Production of 2,3-butanediol by newly isolated Enterobacter cloacae

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Dec 17, 1998 - Abstract Enterobacter cloacae NRRL B-23289 was isolated from local decaying wood/corn soil samples while screening for microorganisms for ...
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App1 Microbio1 Biotechno1 (1999) 52: 321-326

ORIGINAL PAPER

B. C. Saha . R. J. Bothast

Production of 2,3-butanediol by newly isolated Enterobacter cloacae

Received: 17 December 1998

Revision received: 9 March 1999 ; Accepted: 20 March 1999

Abstract Enterobacter cloacae NRRL B-23289 was isolated from local decaying wood/corn soil samples while screening for microorganisms for conversion of L-arabinose to fuel ethanol. The major product of fermentation by the bacterium was meso-2,3-butanediol (2,3-BD). In a typical fermentation, a BD yield of 0.4 g/g arabinose was obtained with a corresponding productivity of 0.63 g/I per hour at an initial arabinose concentration of 50 g/1. The effects of initial arabinose concentration, temperature, pH, agitation, various monosaccharides, and multiple sugar mixtures on 2,3BD production were investigated. BD productivity, yield, and byproduct formation were influenced significantly within these parameters. The bacterium utilized sugars from acid plus enzyme saccharified corn fiber and produced BD (0.35 g/g available sugars). It also produced BD from dilute acid pretreated corn fiber by simultaneous saccharification and fermentation (0.34 g/g theoretical sugars).

Introduction 2,3-Butanediol, otherwise known as 2,3-butylene glycol (2,3-BD), is a valuable chemical feedstock because of its application as a solvent, a liquid fuel, and as a precursor of many synthetic polymers and resins. With a heating value of 27,200 J/g, BD compares favorably with etha-

nol (29,100 J/g) and methanol (22,100 J/g) for use as a liquid fuel and fuel additive (Tran and Chambers 1987). Dehydration of 2,3-BD yields the industrial solvent methyl ethyl ketone (Emerson et al. 1982). Further dehydration yields 1,3-butanediene, which is the starting material for synthetic rubber and is also an important monomer in the polymer industry (Maddox 1996). Methyl ethyl ketone can be hydrogenated to yield high octane isomers suitable for high quality aviation fuels. Diacetyl, formed by catalytic dehydrogenation of the diol, is a highly valued food additive (Magee and Kosaric 1987). A wide variety of chemicals can also be easily prepared from 2,3-BD (Yu and Saddler 1985; Gong et al. 1997). There is an interest in industrial scale production of BD from various agricultural residues as well as logging, pulp and paper, and food industry wastes (Magee and Kosaric 1987). While screening microorganisms for fermentation of arabinose to fuel ethanol from natural sources, we isolated an arabinose-utilizing bacterium. The microorganism was identified as Enterobacter cloacae and its main fermentation product was identified as meso-2,3BD. To date, very little information is available about the production of 2,3-BD from arabinose. In this paper, we report the production of 2,3-BD from arabinose, multiple sugar mixtures, and corn fiber hydrolyzate.

Materials and methods Screening, isolation, and identification of the bacterium

Names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by USDA implies no approval of the product to the exclusion of others that may also be suitable. B. C. Saha ([8]) . R. J. Bothast Fermentation Biochemistry Research Unit, National Center for Agricultural Utilization Research, Agricultural Research Service, United States Department of Agriculture, Peoria, IL 61604, USA e-mail: [email protected] Tel.: + 1-309-6816276 Fax: + 1-309-6816686

The bacterium used in this study was isolated by screening 132 decaying wood/corn soil samples collected from the Peoria, Ill., area. The screening medium contained (per liter): 2 g NaN0 3 , 0.5 g MgS0 4 . 7H 20, 0.5 g NaC1, 0.01 g FeS04 . 7H 2 0, 1.0 g KH 2 P0 4, 0.4 g yeast extract, and 50 g L-arabinose. Arabinose was sterilized separately. The pH of the medium was adjusted to 5.0 with 1 M HC1 before inoculation. The collected soil samples (~0.5 g) were placed in test tubes (1.5 x 15 em, 10 m1 medium with 5% w/v arabinose as carbon source) and incubated at 30°C and 200 rpm for 4 days. After five transfers in liquid culture, samples of culture broth were serially diluted and grown on agar plates (2% w/v agar)

322 containing the screening medium for 2-3 days. Subsequently, single isolated colonies were transferred to test tubes containing ]0 ml of the screening medium. These procedures were repeat;d several times to ensure purity of the culture. The isolate was maintained throughout this study at 4 cC on 2% w/v agar slants with YM medium (0.3% yeast extract and 0.3% malt extract) containing 1% wjv arabinose. A gram-negative test was performed with lysis of cells by 3% KOH (Gregersen 1978). The bacterium was identified using GN MicroPlate (Biolog, Inc., Hayward, Calif.) according to the instructions provided by the manufacturer and using the data base of Microlog GN and fatty acid profile analysis (Sasser 1990). Finally, the species differentiation was made by determining growth and acid production on raffinose, sorbitol, and sucrose and assaying l3-xylosidase activity (Grimont and Grimont 1991). On the basis of the results obtained with the strain in standard tests for the characterization of a bacterium. the isolated organism was identified as a strain of Ell1erobacter cloacae and w';s deposited in the Agricultural Research Service Culture Collection, National Center for Agricultural Utilization Research, Peoria, II1., designated as Ell1erobacter cloacae NRRL B-23289. Fermentation study The medium used for seed culture and fermentation studies was described previously (Slininger et a!. ]982). Sugars were sterilized separately. The pH of the medium was adjusted to pH 5.0 with] M HCI or NaOH before inoculation. An Erlenmeyer flask (125 mI) containing 50 ml medium with arabinose (50 gil) was inoculated with a loopful of cells taken from a stock slant and incubated at 28 cC on a rotary shaker (200 rpm) for 2 days. Fermentation flasks (125-ml Erlenmeyer flask with 50 ml medium) were inoculated with 2 ml of this starter culture and cultivated on a rotary shaker at 200 rpm for 4-5 days. Samples (2 ml) were withdrawn periodically to determine pH, cell growth, residual substrate, and product. Analytical procedures Cell growth was measured in terms of absorbance (A) of the appropriately diluted culture broth at 660 nm; ] A was estimated to be equivalent to 0.442 ± 0.004 g dry weight of the washed cells. Sugars and fermentation products were analyzed by high pressure liquid chromatography (HPLC) using authentic standards (arabinose, glucose, xylose, meso-2, 3-BD, acetoin, and ethanol) by the procedure described previously (Saha and Bothast ]996). Preparation of corn fiber hydrolyzate Wet corn fiber (supplied by Williams Ethanol Services, Pekin, III.) was dried in a forced air oven at 55 cC. Dried corn fiber (10% w/v) was refluxed with 0.5% H 2 S0 4 for] h. The resultant hydrolyzate (prepared by K. Grohmann, USDA, Citrus and Subtropical Products Laboratory, Winter Haven, Fla.) was neutralized with NaOH. A portion of the hydrolyzate was treated with a commer-

cial enzyme mixture (Spezyme GA, Cytolase ]23 from Genencor Internationa], Rochester, NY, and Novozyme ]88 from NovoNordisk BioChem North America. Inc.. Franklinton. NC. each at 2% w/w) at 45 cC for 24 h, autocIa~ed (12] CC,]5 min), and combined with an equal volume of two-fold concentrated sterilized medium and used for growth studies. Another portion of the hydrolyzate was autoclaved (121 cC, ]5 min), combined with an equal volume of two-fold concentrated sterilized medium and enzyme mixture (filter-sterilized), and used for simultaneous saccharification and fermentation (SSF) studies.

Results Analysis of fermentation products E. cloacae NRRL B-23289 was cultivated in the medium containing arabinose at pH 5.0, 30°C, and 200 rpm. The main fermentation product was identified as meso2,3-BD by HPLC analysis. In addition, the organism produced acetoin and small amounts of ethanol.

Effect of substrate concentration on 2,3-BD production The effect of arabinose concentration on 2.3-BD production is shown in Table 1. At low substrate (I % w/v) concentration, the maximum BD yield was lower (0.22 gig substrate) and BD disappeared rapidly with a concomitant increase of acetoin production and an increase in culture pH. The yield of BD was similar at 210% (w/v) arabinose concentration. Cell mass production increased with increasing arabinose concentration, but at 10% (w/v) it was slower and a longer time was required to reach maximum BD concentration. The pH of the culture broth at maximum BD production was found to decrease with increasing arabinose concentrations (Table 1). Initial production of acetoin was delayed with the increase of arabinose concentration.

Effect of temperature and initial pH of growth on 2,3-BD production The effect of temperature (25-40 0c) on growth and 2,3BD production was examined in shake flasks at pH 5.0 and 200 rpm on 50 gil arabinose. The results are sum-

Table I Effect of substrate concentration on 2,3-butanedio] production from arabinose by Enterobacter cloacae NRRL B-23289. Values reported are from duplicate experiments. The growth experiments were performed at pH 5.0, 30 cC, and 200 rpm Arabinose (gil)

]0 20 30 40 50 ]00

Time of maximum butanediol yield (h)

pH at maximum butanediol yield

24 24 24 32 32

6.0 4.8 4.6 4.5 4.3 4.]

72

± ± ± ± ± ±

0.0 0.0 0.] 0.] 0.0 0.0

Cell mass at maximum butanediol yield (gil) 1.6 3.4 4.6 5.3 5.8 4.6

± ± ± ± ± ±

0.0 0.] 0.0 0.2 0.] 0.1

Maximum butanediol

Ethanol

Acetoin

Butanediol yield

(gil)

(gil)

(gig substrate)

0 0 1.8 2.0 2.9 3.9

2.6 2.0 1.0 2.4 1.] 2.6

(gil)

2.2 7.0 11.5 15.2 ]7.8 34.4

± ± ± ± ± ±

0.1 0.0 0.1 0.0 0.4 3.4

± ± ± ±

0.1 0.] 0.0 0.8

± ± ± ± ± ±

0.0 0.0 0.3 0.3 0.2 0.5

0.22 0.35 0.38 0.38 0.36 0.34

± ± ± ± ± ±

0.] 0.0 0.0 0.0 0.0 0.0

323 Table 2 Effect of growth temperature and initial pH of growth on 2,3-butanediol production from arabinose by Enlerobacler cloacae NRRL B-23289. Values reported are from duplicate experiments. Arabinose used, 50 gil Condition

Time of maxImum butanediol yield (h)

pH at maximum butanediol yield

Cell mass at maximum butanediol yield (gil)

Maximum butanediol (gil)

Temperature of growth eC) 25 32 30 32 35 32 40 48

4.8 4.9 5.0 5.0

0.1 0.0 0.0 0.0

4.5 ± 0.0 4.2 ::: 0.1 3.6 ± 0.1 3.2 ± 0.2

1404 17.3 16.7 lOA

pH of growth 5.0 6.0 7.0

4.8 ± 0.0 5.1 ± 0.0 5.5 ± 0.0

4.1 ± 0.0 404 ± 0.0 4.3 ± 0.1

17.2 ± 0.1 20.1 ± 0.2 19.7 ± 0.3

32 32 32

± ± ± ±

marized in Table 2. BD production was optimal at 30°C. During growth of the bacterium, the pH of the culture broth remained almost unchanged. The bacterium grew fastest at 25 0C. Growth of E. cloacae was poor (cell mass 3.2 gil in 48 h), arabinose utilization was slow (56% at 48 h), and the BD produced per gram of arabinose utilized was 0.37 g at 40°C. The rates of BD production were 0.45, 0.54, 0.35, and 0.22 gil per hour at 25, 30, 35, and 40 °e, respectively. The production of acetoin was higher at higher temperatures. The effect of the initial pH (3.0-7.0) of the culture medium on arabinose utilization and 2,3-BD production was investigated at 30 °e and 200 rpm on 50 gil arabinose. The bacterium did not grow at pH 3.0 and growth was poor at 4.0. Yields of BD were 0.34-0.40 gig over the pH range of 5.0-7.0 (Table 2). Optimal yield of BD (0.40 gig) was obtained with initial pH at 6.0. However, at pH 6.0, BD rapidly disappeared after reaching the maximum with a concomitant increase of acetoin production. Acetoin production increased with increasing initial culture pH. Production rates of BD were 0.49, 0.63, and 0.63 gil per hour at pH 5.0, 6.0, and 7.0, respectively. Consequently, a temperature of 30 °e and pH 5.0 were chosen for subsequent fermentations by the bacterium. The time course of the fermentation is shown in Fig. 1. BD production increased with arabinose consumption and then declined when all the arabinose was exhausted. Cell mass continued to increase with arabinose consumption and 2,3-BD production. The bacterium produced 0.43 g BD per gram of arabinose in 39 h. The carbon balance for the arabinose fermentation by E. cloacae at 39 h is 103.6% based on theoretical CO 2 yield.

± ± ± ±

Ethanol

1.2 0.0 1.1 2.3

Butanediol yield

(gil)

Acetoin (gil)

0 1.0 ± 0.0 1.5 ± 0.0 0

104 1.5 204 2.7

0.0 0.0 0.0 l.l

0.29 ± 0.0 0.35 ± 0.0 0.33 ...l- 0.0 0.21 ± 0.0

0 0 0

1.1 ± 0.1 1.2 ± 0.1 2.6 ± 0.3

0.34 ± 0.0 0040 ± 0.0 0.39 ± 0.0

± ± ± ±

(gig substrate)

from 200-400 rpm (data not shown). Maximum 2,3-BD yields were 0.39.0.38,0.36 and 0.36 g per gram substrate in 144, 39, 24, and 24 h at 100, 200, 300, and 400 rpm, respectively. The cell mass concentrations at maximum BD yields were 1.5, 3.3,4.6, and 5.5 gil at 100, 200, 300, and 400 rpm, respectively. Effect of organic acids on 2,3-BD production The effects of acetate, lactate, and succinate (up to 10 gil) on 2,3-BD production were investigated (data not shown). Acetate at a level of 5 gil stimulated both arabinose utilization and 2,3-BD production (14-18% increase). Growth and arabinose utilization by the bacterium were lower in the presence of lactate, but 2,3-BD production increased to about 10% at 10 gil lactate. With 10 gil succinate, growth and sugar utilization patterns were unchanged but 2,3-BD production increased about 8%. 3.5

5

60

3.0

50

2.5

";; Cl

40

CIl

30

eo

E 1.5

g ec0

2

Cii

20 jg CIl

()

1.0

.0 ::J

10

0.5 24

The effect of agitation (100-400 rpm) on the production of 2,3-BD from arabinose was investigated. At 100 rpm, growth and substrate utilization were slow, and growth was faster and more extensive with increasing agitation

~ "0

2.0

3

Effect of agitation on 2,3-BD production

~

48

72

en

0 96

Time (h)

Fig. 1 Time course of 2,3-butanediol production from arabinose by EllIerobacler cloacae NRRL B-23289 at pH 5.0, 30°C and 200 rpm. Substrate used, 50 g(1. Values reported are from duplicate experiments. Symbols: pH, • cell mass (g(1), 0 arabinose, • 2,3butanediol, ... acetoin, • ethanol

324

Utilization of multiple sugar substrates Utilization of glucose, xylose, and arabinose separately and in combination was studied at pH 5.0, 30 °e and 200 rpm. E. cloacae NRRL B-23289 produced 2,3BD from each sugar with the rate of utilization being arabinose > glucose > xylose. The bacterium produced 0.37, 0.38, and 0.43 g BD per gram of glucose, xylose, and arabinose in 63 h, 63 h, and 39 h, respectively (Table 3). When grown on mixed sugars (mixture A, glucose:xylose:arabinose, I: I: I; mixture B, glucose: xylose:arabinose, I:2: I), the bacterium preferentially utilized glucose over xylose, and arabinose over xylose (Fig. 2). Xylose began to disappear only when glucose was totally depleted and only after a considerable proportion of arabinose was consumed. The bacterium produced primarily BD from all sugars. It produced 0.39 g BD per gram substrate in 48 h from both mixture A and mixture B. The bacterium utilized mannose, galactose, fructose, and sucrose and produced 0.37, 0.38, 0.43, and 0.35 g 2,3-BD per gram of substrate in 72, 63, 39, and 48 h, respectively at pH 5.0,30 °e, and 200 rpm.

5

4

4

3

25

20 "2

.9 t5 ::l

'§,

15

(/)

(/)

~3

til

E

C.

2



"0

e0.. (;

2

~

Ui

.0 :::J Cf)

5

24

48

72

Time (h)

Fig. 3 Time courses of glucose. xylose. and arabinose fermentation from dilute acid pretreated plus enzyme saccharifit:d com fiber by Elllerobacler cloacae NRRL B-23289 at pH 5.0, 30'C and 200 rpm. Substrate used, 50 g!l (total). Values reported are from duplicate experiments. Symbols: 0 glucose, D, xylose, 0 arabinose, • 2,3butanediol, ... acetoin

Discussion To our knowledge, this is the first detailed report on the production of 2,3-BD from arabinose. BD is produced during oxygen-limited growth by a fermentative pathway known as the mixed acid-BD pathway (Kosaric et al. 1992). The 2,3-BD pathway and the relative proportions of acetoin and BD serve to maintain the intracellular NAD(NADH balance in changing culture conditions. The theoretical maximum yield of 2,3-BD from monosaccharides is 0.5 gig (Jansen et al. 1984). BD produced by E. cloacae NRRL B-23289 appears to be the primary metabolite excreted as a product of energy metabolism. The yield of BD was not influenced much at substrate concentration (2-10% wlv arabinose). Substrate inhibition of growth occurred only at 100 gil (Table 1). Among the various factors influencing microbial performance, pH is of major importance in 2,3-BD production (Magee and Kosaric 1987). BD production from arabinose by E. cloacae NRRL B-23289 depended on pH and temperature of growth (Table 2). A temperature of 30°C was optimal for BD production. Optimal initial pH for BD production was 6.0. Although 2,3-BD is a product of anaerobic fermentation, aeration is known to enhance its production (Jansen et al. 1984). With the increase of agitation speed, the yield of cell mass was increased and BD production was not suppressed to a similar extent in the case of E. cloacae. Decreasing the agitation speed increased the BD yield but decreased the overall conversion rate due to lower cell concentration. While acetate at high levels may be inhibitory to Klebsiella pneumoniae, low levels of acetate

stimulate BD production (Yu and Saddler 1982). Stormer (1977) noted that acetate in its ionized form induces acetolactate synthase formation, and thereby enhances the catalysis of pyruvate to BD. The production of BD by K. oxytoca NRRL B-199 was enhanced in the presence of low levels (> 8 gil) of lactate (Qureshi and Cheryan 1989). Klebsiella oxytoca ATCC 8724 grew well on xylose with 10 gil succinate and produced additional BD (Eiteman and Miller 1995). The production of BD by E. cloacae NRRL B-23289 was also enhanced by the supplementation of acetate, lactate, and succinate. Since a typical hydrolyzate of lignocellulosic biomass contains a mixture of sugars such as glucose, xylose, and arabinose, we investigated how E. cloacae utilized multiple sugars separately and in mixtures. The result indicates that the bacterium has the capability to convert multiple mixed sugar substrates to BD. The preference for sugar utilized was different when a mixed sugar substrate was used. The bacterium preferentially utilized glucose > arabinose > xylose. The enzymes essential for conversion of xylose to 2,3-BD may be slightly inhibited by the presence of glucose and arabinose in the culture medium. Optimization of the medium components and other process parameters such as pH control and aeration may improve 2,3-BD production. The bacterium demonstrated broad substrate utilization as it produced BD from other sugars such as mannose, galactose, fructose, and sucrose. The 2,3-BD yield (0.350.43 gig sugar) by the newly isolated E. cloacae NRRL B-23289 compares favorably with other 2,3-BD-producing organisms reported in the literature (0.30-0.45 gig sugar) (Maddox 1996). The efficient biological conversion of all the available sugars in agricultural biomass residues to fuels and chemicals is crucial to the efficiency of any process intended to compete economically with petrochemical products (Yu and Saddler 1985). This bacterium utilized all the sugars from dilute acid pretreated enzyme saccharified corn fiber and produced BD (Fig. 3). It also performed well in producing 2,3-BD by a SSF process from dilute acid pretreated corn fiber substrate. The isolated bacterium may be appropriate for developing the commercial production of 2,3-BD from corn fiber and other agricultural residues. Acknowledgement The authors are grateful to Dr. Lawrence K. Nakamura for help in the identification of the bacterium.

References Eiteman MA, Miller JH (1995) Effect of succinic acid on 2,3-butanediol production by Klebsiella oxylOca. Biotechnol Letts 17: 1057-1062 Emerson RR, Flickinger MC, Tsao GT (1982) Kinetics of dehydration of aqueous 2,3-butanediol to methyl ethyl ketone. Ind Eng Chern Prod Res Dev 21: 473-477 Gong CS, Cao N, Tsao GT (1997) Biological production of 2,3butanediol from renewable biomass. In: Saha BC, Woodward J (eds) Fuels and chemicals from biomass. American Chemical Society, Washington, DC, pp 280-293

326 Gregersen TP (1978) Rapid method for distinction of gram-negative from gram-positive bacteria. Eur J Appl Microbiol Biotechnol 5: 123-127 Grimont F, Grimont PAD (1991) The genus Enterobacter. In: Balows A, Truper HG, Dworkin M, Harder W, Schleifer, K-H (eds) The prokaryotes. A handbook on the biology of bacteria: ecophysiology, isolation, identification, applications, vol 3. Springer, New York Berlin Heidelberg, pp 2797-2815 Jansen NB, Flickinger MC, Tsao GT (1984) Production of 2,3butanediol from xylose by Klebsiella oxylOca ATCC 8724. Biotechnol Bioeng 26: 362-368 Kosaric N, Magee RJ, Blaszczyk R (1992) Redox potential measurement for monitoring glucose and xylose conversion by K. pnellmoniae. Chern Biochem Eng Q 6: 145-152 Maddox IS (1996) Microbial production of 2,3-butanediol. In:Roehr M (ed) Biotechnology, vol 6. Products of primary metabolism. VCH, Weinheim, pp 269-291 Magee RJ, Kosaric N (1987) The microbial production of 2,3butanediol. Adv Appl Microbiol 32: 89-161 Qureshi N, Cheryan M (1989) Effect of lactic acid on growth and butanediol production by Klebsiella oxytoca. J Ind Microbiol 4: 453-456

Saha BC, Bothast RJ (1996) Production of L-arabitol from L-arabinose by Candida ell/omaea and Pichia gllilliermondii. Appl Microbiol Biotechnol 45: 299-306 Sasser M (1990) Identification of bacteria by gas chromatography of cellular fattv acids. Technical note 101. Microbial ID Inc.. Newark, Del . Slininger PJ. Bothast RJ. Van Cauwenberge JE. Kurtzman CP (1982) Conversion of ~-xylose to ethan~ by the yeast Pachysolen tannophilus. Biotechnol Bioeng 23: 371-384 Stormer FC (1977) Evidence for regulation of Aerobacter aerogenes pH 6 acetolactate-forming enzyme by acetate ion. Biochem Biophys Res Commun 74: 898-902 Tran AV, Chambers RP (1987) The dehydration of fermentative 2,3-butanediol into methyl ethyl ketone. Biotechnol Bioeng 29: 343-351 Yu EKC, Saddler IN (1982) Enhanced production of 2,3-butanediol by Klebsiella pnellmoniae grown on high sugar concentrations in the presence of acetic acid. Appl Environ Microbiol 44: 777-784 Yu EKC, Saddler IN (1985) Biomass conversion to butanediol by simultaneous saccharification and fermentation. Trends Biotechnol 3: 100-104