Biotechnology Letters 23: 11191123, 2001. 2001 Kluwer Academic Publishers. Printed in the Netherlands. 1119

Optimization of cell growth and poly(3-hydroxybutyrate) accumulation ondate syrup by a Bacillus megaterium strain

S. Omar1, A. Rayes2, A. Eqaab2, I. Vo3 & A. Steinbchel3,1Botany Department, Faculty of Science, Alexandria, Egypt2Biology Department, Faculty of Applied Sciences, Umm Al-Qurra University Makkah, Saudi-Arabia3Institut fr Mikrobiologie,Westflische Wilhelms-Universitt Mnster, Corrensstrae 3, 48149 Mnster, GermanyAuthor for correspondence (Fax: +49-251-8338388; E-mail: steinbu@uni-muenster.de)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 molassessupplemented with NH4Cl. When date syrup and beet molasses were used alone without an additional nitrogensource, a cell density of about 3 g l1 with a PHB content of the cells of 50% (w/w) was achieved. NH4NO3followed by ammonium acetate and then NH4Cl supported cell growth up to 4.8 g l1, whereas PHB accumulationwas increased with NH4Cl followed by ammonium acetate, NH4NO3 and then (NH4)2SO4 to a PHB content ofnearly 42% (w/w). Cultivation of B. megaterium at 30 l scale gave a PHB content of 25% (w/w) of the cells and acell density of 3.4 g l1 after 14 h growth.

Introduction

Polyhydroxyalkanoates (PHAs) are natural, biodegrad-able polyesters, which are accumulated as intracel-lular granules by a large variety of bacteria (Byrom1987, Anderson & Dawes 1990). Besides poly(3-hydroxybutyrate), PHB, approximately 150 other hy-droxyalkanoic acids have been detected as constituentsof PHAs in various bacteria (Steinbchel & Valentin1995). Since the physical and material properties ofPHA resemble those of polypropylene (Barham 1990),commercial production of PHAs is of considerableinterest (Byrom 1987, Emseley 1991). However, thehigher production costs of PHB as compared withthose of plastics based on petrochemicals, have sofar prevented the production and commercialization ofPHB. Many efforts have been made to develop lowcost industrial processes for their production. One ofthe limiting factors for economic PHA production isthe cost of the feedstock, especially the sugar substrate(King 1982): 3 tons of glucose are required to produce1 ton of PHB (Collins 1987). Therefore, less expensivesubstrates, in addition to improved fermentation strate-

gies, low-cost media and easier downstream processesare required and residual carbon sources from agricul-ture like whey or molasses are often used for fermen-tations (Ahn et al. 2001, Tamer et al. 1998a,b, Liuet al. 1998). In addition, optimization of fermentationconditions has substantially enhanced yield and pro-ductivity of many bioprocesses (Chisti & Moo-Young1996).

As with many other fermentations, that of Bacil-lus megaterium is also affected by various parametersincluding carbon and nitrogen sources, supplementa-tion of complex nutrients and organic acids. This wasinvestigated in this study with respect to growth andPHB accumulation in B. megaterium, which was iso-lated from the sludge of a sewage treatment plant inMakkah, Saudi-Arabia. To our knowledge, no reportusing date syrup as sole carbon source for the produc-tion of PHAs has been published, yet. In Saudi-Arabiathe number of date-palm trees exceeds 11 million withan annual production of 500 000 tons of dates.

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Materials and methods

Microorganisms and culture conditions

A strain of Bacillus megaterium isolated from thesludge of a sewage treatment plant in Makkah (Saudi-Arabia) and identified by the DSMZ (Braunschweig,Germany) was grown on mineral salts medium (MSM)described by Schlegel et al. (1961). Fructose andall other carbon sources were used at 8 g l1. Inaddition, NH4Cl was replaced by ammonium ox-alate, NH4NO3, NaNO3, ammonium acetate and(NH4)2SO4 at 0.5 g l1. Date syrup was obtained lo-cally from Wadi Hanifa Dates Factory Ltd (Riyadh,Saudi-Arabia). The typical composition of treated datesyrup was (% ): moisture content 16, ash content 6.8,total solids (w/w) 84, total sugars 79.5, reduced sug-ars 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 com-position of beet molasses (obtained from Pfeifer andLangen, Elsdorf, Germany) was as follows (%): watercontent 23.7, ash content 9.6, total sugar 47, invertedsugars 0.10.5, raffinose 0.31.5, albumen 12, N-freesubstances 7.5 (Schiwek 1995).

Preparation of seed cultureFive ml sterile distilled water was added to bacterialslants (grown for 24 h at 30 C). The resulting suspen-sion was then transferred to 50 ml MSM in a 250 mlErlenmeyer flask and incubated at 30 C and shakenat 140 rpm for 24 h. For cultivation and fermentationtests 5 ml of the seed culture was transferred to 100 mlMSM in a 250 ml flask and agitated at 140 rpm andkept at 30 C.

Fermentation experiments for large scale produc-tion of PHB were done in a 42 l Biostat UD30 stainlesssteal reactor (B. Braun Biotech International, Melsun-gen, Germany) containing 30 l MSM with 2% (w/v)date syrup and 0.5 g l1 NH4Cl. Cultivation parame-ters were as follows: temperature, 30 C; pH, 7; stirrerspeed, 100300 rpm; aeration, 0.9 vvm. Foam waspreferentially controlled by the mechanical foam de-stroyer; only if this was not sufficient, a silicone-basedantifoam was added.

Estimation of growthGrowth of the strain was estimated by drying a certainvolume of fermentation broth to constant weight aftercentrifugation at 3000 g and 4 C.

Analysis of ammoniumAmmonium was estimated in cell free super-natants employing ammonium test bars (MerckAG, Darmstadt, Germany) or a gas-sensitive type152303000 ammonium electrode (Mettler ToledoGmbH, Greifensee, Switzerland).

Analysis of sucroseThe concentration of sucrose was calculated from theamounts of fructose and glucose that were detected incell free supernatants after inversion of sucrose in thepresence of 94 mM sulfuric acid at 70 C for 3 h. Fruc-tose and glucose were analysed by HPLC using an ionexclusion HPX-87H (7.8 300 mm) column at 70 C(Biorad, Richmond, USA) and a RI-71 detector. Thecolumn was equilibrated with 6.5 mM sulfuric acid at0.5 ml min1.

Analysis of accumulated PHAQuantitative determination of PHAs was done by GCaccording to Brandl et al. (1988) and Timm et al.(1990).

Isolation of PHAPHA was isolated from lyophylized cell material byextraction with chloroform in a Soxhlet apparatus andprecipitated by adding 10 vol. ethanol. The precipi-tated PHA was separated from the solvent by filtrationand dried under a constant stream of air.

Results

Date syrup at 5% (w/v) and beet molasses between4 and 5% (w/v) in MSM gave the best growth andPHB accumulation in B. megaterium (results notshown), and these concentrations were therefore usedin all subsequent experiments. When different carbonsources at concentrations equivalent to 8 g fructose l1as well as date syrup and beet molasses (50 g l1)were tested with respect to growth and PHB produc-tion during 48 h incubation at 30 C (Table 1), growth

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Table 1. Effect of different carbon sources added to MSM on celldensity and accumulation of PHB by B. megaterium.

Carbon source Yield CDM PHB content(g l1) (%, w/w of CDM)

Date syrup 3.3 52Beet molasses 3.7 50Glucose 1.2 33Lactose 1.8 26Gluconate (sodium salt) 0.3 24Maltose 0.5 2Fructose 2.2 17Sucrose 0.5 5Xylose 0.4 5

Cultivation conditions: date syrup and beet molasses were usedat 5% (w/v), whereas all other carbon sources were used atconcentrations equivalent to 8 g fructose l1 as described inSchlegels MSM. Ammonium chloride was added at 0.05% (w/v).The cultivations were done in 250 ml Erlenmeyer flasks with100 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 nutri-ents and additional carbon sources to MSM containing5% (w/v) date syrup as carbon source on cell density andaccumulation of PHB by B. megaterium.

Additive Yield CDM PHB content(g l1) (%, w/w of CDM)

Nonea 3.3 33Beef extract 2.9 38Casamino acids 2.1 39Yeast extract 3.3 15Palm oil 3.6 42Succinic acid 2.3 42Propionic acid 0.1 16

aControl: 5% (w/v) date syrup with 0.05% (w/v) NH4Clas nitrogen source.The cultivations were done as described in the legendof Table 1. The additives were applied at 0.2% (w/v).Abbreviations: see legend to Table 1.

and PHB accumulation were significantly higher withdate syrup and beet molasses than with all other car-bon sources tested. Only fructose, lactose and glucoseallowed growth and PHB accumulation (exceeding1 g CDM l1 and 15% PHB of CDM, respectively),whereas gluconic acid, maltose, sucrose and xylosewere not suitable (Table 1).

Supplementation of MSM containing NH4Cl anddate syrup with beef extract, yeast extract, palm oil,casamino acids, succinic acid and propionic acid wasdetermined (Table 2). Only palm oil increased growth

Table 3. Effect of the addition of different nitrogensources to MSM on cell density and accumulation of PHBby B. megaterium.

Nitrogen source Yield CDM PHB content(g l1) (%, w/w of CDM)

Controla 0.5 22NH4Cl 2.4 42NH4 acetate 3.1 40NH4NO3 4.8 38(NH4)2SO4 2.0 34NH4 oxalate 1.7 25NaNO3 1.2 4

aControl: 5% (w/v) date syrup without addition of a ni-trogen source.The cultivation conditions were the same as described inthe legend of Table 1. The nitrogen sources were added at0.05% (w/v). Abbreviations: see legend to Table 1.

slightly, whereas propionic acid had a severe negativeeffect on growth. Accumulation of PHB was enhancedwith succinic acid, palm oil, casamino acids or beefextract (Table 2).

Since the nitrogen source is an important fac-tors for the accumulation of PHB, different salts ofammonium and NaNO3 (Table 3) were tested andprovided at amounts equivalent to the ammonium pro-vided by 0.5 g NH4Cl l1. Growth was high, whenNH4NO3 was used as nitrogen source followed byammonium acetate, NH4Cl, (NH4)SO4, ammoniumoxalate, NaNO3 and finally date syrup (control). Ac-cumulation of PHB in the cells of B. megaterium, onthe other hand, reached its maximum with MSM plus5% (w/v) date syrup and NH4Cl as nitrogen source,followed by ammonium acetate, NH4NO3, (NH4)SO4and finally ammonium oxalate (Table 3). An accumu-lation of only 4% (w/w) PHB was observed, whenNaNO3 was added as sole nitrogen source.

To investigate whether fermentation of B. mega-terium produce high yields of PHB, cultivation of thisisolate was performed in a 42 l capacity fermentor tomonitor and adjust the favorable parameters. Figure 1shows that after the consumption of ammonium (6 h),cell density and PHB accumulation continued to in-crease, resulting in a PHB content of 25% (w/w) anda cell density of 3.4 g l1 within the first 14 h. Thiswas followed by a slight decline during the furthertime course of the fermentation (22 h), when the cellsstarted to form endospores, although sucrose was notfully 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-hydroxybutyricacid); CDM, cellular dry matter.

Discussion

Whereas PHB accumulation has been studied in mostdetail in Ralstonia eutropha (Saito et al. 1995) andin Alcaligenes latus (Grothe et al. 1999), PHB ac-cumulation in B. megaterium has not been studiedin detail, yet. The cultivation conditions of B. mega-terium were systematically optimized using a varietyof inexpensive media suitable for large scale produc-tion of PHB. Many strategies have been developed inthe past with the aim to decrease the PHA productioncosts (Choi & Lee 1999). One was to isolate naturallyoccurring bacterial strains which utilize less expensivecarbon sources as for example molasses that are ob-tained as residual materials from sugar beets or sugarcane (Page 1992) or oils from rhamnose production(Fchtenbusch et al. 2000). In this study, a residualcarbon source abundant in Saudi-Arabia was used tostudy growth and accumulation of PHB in cells ofB. megaterium.

The results of this study indicate important effectsof simple and complex carbon and nitrogen sourceson growth and accumulation of B. megaterium. Datesyrup and beet molasses followed by fructose, lactoseand glucose allowed best growth of the cells, whereasdate syrup, beet molasses, glucose, lactose, sodiumgluconate and fructose gave highest PHB contents,respectively. This is probably due to the numerousorganic 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 bacteriaaccumulate PHA when the cells are cultivated un-der nitrogen-limited conditions. Nevertheless, higheryields of PHA were often obtained, if small amountsof a nitrogen source were added to the medium in thePHA accumulation phase (Wang & Lee 1997). Theeffect of the nitrogen source was also investigated inthis study, and it was shown that the addition of 0.05%(w/v) NH4Cl almost doubled the accumulation of PHBin cells of B. megaterium and that NH4Cl was muchmore suitable than other nitrogen sources. It was re-ported that NH4Cl at 0.05% (w/v) was most suitablefor PHB production in R. eutropha (Schlegel et al.1961).

The supplementation of the mineral salts mediumcontaining date syrup (5% w/v) and NH4Cl (0.05%,w/v) with complex nutrients or additional carbonsources showed that succinic acid, palm oil, casaminoacids and beef extract, respectively, enhanced PHB ac-cumulation, whereas propionic acid and yeast extractdecreased accumulation by nearly 50% in B. mega-terium (Table 3). These results are, to some extent, inaccordance with the results obtained by Page (1992),who reported increased yields of PHB, if A. vinelandiiwas cultivated in media containing in addition to pureor unrefined sugars also complex nitrogen sources.A partial substitution of the sugar content in themedium by small quantities of inexpensive energy-

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rich cosubstrates could lead to increased growth andPHB accumulation, thus reducing the process timesignificantly.

The 30 l fermentation experiment indicated thatPHB production is growth associated in B. megaterium(Figure 1). Due to the depletion of ammonium andprobably other nutrients, sporulation occurred leadingto a decrease of the accumulated PHB and cell har-vest must be accurately timed to prevent loss of theproduced PHB.

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