesize a particular exoenzyme at the extremes of its growth-temperature range generally correlated with its ability to grow at those temperatures on the degradation product from that enzyme’s activity. As an example, T. curvafa cannot grow on maltose at 61°C and this inability is reflected in poor amylase production at that temperature. Conversely, the actinomycete cannot grow on xylose at 38”C, a temperature when xylanase production is only 10% of that observed in the range of SO to 60°C (Stutzenberger & Jenkins 1991).
Acknowledgements We thank E. Thies for excellent technical assistance, and G. Powell and B. Paynter for suggestions in the preparation of this manuscript.
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