Effect of Impeller Speed and pH on the Production of Poly(3

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Effect of Impeller Speed and pH on the Production of Poly(3-hydroxybutyrate) Using Bacillus cereus SPV Sheryl Philip, Sudarshana Sengupta, Tajalli Keshavarz, and Ipsita Roy* Applied Biotechnology Research Group, Department of Molecular and Applied Biosciences, School of Biosciences, University of Westminster, 115 New Cavendish Street, London W1W 6UW, United Kingdom Received December 2, 2008; Revised Manuscript Received February 8, 2009

P(3HB), is one of the most well studied polyhydroxyalkanoates. It is biodegradable, biocompatible, exhibits thermoplastic properties and can be produced from renewable carbon sources. The commercial exploitation of P(3HB) has been mainly held back by its high production costs. Hence, a lot of research is required to optimize P(3HB) fermentation conditions. In this study we have focused on the effects of impeller speed and pH on P(3HB) production in Bacillus cereus SPV. Four different impeller speeds, 50, 125, 250, and 500 rpm, were used. The highest amount of P(3HB) accumulation was achieved using 125 rpm impeller speed (34% dcw) and this was attributed to optimal cell growth rate. Also, pH-stat fermentations were carried out at pH 3.0, 6.8, and 10. This study confirmed that lack of P(3HB) degradation during unbuffered Bacillus fermentations is due to the low pH conditions. This observation is crucial for the industrial exploitation of the genus Bacillus for the production of P(3HB).

Introduction Polyhydroxyalkanoates (PHAs) are a family of linear polyesters of 3, 4, 5, and 6-hydroxyacids, synthesized by a wide variety of bacteria through the fermentation of sugars, lipids, alkanes, alkenes, and alkanoic acids. They are found as discrete cytoplasmic inclusions in bacterial cells. Once extracted from the cells, PHAs exhibit thermoplastic and elastomeric properties. PHAs are recyclable, are natural materials, and can be easily degraded to carbon dioxide and water. Hence, they are excellent replacements to petroleum-derived plastics in terms of processability, physical characteristics, and biodegradability.1 In addition, these polymers are biocompatible and find several applications in medicine. Hence, PHAs are increasingly being exploited for commercial purposes.2 The most commonly synthesized PHA is poly(3-hydroxybutyrate), P(3HB). P(3HB) is a crystalline polymer with a melting temperature of 177 °C and has found many applications in the industry. Biomer produces P(3HB) from Alcaligenes latus to manufacture articles such as combs, pens, and bullets. P(3HB) is biocompatible, optically pure and possesses piezoelectricity, which helps in osteoinduction, the process of inducing osteogenesis.3,4 P(3HB) has thus been used in bone tissue engineering, as bone plates, osteosynthetic materials, and surgical sutures.5 When hydroxyapatite particles are incorporated into P(3HB), a bioactive and biodegradable composite is formed and this can be used in hard tissue regeneration. The combination of hydroxyapatite or Bioglass with P(3HB) is particularly beneficial in bone tissue engineering because these composites possess mechanical strength similar to that of human bones.6-8 P(3HB) can be used as implant patches that are good scaffolds for tissue regeneration in low pressure systems.9 P(3HB)-based devices have also been found useful in the slow release of drugs and hormones. P(3HB) can be used to make the nonwoven cover stock and the plastic film moisture barriers in nappies and * To whom correspondence should be addressed. Tel.: +44-20-79115000, ext. 3567. Fax: +44-20-79115087. E-mail: [email protected].

sanitary towels, along with some specialty paramedical film applications in hospitals.2,10 The widespread commercialization of P(3HB) has been held back by its high production costs compared to petroleum derived plastics. Hence, it is of vital consequence that cost-effective processes, including utilization of alternative substrates and lowcost media, be found that can reduce these costs and increase the range of P(3HB) applications in the industry. To achieve the ideal fermentation process for P(3HB) production, a large number of fermentation parameters including pH, dissolved oxygen tension, impeller speed, and temperature of the culture need to be optimized to enhance yield and productivity of P(3HB). The extraction method employed to isolate the polymer from the bacterial cells is another factor that determines both the cost-effectiveness and the pharmacological purity of the polymer during large scale production. A number of separation processes have been described by several authors for polymer recovery. However, the most popular method involves using boiling chloroform in a soxhlet.11 Although Bacillus sp. has long been reported to produce PHAs,12,13 most PHA production and optimization has focused on Gram negative bacteria and limited studies have been carried out in Bacillus species.14-16 One major advantage of PHA production using the Gram positive species is the absence of the immunogenic lipopolysaccharides. Lipopolysaccharides constitute the major impurity in purified PHAs from Gram negative bacteria and are known to induce strong immunogenic reactions. Hence, their absence in PHA isolated from Gram positive bacteria, such as the genus Bacillus, is a major advantage in the context of medical applications of the PHAs.17 Wu et al. (2001), scaled up the production of P(3HB) by Bacillus sp. JMa5, using a molasses medium, which resulted in polymer accumulation of 25-35% dry cell weight. They found that using a high carbon/nitrogen ratio or a high carbon/ phosphorus ratio and low oxygen supply elicited sporulation in Bacillus sp. JMa5, even though, under these conditions, P(3HB)

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production is increased in other bacterial species. The sporulation of Bacillus sp. JMa5 contributed to a reduced P(3HB) yield.16 Bacillus cereus SPV, when grown using glucose as the carbon source, in the modified Kannan and Rehacek medium, produces P(3HB).18 The production of P(3HB) in this strain has been scaled up and Bacillus cereus SPV accumulates about 29% of its dry cell weight as P(3HB) in 48 h. Further, the cells were able to accumulate P(3HB) at a concentration of 38% of dry cell weight when a simple glucose feeding strategy was adopted in a fed batch fermentation. Once maximal P(3HB) accumulation was achieved, the P(3HB) did not degrade and this was hypothesized to be due to acidic pH conditions. Different extraction procedures were tried out such as the soxhlet method, the chloroform method and the chloroform-hypochlorite dispersion method. It was found that the chloroform extraction method produced the highest crude yield of P(3HB), although the purest form of P(3HB) was obtained from the Soxhlet method.19 There have been limited studies on the optimization of P(3HB) production, at the bioreactor level, using any Bacillus sp. In this study, the effect of impeller speed and pH on P(3HB) production, using Bacillus cereus SPV, was investigated for the first time. The results obtained can be used toward the economization of P(3HB) production using this Bacillus sp. and other members of this genus.

2. Materials and Methods Chemicals. All chemicals were obtained from Sigma-Aldrich Company Ltd. (Dorset, England). Yeast extract was purchased from DIFCO (BD UK Ltd. Oxford, U.K.). Analytical grade reagents used for GC-MS was obtained from Sigma-Aldrich Company Ltd. Bacterial Strain and Maintenance. Bacillus cereus SPV was obtained from the University of Westminster culture collection. This strain has been used for the current study. Stock cultures were grown at 30 °C in nutrient broth (containing g/L: “Laboratory-Lemco” powder, 1.0; yeast extract, 2.0; peptone, 5.0; sodium chloride, 5.0) and stored at 4 °C after growth on nutrient agar (containing (g/L): “LaboratoryLemco” powder, 1.0; yeast extract, 2.0; peptone, 5.0; sodium chloride, 5.0; agar, 15.0). Media and Culture Conditions. Preparation of Inoculum. The inoculum was prepared in 250 mL Erlenmeyer flasks containing 30 mL of sterile nutrient broth (which were inoculated with single colonies from the nutrient agar plates grown overnight). A total of 140 mL of inoculum was prepared for inoculation into the fermenters. Flasks were incubated at 30 °C for 24 h on a rotary shaker at 250 rpm. Fermentation (2 L). A semidefined P(3HB) production medium, Kannan and Rehacek medium,20 was used with a slight modification (in g/L): glucose, 20; Difco yeast extract, 2.5; potassium chloride, 3; ammonium sulfate, 5.0; 100 mL of defatted soybean dialysate (prepared from 10 g of defatted soybean meal in 1000 mL of distilled water for 24 h at 4 °C). The production of P(3HB) in Bacillus cereus SPV was carried out in 2 L fermenters with a 1.4 L working volume. The fermenters were sterilized with 1 L distilled water and the salts were dissolved in 400 mL of distilled water and autoclaved separately. The autoclaved salts were added aseptically to the fermenters and the pH was set to 6.8 using 1 N NaOH before inoculation with the 24 h inoculum. For impeller speed studies, the impeller speed was set to and maintained at 500, 250, 125, and 50 rpm through out the fermentation for the respective runs. For pH studies, the pH was adjusted to and maintained at 3.0, 6.8, and 10.0. The % DOT was set to 100 before each run. Other fermentation parameters, such as carbohydrate concentration and optical density, for measuring cell growth, were monitored throughout the run. A constant airflow rate of 1.0 vvm and a constant

Figure 1. The (a) 1H and (b) 13C spectra of the P(3HB) polymer obtained from Bacillus cereus SPV when grown on glucose as the sole carbon source.

temperature of 30 °C were maintained. A polarographic oxygen-sensing probe was used to measure % DOT (Ingold, Mettler-Toledo Ltd., Beaumont Leys, Leicester, U.K.). Antifoam (FG-10; Dow corning, Edison, NJ) at a concentration of 1:10 (v/v) in water was added when necessary. Samples were withdrawn aseptically at fixed intervals to determine the amount of P(3HB) using GC. Determination of Carbohydrate Concentration. Samples were collected from the fermenter at 4 h intervals to determine carbohydrate concentration. The total carbohydrate content was determined by the Phenol-Sulfuric Acid Method.21 Extraction of P(3HB). A total of 1 g of freeze-dried cells was treated with a solution containing 50 mL of chloroform and 50 mL of a 30% sodium hypochlorite solution in water. After treatment for 1 h at 37 °C, the cells were centrifuged at 2500 g for 10 min. This resulted in three separate phases. The bottom chloroform phase containing P(3HB) was recovered and the polymer recovered by reprecipitation with 10 volumes of ice-cold methanol. NMR. 13C and 1H NMR analysis was carried out to identify the polymer chemical structure. The sample for analysis was prepared by dissolving 20 mg of the polymer in 1 mL of deuterated chloroform (CDCl3). The analysis was carried out at Department of Chemistry, UCL, U.K.

Production of Poly(3-hydroxybutyrate)


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Figure 2. Growth and P(3HB) accumulation of Bacillus cereus SPV in a 2 L fermenter with an impeller speed of 500 rpm. Changes in pH (blue triangle), optical density (black circle), carbohydrate concentration (green diamond), and dissolved oxygen tension (brown triangle) were monitored when Bacillus cereus SPV was grown in Kannan and Rehacek medium. All the measurements were carried out in duplicates and the standard deviation was (0-1%.

Figure 3. (a) Growth and P(3HB) accumulation of Bacillus cereus SPV in a 2 L fermenter with an impeller speed of 250 rpm. Changes in pH (blue triangle), optical density (black circle), PHB, % dry cell weight (red star), carbohydrate concentration (green diamond), and dissolved oxygen tension (brown triangle) were monitored when Bacillus cereus SPV was grown in Kannan and Rehacek medium. All the measurements were carried out in duplicates and the standard deviation was (0-1%. (b) Temporal variation of polymer accumulation in Bacillus cereus SPV in a 2 L fermenter with an impeller speed of 250 rpm.

Results Nuclear Magnetic Resonance (NMR). Bacillus cereus SPV accumulates P(3HB) when grown in the Kannan and Rehacek medium, using glucose as the sole carbon source. For confirmation of the chemical structure of the polymer, NMR analysis

was carried out using the polymer dissolved in deuterated chloroform. The 1H and 13C spectra of the polymer are shown in Figure 1. The 1H NMR spectral analysis revealed the presence of 3, 2, and 1 protons at chemical shifts 1.2, 2.4-2.6, and 5.3 ppm,

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Figure 4. (a) Growth and P(3HB) accumulation of Bacillus cereus SPV in a 2 L fermenter with an impeller speed of 125 rpm. Changes in pH (blue triangle), optical density (black circle), PHB, % dry cell weight (red star), carbohydrate concentration (green diamond), and dissolved oxygen tension (brown diamond) were monitored when Bacillus cereus SPV was grown in Kannan and Rehacek medium. All the measurements were carried out in duplicates and the standard deviation was (0-1%. (b) Temporal variation of polymer yield in Bacillus cereus SPV in a 2 L fermenter with an impeller speed of 125 rpm.

respectively (Figure 1a). The molecular structure of the polyester, as indicated by chemical shifts, includes a -CH2-CHbackbone with a CH3 group as shown in Figure 1a. The 13C NMR spectrum of the polymer on the other hand showed chemical shifts at 19.74, 40.84, 67.64, and 169.07 ppm, which showed the presence of CH3, CH2, CH, and CdO groups, respectively (Figure 1b). Effect of Impeller Speed on P(3HB) Production in Bacillus cereus SPV. Four different fermentations were carried out in 2 L stirred tank reactors using the modified Kannan and Rehacek medium for PHA production.20 Glucose at 20 g/L was used as the carbon source. Different impeller speeds of 500 (Figure 2), 250 (Figure 3), 125 (Figure 4), and 50 rpm were used. The initial pH of the production medium was adjusted to 6.8 in all cases. The cultures were grown for 72 h and 10 mL samples were collected at intervals of 4 h to carry out GC analysis to quantify P(3HB) yield. Figure 2 shows the profile of a 2 L fermentation using an impeller speed of 500 rpm. During this fermentation, the cells were found to grow steadily and reach stationary phase by the 30th hour. It was observed that relative to the other fermentions in this study, a very high value of OD, that is, 5, was achieved.

The Dissolved Oxygen Tension (% DOT) levels dropped from 100% at the beginning of the fermentation to 30% at the 20th hour and then rose back to 100 after 40 h. The fermentation profile also showed that the carbohydrate concentration in the media dropped down from 20 g/L to 5 g/L by the 37th hour, indicating a good level of utilization of the carbon source. The pH dropped from 6.8 at the beginning of the fermentation to a very low value of 2.5 by the end of the run. However, the samples collected at 4 h intervals showed that there was no P(3HB) production at all during this fermentation. Figure 3a represents the profile of a 2 L fermentation with an impeller speed of 250 rpm. In this case, the cells entered stationary phase much earlier than the previous condition (by the 22nd hour of growth). A maximal OD of 2.7 was achieved, indicating lesser growth than the previous condition. P(3HB) formation began after the seventh hour of fermentation. The onset of P(3HB) accumulation coincided with a drop in % DOT levels. The % DOT levels began to rise again after 48 h of growth. The carbohydrate concentration began to decrease with the onset of P(3HB) formation and decreased from 20 to 4 g/L by the 43rd hour. At the beginning of the fermentation, the pH was set to 6.8, and by the eighth hour, a pH minimum of 4.5



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Figure 5. (a) Growth and P(3HB) accumulation of Bacillus cereus SPV in a 2 L fermenter set to and maintained at a pH of 6.8. Changes in pH (blue triangle), optical density (black circle), PHB, % dry cell weight (red star), carbohydrate concentration (green diamond), and dissolved oxygen tension (brown diamond) were monitored when Bacillus cereus SPV was grown in Kannan and Rehacek medium. All the measurements were carried out in duplicates and the standard deviation was (0-1%. (b) Temporal variation of polymer yield in Bacillus cereus SPV in a 2 L fermenter set to and maintained at a pH of 6.8.

was reached. P(3HB) accumulation continued to increase during the fermentation and reached a maximum of 29% dry cell weight at 48 h of growth. Once maximal polymer concentration was achieved, it remained constant and no further degradation was observed (Figure 3b). Figure 4a is the fermentation profile of a 2 L fermenter with an impeller speed of 125 rpm. During this fermentation, the cells entered their stationary phase at the 48th hour. A maximum OD of 4.2 was observed and in this case the % DOT levels reached a minimum of 10% at 24 h and then rose to 100% by the 48th hour. Like in the previous fermentation, the initiation of polymer production coincided with a corresponding decrease in the glucose concentration, which kept decreasing from 20 to 7 g/L until the 48th hour. The pH dropped from 6.8 to a minimum of 4.5 within the 15th hour. P(3HB) formation started during the sixth hour of growth and reached a maximum of 34% dry cell weight at 48 h, after which the polymer content remained constant without degradation (Figure 4b). Growth of the culture was poor at 50 rpm and P(3HB) production was negligible (results not shown). Effect of pH on P(3HB) Production in Bacillus cereus SPV. Three different batch fermentations of Bacillus cereus SPV were carried out in 2 L stirred tank reactors to study the effect of pH on P(3HB) production. The impeller speed was kept constant at 125 rpm in all cases since this was found to be the optimum impeller speed in the study described above. The pH

of the production medium was maintained at 3, 6.8, and 10, respectively. Figure 5a shows the profile of the 2 L fermentation where the pH was maintained at 6.8 throughout the run. During this fermentation, the cells were found to grow steadily and reach their stationary phase at the 24th hour. A maximum OD of 5.9 was achieved in this run, the highest value observed in all the fermentations carried out in this study. The % DOT level dropped from 100 to 20 within 40 h and remained at that level for the remainder of the run. The carbohydrate concentration dropped from 20 to 7 g/L within 48 h. A maximum P(3HB) yield of 23% dry cell weight was achieved at 45 h. However, the polymer started degrading after 50 h at an average rate of 0.25 mg/mL/hour (Figure 5b). Figure 6a shows the fermentation profile of a 2 L fermenter when the pH was set to and maintained at 3.0 throughout the run. During this fermentation, the cells took about 10 h to adapt to the acidic pH and the maximum optical density was very low, i.e., 0.05. Hence, pH 3.0 is not a favorable environment for cell growth. The %DOT levels remained constant at and around 100% during the course of the run, as expected, since minimal cell growth had occurred. Surprisingly, the carbohydrate concentration dropped down from 20 g/L to 12.5 g/L by the 70th hour. Figure 7a represents the fermentation profile of a 2 L fermenter where the pH was set and maintained at 10.0

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Figure 6. (a) Growth and P(3HB) accumulation of Bacillus cereus SPV in a 2 L fermenter set to and maintained at a pH of 3.0. Changes in pH (blue triangle), optical density (black circle), PHB, % dry cell weight (red star), carbohydrate concentration (green diamond), and dissolved oxygen tension (brown diamond) were monitored when Bacillus cereus SPV was grown in Kannan and Rehacek medium. All the measurements were carried out in duplicates and the standard deviation was (0-1%. (b) A temporal variation of polymer yield in Bacillus cereus SPV in a 2 L fermenter set to and maintained at a pH of 3.0.

throughout the run. In this profile, a maximum OD of 1.22 was achieved. The % DOT levels remained constant until the 24th hour, where the cells were possibly adapting to the high pH conditions. The % DOT levels then dropped from 100 to 5% by the 64th hour, the initiation of the descent was coincidental with a rapid increase in OD levels indicating rapid cell growth. Also, as expected, the carbohydrate concentration dropped from 20 to 7 g/L within 50 h alongside the drop in the % DOT value. P(3HB) accumulation was low and amounted to a maximum value of 16% dry cell weight, achieved at 48 h. This was followed by immediate degradation of the polymer at an average rate of 0.3 mg/mL/h, comparable to the degradation rate found at pH 6.8 (Figure 7b). A summary of the fermentation conditions maintained in this study, amount of maximum P(3HB) accumulated and time of maximal P(3HB) accumulation are tabulated in Table 1.

Discussion P(3HB) production using Bacillus cereus SPV was carried out using the unique Kannan and Rehacek medium containing glucose as the sole carbon source under different fermentation conditions. This is a nitrogen limiting medium and has been shown previously to promote P(3HB) accumulation.1 The purified polymer was characterized using NMR spectroscopy. This data confirmed that the chemical structure of the polymer was that of P(3HB). In addition, the characteristic signals for

other hydroxyalkanoic acids were absent confirming the homopolymeric nature of the polymer from Bacillus cereus SPV. Ever since P(3HB) was discovered in 1926 by Lemoigne in Bacillus M, there has been a large amount of research on the production and characterization of this polymer. However, most of this research has been carried out on Gram negative bacteria. As discussed in the introduction, polymer production using Gram positive bacteria have an advantage for medical applications since these bacteria lack the lipopolysaccharides (LPS) present in Gram negative bacteria.17 Hence, in this work we have made one of the first attempts to understand the effect of variation in fermentation parameters on the production of P(3HB) using Bacillus sp. The two main factors we have focused on in this study are impeller speed and pH. At a high impeller speed of 500 rpm (Figure 2), initially, the carbohydrate consumption rate was relatively slow followed by a rapid decrease in carbohydrate levels. The cells grew well under these conditions, however, there was no production of P(3HB) throughout the fermentation. This might be due to changes in cellular metabolism induced by the shear related to high stirrer speed. The available resources were utilized mainly for growth rather than for polymer production and possibly toward other stress induced metabolic pathways. When the effect of impeller speed on lipase production was studied in a Brazilian wild type strain of Yarrowia lipolytica, it was observed that at a high impeller speed of 400 rpm, a decrease in the maximum lipase activity was observed due to the shear stress promoted



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Figure 7. (a) Growth and P(3HB) accumulation of Bacillus cereus SPV in a 2 L fermenter set to and maintained at a pH of 10.0. Changes in pH (blue triangle), optical density (black circle), PHB, % dry cell weight (red star), carbohydrate concentration (green diamond), and dissolved oxygen tension (brown diamond) were monitored when Bacillus cereus SPV was grown in Kannan and Rehacek medium. All the measurements were carried out in duplicates and the standard deviation was (0-1%. (b) A temporal variation of polymer yield in Bacillus cereus SPV in a 2 L fermenter set to and maintained at a pH of 10.0. Table 1. Summary of the Fermentation Conditions Maintained, Amount of Maximum P(3HB) Accumulated, and Time of Maximal P(3HB) Accumulation in this Studya impeller condition speed (rpm) 1 2 3 4 5 6 a

500 250 125 125 125 125

pH unbuffered unbuffered unbuffered 6.8 3.0 10.0

maximum time of P(3HB) maximum polymer yield (% dcw) accumulation (hrs) ND 29 34 23 21 16

ND 48 48 45 25 48

ND: not detected.

by the impellers.22 Studies carried out on KluyVeromyces marxianus also showed that, at revolution speeds beyond 450 rpm, inulinase production decreased. Bernard et al., 2005, found that the sensitivity to shear stress is an intrinsic characteristic of a microorganism and is a limiting factor in the optimization and scale up processes.23 In contrast, almost no growth of the bacteria was observed in the fermentation with an impeller speed of 50 rpm Hence, at this speed, there is not enough mixing of the culture to provide the essential minimal oxygen levels and nutrients to allow growth. P(3HB) polymer production was thus observed only under two impeller speed conditions, that is, 125 and 250 rpm. The highest accumulation of P(3HB) occurred in the fermenter with an impeller speed of 125 rpm, 34% dry cell weight of P(3HB), whereas at 250 rpm, 29% dry cell weight of P(3HB) was

accumulated. P(3HB) polymer accumulation reached its peak during the stationary phase in both the fermentations, indicating nongrowth related accumulation. The pH of the cultures in both these fermentations dropped from 6.8 to 4.5 during the course of the fermentation. As is clearly evident from the fermentation profiles (Figures 3a and 4a), the main difference in the two fermentations were in the variation of the OD value and the levels of P(3HB) accumulation. The culture in the fermentation at 250 rpm grew at a much faster rate and reached stationary phase much earlier (24 h), whereas in the fermentation with an impeller speed of 125 rpm, the culture grew relatively slower and reached stationary phase after 48hrs of growth. This result indicates that a relatively slower cell growth rate observed at 125 rpm impeller speed results in enhanced P(3HB) production. Fast cell growth possibly results in the utilization of all the available resources for growth leaving reduced resources for polymer production. However, a relatively slower growth rate allows the division of resources toward cell growth and polymer production, an ideal balance. In addition, it was evident that the % DOT level in the 125 rpm fermentation was higher than that in the 250 rpm fermentation (10% vs almost 0%), during P(3HB) production. Hence, at 125 rpm impeller speed, the balance between cell growth and the % DOT has led to a relatively higher % DOT value. We can thus postulate that a certain minimum level of % DOT is desirable for PHA production by Bacillus cereus SPV. In the 500 rpm fermentation, the minimum % DOT was much higher (30%), resulting in no P(3HB) production. Thus, beyond a certain maximum value, higher % DOT values are possibly

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deleterious for P(3HB) production. Gong et al., 2008, found a 12-fold increase in PHA production when the dissolved oxygen was decreased from 40 to 20%.24 These observations must, however, also include the effects of shear force due to changing impeller speed. Thus, both % DOT and shear force affect P(3HB) production in Bacillus cereus SPV. This is the first detailed study of the effect of impeller speed on the production of P(3HB) using a Bacillus sp. Another interesting observation was that once the maximum level of P(3HB) had been accumulated (Figures 3b and 4b), the polymer concentration remained constant up to 72 h without degradation. This lack of P(3HB) degradation observed in Bacillus cereus SPV has been attributed to the acidic environment in the fermentation during maximal P(3HB) production. The pH was found to drop from 6.8 to 4.5 during the fermentation, possibly due to the production of acetic acid, lactic acid and pyruvic acid.25 This finding was similar to that made by Kominek and Halvorson, 1965, who observed that a low pH environment inhibits polymer degradation and spore formation in Bacillus cereus T.26 Valappil et al., 2007, also monitored the fermentation run for sporulation using the SchaefferFulton method and no sporulation was observed.17 However, this observation was in contrast to reports by Wu et al., 2001, who observed that low dissolved oxygen levels led to the degradation of P(3HB) in Bacillus JMa5, to be utilized for sporulation.16 Because pH is thought to be such a crucial parameter in P(3HB) production by Bacillus, the second part of this study looked at the effect of pH on P(3HB) production in Bacillus cereus SPV. The different fermentations with production medium buffered to pH 3, 6.8, and 10, respectively, led to a maximum P(3HB) yield of 21, 23, and 16% dcw, respectively. Hence, maximum yield was obtained at pH 6.8. This was followed by polymer degradation in all three fermentations (Figures 5b, 6b, and 7b). However, at pH 3, the average rate of degradation was found to be much slower (0.06 mg/mL/ h), Figure 6b, as compared to that at pH 6.8 (0.25 mg/mL/ h), and pH 10 (0.3 mg/mL/h). This indicates that lower the pH of the medium, the lower is the rate of degradation of the polymer. In fact, in the previous fermentations where the pH was not controlled and allowed to fall to a value of 4.5, the polymer produced did not show any further degradation (Figures 3b and 4b). The low rate of degradation observed during the fermentation at pH 3 is possibly due the added stress encountered by the bacteria, a nonacidophile, being forced to grow at pH 3. These observations confirm our previous hypothesis, that low pH conditions during maximal P(3HB) production leads to the lack of polymer degradation in Bacillus cereus SPV.19 This is possibly due to the fact that P(3HB) degradation leads to the production of 3-hydroxybutyrate, an acid, hence by the law of mass action, under low pH conditions the polymer degradation is inhibited. This feature of Bacillus fermentations will be crucial during the commercial production of P(3HB) using this genus. Further, looking at other features of the pH stat fermentations, where pH was maintained at 3.0, the pH was too acidic to support any reasonable cell growth and a maximum optical density of 0.05 was achieved as compared to 5.8 and 1.2 achieved during the fermentations maintained at pH 6.8 and 10, respectively. As expected, there was much lower glucose consumption and negligible % DOT decrease in the pH 3 fermentation when compared to the other two pH stat fermentation systems. The drop in carbohydrate levels could be attributed to acidic hydrolysis of the glucose. P(3HB) production did occur

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even under these stressful conditions and the highest amount of P(3HB) was produced at 25 h which amounted to 21% of the dry cell weight. The cells grew best at pH 6.8, as is evident from the high maximum OD value obtained, however, even at pH 10 the cells grew relatively well after an initial lag phase. The cell growth profile was however quite unusual at pH 10, with a sudden decrease in the OD value once the maximum value had been achieved, indicating rapid cell death. Maximum polymer accumulation was observed at 25 h in the pH 3 fermentation (21% dcw), whereas both the 6.8 (23% dcw) and 10 (16% dcw) pH-stat fermentations exhibited maximal accumulation around 45-48 h.

Conclusions Thus, in conclusion, this study has led to an enhanced knowledge and understanding of the effect of impeller speed and pH on the production of P(3HB) using Bacillus cereus SPV. It has been found that, for 2 L fermentations, an impeller speed of 125 rpm is the optimal condition leading to an ideal balance of cell growth and polymer production. Further enhancement of impeller speed leads to a much more rapid growth rate leading to a relatively imbalanced condition and lower levels of polymer production. Also, it has been found that polymer degradation can be prevented in Bacillus fermentations under uncontrolled pH conditions, which led to final pH values as low as 4.5, rather than in pH stat fermentations. This observation can revolutionize large production of the polymer using Bacillus sp. at an industrial scale. This study is, thus, a significant first step forward toward the development of an optimized production of P(3HB) using the genus Bacillus. The lack of LPS in the polymer produced by this genus and the ability of this genus to utilize a variety of carbon sources, possibly even lignocellulosics, indicates a bright future for the commercialization of this fermentation process. Acknowledgment. S.P. was supported financially by the University of Westminster, London, U.K.

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Effect of Impeller Speed and pH on the Production of Poly(3

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