Biotechnology Letters 23: 11191123, 2001. 2001 Kluwer Academic Publishers. Printed in the Netherlands.

1119

Optimization of cell growth and poly(3-hydroxybutyrate) accumulation on date syrup by a Bacillus megaterium strain
S. Omar1 , A. Rayes2 , A. Eqaab2 , I. Vo3 & A. Steinbchel3,
1 Botany

Department, Faculty of Science, Alexandria, Egypt Department, Faculty of Applied Sciences, Umm Al-Qurra University Makkah, Saudi-Arabia 3 Institut fr Mikrobiologie,Westflische Wilhelms-Universitt Mnster, Corrensstrae 3, 48149 Mnster, Germany Author for correspondence (Fax: +49-251-8338388; E-mail: steinbu@uni-muenster.de)
2 Biology

Received 27 March 2001; Revisions requested 12 April 2001; Revisions received 14 May 2001; Accepted 14 May 2001

Key words: Bacillus megaterium, date syrup, molasses, poly(3-hydroxybutyrate)

Abstract Optimal growth and PHB accumulation in Bacillus megaterium occurred with 5% (w/v) date syrup or beet molasses supplemented with NH4 Cl. When date syrup and beet molasses were used alone without an additional nitrogen source, a cell density of about 3 g l1 with a PHB content of the cells of 50% (w/w) was achieved. NH4 NO3 followed by ammonium acetate and then NH4 Cl supported cell growth up to 4.8 g l1 , whereas PHB accumulation was increased with NH4 Cl followed by ammonium acetate, NH4 NO3 and then (NH4 )2 SO4 to a PHB content of nearly 42% (w/w). Cultivation of B. megaterium at 30 l scale gave a PHB content of 25% (w/w) of the cells and a cell density of 3.4 g l1 after 14 h growth.

Introduction Polyhydroxyalkanoates (PHAs) are natural, biodegradable polyesters, which are accumulated as intracellular granules by a large variety of bacteria (Byrom 1987, Anderson & Dawes 1990). Besides poly(3hydroxybutyrate), PHB, approximately 150 other hydroxyalkanoic acids have been detected as constituents of PHAs in various bacteria (Steinbchel & Valentin 1995). Since the physical and material properties of PHA resemble those of polypropylene (Barham 1990), commercial production of PHAs is of considerable interest (Byrom 1987, Emseley 1991). However, the higher production costs of PHB as compared with those of plastics based on petrochemicals, have so far prevented the production and commercialization of PHB. Many efforts have been made to develop low cost industrial processes for their production. One of the limiting factors for economic PHA production is the cost of the feedstock, especially the sugar substrate (King 1982): 3 tons of glucose are required to produce 1 ton of PHB (Collins 1987). Therefore, less expensive substrates, in addition to improved fermentation strate-

gies, low-cost media and easier downstream processes are required and residual carbon sources from agriculture like whey or molasses are often used for fermentations (Ahn et al. 2001, Tamer et al. 1998a,b, Liu et al. 1998). In addition, optimization of fermentation conditions has substantially enhanced yield and productivity of many bioprocesses (Chisti & Moo-Young 1996). As with many other fermentations, that of Bacillus megaterium is also affected by various parameters including carbon and nitrogen sources, supplementation of complex nutrients and organic acids. This was investigated in this study with respect to growth and PHB accumulation in B. megaterium, which was isolated from the sludge of a sewage treatment plant in Makkah, Saudi-Arabia. To our knowledge, no report using date syrup as sole carbon source for the production of PHAs has been published, yet. In Saudi-Arabia the number of date-palm trees exceeds 11 million with an annual production of 500 000 tons of dates.

1120 Materials and methods Microorganisms and culture conditions A strain of Bacillus megaterium isolated from the sludge of a sewage treatment plant in Makkah (SaudiArabia) and identied by the DSMZ (Braunschweig, Germany) was grown on mineral salts medium (MSM) described by Schlegel et al. (1961). Fructose and all other carbon sources were used at 8 g l1 . In addition, NH4 Cl was replaced by ammonium oxalate, NH4 NO3 , NaNO3 , ammonium acetate and (NH4 )2 SO4 at 0.5 g l1 . Date syrup was obtained locally from Wadi Hanifa Dates Factory Ltd (Riyadh, Saudi-Arabia). The typical composition of treated date syrup was (% ): moisture content 16, ash content 6.8, total solids (w/w) 84, total sugars 79.5, reduced sugars 4.9, inverted sugars 74.8, total proteins (as N) 3.1, total lipids (fats) 2, pectin content (as calcium pectate) 1.5 (all in %), vitamin C 0.19, minerals: sodium 13, potassium 203, iron 7.8, magnesium 143, calcium 338 (all in mg 100 g1 ) (Mustafa et al. 1982). The composition of beet molasses (obtained from Pfeifer and Langen, Elsdorf, Germany) was as follows (%): water content 23.7, ash content 9.6, total sugar 47, inverted sugars 0.10.5, rafnose 0.31.5, albumen 12, N-free substances 7.5 (Schiwek 1995). Preparation of seed culture Five ml sterile distilled water was added to bacterial slants (grown for 24 h at 30 C). The resulting suspension was then transferred to 50 ml MSM in a 250 ml Erlenmeyer ask and incubated at 30 C and shaken at 140 rpm for 24 h. For cultivation and fermentation tests 5 ml of the seed culture was transferred to 100 ml MSM in a 250 ml ask and agitated at 140 rpm and kept at 30 C. Fermentation experiments for large scale production of PHB were done in a 42 l Biostat UD30 stainless steal reactor (B. Braun Biotech International, Melsungen, Germany) containing 30 l MSM with 2% (w/v) date syrup and 0.5 g l1 NH4 Cl. Cultivation parameters were as follows: temperature, 30 C; pH, 7; stirrer speed, 100300 rpm; aeration, 0.9 vvm. Foam was preferentially controlled by the mechanical foam destroyer; only if this was not sufcient, a silicone-based antifoam was added. Estimation of growth Growth of the strain was estimated by drying a certain volume of fermentation broth to constant weight after centrifugation at 3000 g and 4 C. Analysis of ammonium Ammonium was estimated in cell free supernatants employing ammonium test bars (Merck AG, Darmstadt, Germany) or a gas-sensitive type 152303000 ammonium electrode (Mettler Toledo GmbH, Greifensee, Switzerland). Analysis of sucrose The concentration of sucrose was calculated from the amounts of fructose and glucose that were detected in cell free supernatants after inversion of sucrose in the presence of 94 mM sulfuric acid at 70 C for 3 h. Fructose and glucose were analysed by HPLC using an ion exclusion HPX-87H (7.8 300 mm) column at 70 C (Biorad, Richmond, USA) and a RI-71 detector. The column was equilibrated with 6.5 mM sulfuric acid at 0.5 ml min1 . Analysis of accumulated PHA Quantitative determination of PHAs was done by GC according to Brandl et al. (1988) and Timm et al. (1990). Isolation of PHA PHA was isolated from lyophylized cell material by extraction with chloroform in a Soxhlet apparatus and precipitated by adding 10 vol. ethanol. The precipitated PHA was separated from the solvent by ltration and dried under a constant stream of air.

Results Date syrup at 5% (w/v) and beet molasses between 4 and 5% (w/v) in MSM gave the best growth and PHB accumulation in B. megaterium (results not shown), and these concentrations were therefore used in all subsequent experiments. When different carbon sources at concentrations equivalent to 8 g fructose l1 as well as date syrup and beet molasses (50 g l1 ) were tested with respect to growth and PHB production during 48 h incubation at 30 C (Table 1), growth

1121
Table 1. Effect of different carbon sources added to MSM on cell density and accumulation of PHB by B. megaterium. Carbon source Yield CDM (g l1 ) 3.3 3.7 1.2 1.8 0.3 0.5 2.2 0.5 0.4 PHB content (%, w/w of CDM) 52 50 33 26 24 2 17 5 5 Table 3. Effect of the addition of different nitrogen sources to MSM on cell density and accumulation of PHB by B. megaterium. Nitrogen source Controla NH4 Cl NH4 acetate NH4 NO3 (NH4 )2 SO4 NH4 oxalate NaNO3 Yield CDM (g l1 ) 0.5 2.4 3.1 4.8 2.0 1.7 1.2 PHB content (%, w/w of CDM) 22 42 40 38 34 25 4

Date syrup Beet molasses Glucose Lactose Gluconate (sodium salt) Maltose Fructose Sucrose Xylose

a Control: 5% (w/v) date syrup without addition of a ni-

Cultivation conditions: date syrup and beet molasses were used at 5% (w/v), whereas all other carbon sources were used at concentrations equivalent to 8 g fructose l1 as described in Schlegels MSM. Ammonium chloride was added at 0.05% (w/v). The cultivations were done in 250 ml Erlenmeyer asks with 100 ml medium for 48 h at 30 C and 150 rpm. Abbreviations: MSM, mineral salts medium; CDM, cellular dry matter; PHB, poly(3-hydroxybutyric acid). Table 2. Effect of the addition of some complex nutrients and additional carbon sources to MSM containing 5% (w/v) date syrup as carbon source on cell density and accumulation of PHB by B. megaterium. Additive Nonea Beef extract Casamino acids Yeast extract Palm oil Succinic acid Propionic acid Yield CDM (g l1 ) 3.3 2.9 2.1 3.3 3.6 2.3 0.1 PHB content (%, w/w of CDM) 33 38 39 15 42 42 16

trogen source. The cultivation conditions were the same as described in the legend of Table 1. The nitrogen sources were added at 0.05% (w/v). Abbreviations: see legend to Table 1.

a Control: 5% (w/v) date syrup with 0.05% (w/v) NH Cl 4

as nitrogen source. The cultivations were done as described in the legend of Table 1. The additives were applied at 0.2% (w/v). Abbreviations: see legend to Table 1.

and PHB accumulation were signicantly higher with date syrup and beet molasses than with all other carbon sources tested. Only fructose, lactose and glucose allowed growth and PHB accumulation (exceeding 1 g CDM l1 and 15% PHB of CDM, respectively), whereas gluconic acid, maltose, sucrose and xylose were not suitable (Table 1). Supplementation of MSM containing NH4 Cl and date syrup with beef extract, yeast extract, palm oil, casamino acids, succinic acid and propionic acid was determined (Table 2). Only palm oil increased growth

slightly, whereas propionic acid had a severe negative effect on growth. Accumulation of PHB was enhanced with succinic acid, palm oil, casamino acids or beef extract (Table 2). Since the nitrogen source is an important factors for the accumulation of PHB, different salts of ammonium and NaNO3 (Table 3) were tested and provided at amounts equivalent to the ammonium provided by 0.5 g NH4 Cl l1 . Growth was high, when NH4 NO3 was used as nitrogen source followed by ammonium acetate, NH4 Cl, (NH4 )SO4 , ammonium oxalate, NaNO3 and nally date syrup (control). Accumulation of PHB in the cells of B. megaterium, on the other hand, reached its maximum with MSM plus 5% (w/v) date syrup and NH4 Cl as nitrogen source, followed by ammonium acetate, NH4 NO3 , (NH4 )SO4 and nally ammonium oxalate (Table 3). An accumulation of only 4% (w/w) PHB was observed, when NaNO3 was added as sole nitrogen source. To investigate whether fermentation of B. megaterium produce high yields of PHB, cultivation of this isolate was performed in a 42 l capacity fermentor to monitor and adjust the favorable parameters. Figure 1 shows that after the consumption of ammonium (6 h), cell density and PHB accumulation continued to increase, resulting in a PHB content of 25% (w/w) and a cell density of 3.4 g l1 within the rst 14 h. This was followed by a slight decline during the further time course of the fermentation (22 h), when the cells started to form endospores, although sucrose was not fully consumed, yet.

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Fig. 1. Time course of a batch fermentation with B. megaterium in mineral salts medium containing 2% (w/v) date syrup as sole carbon source. Cultivation conditions: temperature, 30 C; pH, 7; agitation, 100300 rpm; aeration, 0.9 vvm. Abbreviations: PHB, poly(3-hydroxybutyric acid); CDM, cellular dry matter.

Discussion Whereas PHB accumulation has been studied in most detail in Ralstonia eutropha (Saito et al. 1995) and in Alcaligenes latus (Grothe et al. 1999), PHB accumulation in B. megaterium has not been studied in detail, yet. The cultivation conditions of B. megaterium were systematically optimized using a variety of inexpensive media suitable for large scale production of PHB. Many strategies have been developed in the past with the aim to decrease the PHA production costs (Choi & Lee 1999). One was to isolate naturally occurring bacterial strains which utilize less expensive carbon sources as for example molasses that are obtained as residual materials from sugar beets or sugar cane (Page 1992) or oils from rhamnose production (Fchtenbusch et al. 2000). In this study, a residual carbon source abundant in Saudi-Arabia was used to study growth and accumulation of PHB in cells of B. megaterium. The results of this study indicate important effects of simple and complex carbon and nitrogen sources on growth and accumulation of B. megaterium. Date syrup and beet molasses followed by fructose, lactose and glucose allowed best growth of the cells, whereas date syrup, beet molasses, glucose, lactose, sodium gluconate and fructose gave highest PHB contents, respectively. This is probably due to the numerous organic nitrogen compounds present in date syrup. In contrast, only little PHB was accumulated when

the cells were cultivated in MSM containing xylose, sucrose or maltose. Most PHA producing bacteria accumulate PHA when the cells are cultivated under nitrogen-limited conditions. Nevertheless, higher yields of PHA were often obtained, if small amounts of a nitrogen source were added to the medium in the PHA accumulation phase (Wang & Lee 1997). The effect of the nitrogen source was also investigated in this study, and it was shown that the addition of 0.05% (w/v) NH4 Cl almost doubled the accumulation of PHB in cells of B. megaterium and that NH4 Cl was much more suitable than other nitrogen sources. It was reported that NH4 Cl at 0.05% (w/v) was most suitable for PHB production in R. eutropha (Schlegel et al. 1961). The supplementation of the mineral salts medium containing date syrup (5% w/v) and NH4 Cl (0.05%, w/v) with complex nutrients or additional carbon sources showed that succinic acid, palm oil, casamino acids and beef extract, respectively, enhanced PHB accumulation, whereas propionic acid and yeast extract decreased accumulation by nearly 50% in B. megaterium (Table 3). These results are, to some extent, in accordance with the results obtained by Page (1992), who reported increased yields of PHB, if A. vinelandii was cultivated in media containing in addition to pure or unrened sugars also complex nitrogen sources. A partial substitution of the sugar content in the medium by small quantities of inexpensive energy-

1123 rich cosubstrates could lead to increased growth and PHB accumulation, thus reducing the process time signicantly. The 30 l fermentation experiment indicated that PHB production is growth associated in B. megaterium (Figure 1). Due to the depletion of ammonium and probably other nutrients, sporulation occurred leading to a decrease of the accumulated PHB and cell harvest must be accurately timed to prevent loss of the produced PHB.
nological rhamnose production. Appl. Microbiol. Biotechnol. 53: 167172. Grothe R, Moo-Young M, Chisti Y (1999) Fermentation optimization for the production of poly( -hydroxybutyric acid) microbial thermoplastic. Enzyme Microbiol. Technol. 25: 132141. King PP (1982) Biotechnology. An industrial view. J. Chem. Technol. Biotechnol. 32: 28. Liu F, Li WQ, Ridgway D, Gu TY, Shen ZY (1998) Production of poly-beta-hydroxybutyrate on molasses by recombinant Escherichia coli. Biotechnol. Lett. 20: 345348. Mustafa AI, Hamad AM, Wahdan AN, Al-Kahtani MS (1982) Extraction of date syrup (Dibs) and its utilization in bakery products and juice. In: Proceedings of the First Symposium on the Date Palm in Saudi Arabia. Al Hassa, Saudi-Arabia: King Faisal University, pp. 534543. Page WJ (1992) Production of polyhydroxyalkanoates by Azotobacter vinelandii UWD in beet molasses culture. FEMS Microbiol. Rev. 103: 149158. Saito Y, Nakamura S, Hiramitsu M, Doi Y (1995) Microbial synthesis and properties of poly(3-hydroxybutyrate-co-4hydroxybutyrate). Polymer Int. 39: 169174. Schiwek H (1995) Zusammensetzung von Zuckerrbenmelasse. Zuckerindustrie 120: 273282. Schlegel HG, Kaltwasser H, Gottschalk G (1961) Ein Submersverfahren zur Kultur wasserstoffoxidierender Bakterien: Wachstumsphysiologische Untersuchungen. Arch. Mikrobiol. 38: 209222. Steinbchel A, Valentin HE (1995) Diversity of bacterial polyhydroxyalkanoic acids. FEMS Microbiol. Lett. 128: 219228. Tamer IM, Moo-Young M, Chisti J (1998a) Disruption of Alcaligenes latus for recovery of poly( -hydroxybutyric acid): comparison of high-pressure homogenization, bead milling, and chemically induced lysis. Int. Eng. Chem. Res. 37: 18071814. Tamer IM, Moo-Young M, Chisti J (1998b) Optimization of poly( hydroxybutyric acid) recovery from Alcaligenes latus: combined mechanical and chemical treatments. Bioprocess. Eng. 19: 459 468. Timm A, Byrom D, Steinbchel A (1990) Formation of blends of various poly(3-hydroxyalkanoic acids) by a recombinant strain of Pseudomonas oleovorans. Appl. Microbiol. Biotechnol. 33: 296301. Wang FL, Lee SY (1997) Poly(3-hydroxybutyrate) production with high productivity and high polymer content by a fed-batch culture of Alcaligenes latus under nitrogen limitation. Appl. Environ. Microbiol. 63: 37033706.

References
Ahn WS, Park SJ, Lee SY (2001) Production of poly(3hydroxybutyrate) from whey by cell recycle fed-batch culture of recombinant Escherichia coli. Biotechnol. Lett. 23: 235240. Anderson AJ, Dawes EA (1990) Occurrence, metabolism, metabolic role and industrial uses of bacterial polyhydroxyalkanoates. Microbiol. Rev. 47: 144149. Barham PJ (1990) Physical properties of poly(hydroxybutyrate) and poly(hydroxybutyrate-co-hydroxyvalerate). In: Dawes EA, ed. Novel Biodegradable Microbial Polymers. Dordrecht: Kluwer Academic Publishers, pp. 8196. Brandl H, Gross RA, Lenz RW, Fuller RC (1988) Pseudomonas oleovorans as a source of poly( -hydroxyalkanoates) for potential applications as biodegradable polyesters. Appl. Environ. Microbiol. 54: 19771982. Byrom D (1987) Polymer synthesis by microorganisms: technology and economics. Trends Biotechnol. 5: 245250. Chisti Y, Moo-Young M (1996) Bioprocess intensication through bioreactor engineering. Trans. Inst. Chem. Eng. 74: 575583. Choi J, Lee SY (1999) Factors effecting the economics of polyhydroxyalkanoate production by bacterial fermentation. Appl. Microbiol. Biotechnol. 51: 1321. Collins SH (1987) Choice of substrate in polyhydroxybutyrate synthesis. Spec. Publ. Soc. Gen. Microbiol. 21: 161168. Emseley J (1991) Biodegradable plastics. New Sci. 50: 14. Fchtenbusch B, Wullbrandt D, Steinbchel A (2000) Production of polyhydroxyalkanoic acids by Ralstonia eutropha and Pseudomonas oleovorans from an oil remaining from biotech-