Journal of Applied Microbiology ISSN 1364-5072

ORIGINAL ARTICLE

Potential of Bacillus sp. to produce polyhydroxybutyrate from biowaste

T. Kumar1,2, M. Singh1,2, H.J. Purohit3 and V.C. Kalia1
1 Microbial Biotechnology and Genomics, Institute of Genomics and Integrative Biology (IGIB), CSIR, Delhi University Campus, Delhi, India 2 Department of Biotechnology, University of Pune, Pune, India 3 Environmental Genomics Unit, National Environmental Engineering Research Institute (NEERI), CSIR, Nehru Marg, Nagpur, India

Keywords Bacillus, biowaste, pea-shell, polyhydroxybutyrate, polyhydroxyalkanoate. Correspondence Vipin C. Kalia, Microbial Biotechnology and Genomics, Institute of Genomics and Integrative Biology (IGIB); CSIR, Delhi University Campus, Mall Road, Delhi 110007, India. E-mail: vckalia@igib.res.in, vc_kalia@yahoo.co.in

Abstract Aim: To test the Bacillus strains for their abilities to produce polyhydroxybutyrate (PHB) from different sugars and biowaste (Pea-shells). Methods and Results: Six Bacillus strains were checked for their ability to produce PHB from GM2 medium supplemented with different sugars at the rate of 1% (w v) and from biowaste and GM2 (BW : M) combinations (3 : 7, 1 : 1, 7 : 3). Glucose supplemented GM2 medium resulted in maximum PHB production of 435 mg l)1 constituting 3162% w w of the total cell dry mass. Substituting GM2 medium to the extent of 50% with biowaste (pea-shell slurry) resulted in 9451205 mg l)1 PHB (5565% w w). Optimization for additional nitrogen supplementation, inoculum size resulted in a nal PHB production of 30103370 mg l)1 equivalent to 300 g kg)1 biowaste (dry wt). Conclusion: The Bacillus strains were able to produce PHB from biowaste (Pea-shells) as cheap source of substrate. Signicance and Impact of the Study: This is the rst report on usage of pea-shells as feed for PHB production, opening new possibilities for its use for production of PHB and Bacillus as potential candidate for the purpose.

2008 1484: received 29 August 2008, revised 11 October 2008 and accepted 8 November 2008
doi:10.1111/j.1365-2672.2009.04160.x

Introduction For sustainable production systems, the focus has shifted from petroleum based plastics, which are biologically inert to biodegradable plastics. While the conventional synthetic plastics contribute to serious pollution problems, bacterial polyhydroxybutyrate (PHB), a class of polyhydroxyalkanoates (PHA) has attracted much attention as environmentally degradable plastics. On the basis of a very high PHA content (7075% w w) in dry cell mass (DCM) from microbial fermentation of pure glucose and organic acids, it is estimated that the high production cost is mainly attributed to the carbonaceous raw materials (>45%) and polymer recovery (>26%) (Du et al. 2004). These are some of the important limitations still persisting in the bulk production of bioplastics (Lee 2006). Much effort has been devoted to reduce the cost of PHA production by the isolation of better bacterial strains (Singh and Mallick 2008), development of

more efcient fermentation and recovery processes and utilization of biowastes as carbonaceous raw materials (Akiyama et al. 2003; Reddy et al. 2003; Du et al. 2004). Using genomic approach, microbes such as Burkholderia fungorum, Novosphingobium aromaticivorans and Microbulbifer degradens with potential to produce H2 and PHA and ability to grow on wastes have been reported (Kalia et al. 2003). Approximately 800010 000 tonnes per year of pea shells are disposed of as waste at a fruit and vegetable processing unit in Delhi. Production of biofuels such as H2 and CH4 from pea-shells as feed has already been reported (Kalia and Joshi 1995). The present work was carried out to evaluate and optimize the PHB production from pea-shell biowaste with the help of Bacillus strains, some of which were recently reported to produce PHA and H2 by dark fermentative process (Porwal et al. 2008). This study will help to further enhance the efciency of biodegradation process and reduce pollution.
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Material and methods Isolation and selection of micro-organisms Bacillus cereus EGU3 (DQ487039) was isolated from contaminated pickle on Nutrient Agar (NA, nutrient medium HiMedia, 13 g l)1, Agar 2%) plates at pH 12 and 05% NaCl. Bacillus cereus EGU43 (DQ508969), B. cereus EGU44 (DQ508970), B. thuringiensis EGU45 (DQ508971) and Bacillus sp. EGU75 (EF633209) were isolated from sludge from Madras Renery Limited Efuent Treatment Plant (ETP), Chennai, on NA plates at pH 7 and 3% NaCl. Bacillus cereus EGU520 (DQ508978) was isolated at pH 10% and 05% NaCl from fermenting potato peels inoculated with cattle dung. Accession numbers of the isolates used in this study and their corresponding 16S rDNA gene sequences can be viewed at http:// www.ncbi.nlm.nih.gov/. All the strains were subsequently maintained on NA at pH 70% and 05% NaCl. The six strains presented here are among the high PHB producers selected out of 100 strains screened for this characteristic. Bacterial growth and PHB production on different sugars Inoculum for PHB production was prepared by inoculating single colony from NA plate into 10 ml Nutrient broth, at 37C for 1620 h at 200 rev min)1. The inoculum size varied from 1 lg cell protein ml)1 medium to 10 lg cell protein ml)1 medium for sugar supplemented GM2 medium (g l)1 of distilled water: Yeast extract, 10; K2HPO4, 10; MgSO47H2O, 05; Glucose, 10; pH-72) (Jan et al. 1996) and 1, 10, 100 and 1000 lg cell protein ml)1 of GM2 medium and biowaste combinations. PHB production was monitored for all the six strains on GM2 medium (400 ml in 1000 ml conical asks) supplemented separately at the rate of 1% (w v) with ve different sugars (glucose or fructose or maltose or sucrose or lactose), and incubated at 37C at 200 rev min)1 Aliquots of 200 ml were analysed for DCM and PHB after 24 and 48 h of incubation. Growth of different bacterial strains on GM2 medium supplemented with 1% (w v) different sugars (listed above) and minimal medium (g l)1 of distilled water): Na2HPO4, 60; KH2PO4, 30; NaCl, 05; NH4Cl, 10; MgSO4, 1 mmol l)1 (1 ml); CaCl2, 01 mmol l)1 (1 ml); pH-70 supplemented with 1% (w v) glucose or lactose or sucrose incubated at 37C at 200 rev min)1 for 48 h was monitored at 600 nm on Perkin Elmer Lambda 35 spectrophotometer. PHB production from biowaste Green pea shells (10% total solids) were used in the experiments. Pea shell slurry (PSS), 4 l at 2% TS was
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inoculated with acidogens (fresh cattle dung slurry; 3% TS) (1 vol.) to slurry (9 vol.) and incubated at 37C for 2 days (Kalia and Joshi 1995). Slurry obtained at this stage was sieved and the ltrate (25 l) was centrifuged at 5600 g for 20 min. The centrifuged ltrate was stored at 4C. Eight hundred milliliters of the following combinations of the centrifuged PSS and GM2 medium (BW : M = 3 : 7, 1 : 1 and 7 : 3) were tested for PHB production. The pH of the medium was adjusted to 72 with 1N NaOH at the time of inoculation with different Bacillus strains at the rate of 10 lg cell protein ml)1 of the combination (BW : M) medium. These combinations were compared with BW : M supplemented with casein enzyme hydrolysate (CEH), pancreatic digest of casein (equivalent to tryptone) at the rate of 02 g l)1. Further optimization for the effect of CEH on PHB production from BW : M (1 : 1) was done by adding it at the rate of 02, 04 and 08 g l)1 and compared with control (no supplementation). BW : M (1 : 1) supplemented with 04 g l)1 CEH was further tested for the effect of inoculum size on PHB production by adding them at the rate of 10, 100 and 1000 lg cell protein ml)1 of feed. All the incubations were done at 37C for 96 h at 200 rev min)1. Aliquots of 200 ml for biomass estimation and PHB analyses were withdrawn regularly at 24 h intervals. Analytical procedure Samples (200 ml) were analysed for DCM and PHB production. Aliquots (200 ml) were centrifuged at 5600 g for 20 min. The pellet was washed with 10 ml saline solution (NaCl, 09 g l)1) and recentrifuged. The pellet was dried at 85 C for 36 h and weighed to estimate DCM. Samples for PHB analysis were processed and esters of propanol and b-hydroxylacids from PHB hydrolysis were analysed with GC tted with 10% Reoplex 400 (Riis and Mai 1988). Total Solids (TS%) were estimated by heating a sample of pea shells at 110C for 24 h. Gravimetric estimation of the polymer yield was done from dry cell mass by extraction of the polymer using chloroform and methanol (Khardenavis et al. 2007). This analysis was done only while estimating the effect of inoculum size on PHB production. Results Sugar utilization and PHB production The growth of six Bacillus strains varied considerably on different sugar supplemented GM2 medium. The growth of all the strains was high on glucose, fructose and maltose, moderate on sucrose and relatively poor on lactose

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(a) 25 OD at 600 nm 2 15 1 05 0 (b) 25 OD at 600 nm 2 15 1 05 0 (c) 25 OD at 600 nm 2 15 1 05

0 (d) 25 OD at 600 nm 2 15 1 05 0 (e) 2 OD at 600 nm 15 1 05 0 0 2 4 6 8 10 24 26 28 30 34 48 Time (h)

Figure 1 Growth patterns of Bacillus strains on medium (GM2) supplemented with different sugars. A: Glucose; B: Fructose; C: Maltose; D: Lactose; E: Sucrose inoculated with: Bacillus sp. EGU75 h, B. cereus EGU3 r, B. cereus EGU43 d, B. cereus EGU44 , B. cereus EGU520 4 and B. thuringiensis EGU45 ).

medium containing lactose or sucrose or glucose revealed very poor growth (specic growth rate ranging from 019 to 039 h)1) on lactose. Hence, the slow growth observed on lactose supplemented GM2 medium was perhaps due to other carbon sources present in yeast extract (one of the components of GM2). The growth on minimal medium with glucose or sucrose was in the normal range (Specic growth rate ranging from 057 to 063 h)1 and 015 to 078 h)1 respectively). Hence, we concluded that these strains have the ability to utilize multiple sugars, as carbonaceous source. PHB producing abilities of different Bacillus strains varied from 190 to 435 mg l)1 on glucose supplemented GM2 medium. PHB constituted 3162% of the total DCM (Table 1). It may be remarked here at this stage that PHB production was recorded only up to 48 h. Bacillus cereus EGU44 and B. thuringiensis EGU45 could be rated among the high PHB producers. With the change of sugar from glucose to fructose, there was a dramatic change in the PHB production yield of all the strains. Except for Bacillus sp. EGU75, where there was a marked enhancement in its PHB production from 190 mg l)1 with glucose to 385 mg l)1 with fructose, i.e. an improvement of two fold. It also led to a higher yield of PHB which comprised of 75% of the DCM. All other strains showed a decline in PHB production to the extent of 4497%. The maximum reduction in PHB production yield was recorded with B. cereus EGU3, where the PHB production was practically negligible. On switching on to maltose as sugar supplement, the abilities of all the 6 Bacillus strains to produce PHB showed further decline, so much so that only 843% of the PHB could be realized in comparison to that with glucose. The only exception was B. cereus EGU43, which showed only 36% loss with a nal PHB production of 220 mg l)1 and a yield of 48% w w. A similar pattern of decline in PHB production and yield was seen with all the strains on sucrose as sugar supplement. Incidentally, B. cereus EGU44 and B. cereus EGU520 showed almost similar PHB production on fructose and sucrose. Although these strains have abilities to grow and convert sugars to PHB with different efciencies, however glucose turned out to be the best sugar supplement followed by fructose. PHB could not be detected on lactose containing GM2 medium, where growth itself was very poor. PHB production from biowaste Production of PHB by six Bacillus strains from combinations of BW : M in different ratios, with increasing contribution of biowaste is presented in Table 2. PHB production from BW : M in 3 : 7 ratio was in the range of 140650 mg l)1, which constituted 1247% w w of the
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(Fig. 1). Bacillus cereus EGU520 grew well on all the sugar supplemented medium. Bacillus cereus EGU3 could utilize glucose and fructose better than others whereas B. thuringiensis EGU45 and Bacillus sp EGU75 grew well on maltose and sucrose, respectively. Since Bacillus strains are not known to grow well on lactose as carbon source, a comparison of growth of all the 6 strains on minimal

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Table 1 Effect of sugar on polyhydroxybutyrate production by different Bacillus strains Organism Medium (GM2) supplemented with sugars (1% w v)
Medium (GM2) supplemented with sugars (1% w v) Glucose PHB (mg l)1) 435 415 350 345 325 190 Yield (% w w) 50 50 62 50 38 31 Fructose PHB (mg l)1) 175 205 90 195 10 385 Yield (% w w) 25 40 25 28 2 75 Maltose PHB (mg l)1) 70 185 65 220 90 15 Yield (% w w) 18 35 13 48 12 13 Sucrose PHB (mg l)1) 170 110 80 40 70 25 Yield (% w w) 35 29 21 12 33 13 Lactose PHB (mg l)1) nd nd nd nd nd nd Yield (% w w) na na na na na na

Organism Bacillus cereus EGU44 B. thuringiensis EGU45 B. cereus EGU520 B. cereus EGU43 B. cereus EGU3 Bacillus sp. EGU75

Values are based on two sets of experiments observed after 48 h of incubation. SD was less than 5%. nd, not detectable;na, not applicable.

Table 2 Production of polyhydroxybutyrate from biowaste by Bacillus strains

Biowaste*: Medium (BW : M) 3:7 1:1 7:3 3:7 1:1 7:3

Without casein enzyme hydrolysate

With casein enzyme hydrolysate

Organism Bacillus sp. EGU75 B. cereus EGU44 B. cereus EGU3 B. thuringiensis EGU45 B. cereus EGU520 B. cereus EGU43

PHB Yield PHB Yield PHB Yield PHB Yield PHB Yield PHB Yield (mg l)1) (% w w) (mg l)1) (% w w) (mg l)1) (% w w) (mg l)1) (% w w) (mg l)1) (% w w) (mg l)1) (% w w) 650 490 455 390 325 140 35 47 25 21 17 12 190 340 215 510 370 165 8 18 15 25 17 13 185 170 225 175 190 165 9 17 12 9 18 15 870 285 955 905 430 1485 47 21 66 54 39 72 705 945 930 1205 855 800 22 65 61 55 43 30 255 75 680 165 250 350 22 5 48 9 24 25

Values are based on two sets of experiments observed after 72 h of incubation. SD was less than 5%. *Pea shell slurry. GM2 medium.

DCM. In spite of reducing the contribution of medium (containing glucose) by 30% in the feed, we could observe that Bacillus strains were still able to utilize biowaste to same extent in the case of B. cereus EGU44, B. thuringiensis EGU45 and B. cereus EGU520. A 1434 fold increase in PHB production with B. cereus EGU3 and Bacillus sp. EGU75 on BW : M compared to that on GM2, respectively indicated that pea-shell waste was proving to be a good feed material. Bacillus cereus EGU43 was the only strain which showed a dramatic loss of 59% in PHB production on replacing 30% of the medium. Since the overall objective was to maximize the contribution of biowaste in the feed, we checked the abilities of the six Bacillus strains on higher BW : M ratio of 1 : 1 and 7 : 3. Increasing the contribution of waste from 30% to 50%, i.e. BW : M = 1 : 1 resulted in 13 to 24% increase in PHB production with B. cereus EGU520, B. cereus EGU43 and B. thuringiensis EGU45. Incidentally
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in these three cases, PHB yields were only marginally affected and continued to be in the range 13% to 25% w w, indicating thereby that the waste to PHB conversion efciency of these Bacillus strains was maintained. On the other hand, with the other three Bacillus strains Bacillus sp. EGU75, B. cereus EGU44 and B. cereus EGU3, an increased contribution of biowaste in the feed resulted in 3171% reduction in PHB production. It appears that there had been a metabolic shift from PHB pathway to other metabolism such that PHB yields reduced from 25% to 47% (w w) at BW : M = 3 : 7 to 818% at BW : M = 1 : 1 with Bacillus sp. EGU75, B. cereus EGU44 and B. cereus EGU3 (Table 2). Further increase in biowaste contribution to BW : M = 7 : 3 continued to show a decline in PHB production by B. cereus EGU44, B. thuringiensis EGU45 and B. cereus EGU520. The yields decreased to a very low level of 918% equivalent to 170 190 mg l)1. With the other three Bacillus strains, the PHB

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production was almost similar to that recorded at BW : M = 1 : 1 level. It may be concluded here that the ability of Bacillus strains to utilize biowaste for PHB production varies: Bacillus sp. EGU75, B. cereus EGU44 and B. cereus EGU3 were most efcient on BW : M = 3 : 7 level; B. thuringiensis EGU45 and B. cereus EGU520 found BW : M = 1 : 1 as conducive and B. cereus EGU43 proved to be the most efcient on GM2 medium supplemented with glucose. Effect of nitrogen supplementation on PHB production PHB biosynthesis is greatly affected by C : N ratio (Wang et al. 2007) and the improved production has been achieved by supplementation of a small amount of complex nitrogen source such as soybean hydrolysate. In view of these informations, the effect of CEH as nitrogen supplement 002% (w v) on different BW : M combinations was checked. It was found to greatly enhance the PHB production and yields to various extents (Table 2). At 30% level of biowaste in the feed (BW : M = 3 : 7), a 25 81% increase in PHB production accompanied by a net enhancement of 1260% in PHB yield compared to the non CEH supplemented BW : M (3 : 7) as feed was recorded. The only exception was B. cereus EGU44, where the PHB producing abilities declined from 490 to 285 mg l)1 on CEH supplementation. It was also accompanied by decrease in PHB yield from 47% (w w) to 21% (w w) (Table 2). Bacillus cereus EGU43 resulted in maximum PHB production of 1485 mg l)1 and a yield of 72% (w w) on CEH supplemented BW : M = 3 : 7 as feed. Its interesting to mention that this strain had a very low PHB production level of 140 mg l)1 on BW:M combination without CEH supplementation. At higher contribution of biowaste in the BW: M combinations as feed, supplemented with CEH proved effective up to BW : M = 1 : 1 level. The trend of improvement in PHB production could not be sustained at BW : M = 7 : 3 level in spite of CEH supplementation.

It may be remarked that at 70% biowaste contribution in the feed, CEH supplementation led to higher yields in most of the cases in comparison to nonsupplemented BW : M combination. An overall analysis of different combinations of BW : M, with CEH supplementation revealed that different Bacillus strains responded differently to these conditions. Bacillus cereus EGU43 proved best at BW : M = 3 : 7 combination; 1485 mg l)1, 72% w w PHB; B. thuringiensis EGU45; B. cereus EGU44 and B. cereus EGU3 gave best yields at BW : M = 1 : 1 combinations (9301205 mg l)1; 5565% w w PHB). Although B. thuringiensis EGU45 and B. cereus EGU44 also had their highest PHB yield at BW : M = 1 : 1 combinations, B. cereus EGU3 had its highest PHB yield at BW : M = 3 : 7 combination (955 mg l)1; 66% w w PHB). Since the overall objective was to maximize the contribution of biowaste in the feed, the following combinations of biowaste (BW : M with CEH supplementation at 1 : 1 level) and bacteria (B. cereus EGU44 and B. thuringiensis EGU45) were selected for further optimization. Optimization of nitrogen supplementation on PHB production The effect of concentration of CEH supplementation as nitrogen supplement seemed to be very clear cut on PHB production by Bacillus strains (Table 3). A comparison of CEH supplementation at 02, 04 and 08 g l)1 showed that there was a marked increase in PHB production in comparison to control and maximum yield was detected after 72 h of incubation. Maximum enhancement in PHB production was observed with 04 g l)1 (004% w v) supplementation, where the PHB production achieved was 19452075 mg l)1 and a yield of 7172% w w. These enhancements were 27- to 41-fold compared to control and 17- to 2-fold compared to 02 g l)1 CEH. A further addition of CEH at 08 g l)1 proved detrimental to PHB production since C : N ratio might have been dramatically

Table 3 Effect of supplemental nitrogen concentration on polyhydroxybutyrate production from biowaste*

Biowaste: GM2 Medium with casein enzyme hydrolysate (g l)1) Control PHB (mg l)1) 340 510 Yield (% w w) 18 25 02 PHB (mg l)1) 945 1205 Yield (% w w) 65 55 04 PHB (mg l)1) 1945 2075 Yield (% w w) 72 71 08 PHB (mg l)1) 745 630 Yield (% w w) 49 29

Organism Bacillus cereus EGU44 B. thuringiensis EGU45

Values are based on two sets of experiments observed after 72 h of incubation. The inoculum was 10 lg cell protein ml)1 for all cultivations. SD was less than 5%. *BW (Pea shell slurry) : M (GM2 Medium) = 1 : 1.
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reduced. The nal PHB yield of 630 and 745 mg l)1 were however, still better than the control. Effect of inoculum size on PHB production The initial observation on the effect of inoculum size on PHB production was recorded on GM2 medium, where 1 lg cell protein ml)1 was found to be equally effective in comparison to 10 lg cell protein ml)1. However, with biowaste in the medium a preliminary assay had shown that 10 lg cell protein ml)1 was better than 1 lg cell protein ml)1. An increase in inoculum size from 10 lg cell protein ml)1 to 100 lg cell protein ml)1 of feed helped to improve the PHB production to 30103370 mg ml)1 i.e. an improvement of 3242% (Table 4). Through gravimetric analysis 22502500 mg PHB l)1 could be extracted, which was equivalent to 75% of that recorded with GC analysis. A further increase in inoculum size to 1000 lg cell protein ml)1 did not lead to any signicant change in PHB yield (Table 4). It may be concluded that although the PHB production has improved, however, the PHB metabolic efciency in terms of yield had not been maintained at a high level of 7172% (w w) observed at low microbial populations. The effort for 10-fold greater inoculum size was not compensated by an equal increase in PHB production. It reected that there was a potential for converting waste into PHB but we may have to look for some other parameters also to further improve the yields. Discussion In spite of the large number of environmental benets associated with bioplastics, production cost continues to be the main issue. It has been estimated that around 45% of the cost of production is associated with carbonaceous raw materials (Du et al. 2004). It is thus worth concentrating our efforts on searching for a cheaper raw material. Biowastes rich in organic matter, particularly carbohydrates and fatty acids are the obvious targets for

such alternatives as has been widely attempted by a large number of researchers worldwide. An equally important component is the micro-organism involved in this bioconversion. A large number of organisms have been reported to produce PHA from pure substrates and even biowastes. We have chosen pea-shells as biowastes, due to their availability in large quantities (800010 000 tonnes per annum) and their relatively high convertibility into H2 and CH4 (Kalia and Joshi 1995). Production of PHB from such a biowaste is expected to increase biodegradation efciency by providing value added products. On the other hand, we have chosen Bacillus strains since our screening of over 100 different microbial strains belonging to different genera and species, revealed Bacillus species among the high PHB producers. We have previously also shown Bacillus strains to produce H2 from glucose and biowastes (Kalia and Purohit 2008). Bacillus has the potential to be likely candidates in the future for the production of PHB (Labuzek and Radecka 2001; Borah et al. 2002) and H2 because of its unique properties such as abilities to utilize cellulose as a carbon and energy source, produce laccase, survival under unfavourable conditions, etc. (Kalia and Purohit 2008; Porwal et al. 2008). A survey of the published literature available in public domain reveals that B. megaterium could produce 176 g l)1 PHB (52% w w) at 48 h incubation period from date syrup beet molasses (Omar et al. 2001). In our work, being reported here, we have achieved a PHB production level of 337 g l)1 (41% w w) at 72 h incubation period from pea-shells as waste. Although sugar, starch and oil based wastes such as rice bran (Huang et al. 2006), vegetable oils (Kahar et al. 2004; Thakor et al. 2005), palm kernel oil (Bhubalan et al. 2008), whey, food scraps, agro industrial byproducts (Khardenavis et al. 2007), sugarcane liquor medium (Jiang et al. 2008) and palm oil mill efuent (POME) (Alias and Tan 2005) are reported substrates for PHB production. Pea shell is a non starchy and non sugary waste available in very large quantities and is being reported here for the rst time for its utilization for PHB
Table 4 Effect of inoculum size on polyhydroxybutyrate production on biowaste*

Inoculum on biowaste: GM2 Medium (lg cell protein ml)1) 10 PHB (mg l)1) 1945 2075 Yield (% w w) 72 71 100 PHB (mg l)1) 3370 3010 Yield (% w w) 41 41 1000 PHB (mg l)1) 3035 2435 Yield (% w w) 42 40

Organism Bacillus cereus EGU44 B. thuringiensis EGU45

Values obtained are based on two sets of experiments observed after 72 h of incubation. The medium contains casein enzyme hydrolysate at 04 g l)1 concentration for all cultivations. SD was less than 5% *BW : M = 1 : 1.
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production equivalent to 300 g kg)1 dry mass based on the following observations: at 2% TS PSS, in 1 : 1 ratio (BW : M), it amounts to 5 g glucose (in GM2 medium) and 10 g dry mass of pea-shells per litre of feed. With GM2, we observed a value of 500 mg PHB l)1, the total contribution towards PHB production in BW : M can be observed to be 250 mg l)1. At this rate, the contribution of biowaste supplemented with CEH can be calculated to be 3370 ) 250 = 3120 mg PHB 10 g pea-shells on dry wt basis. It leads to a nal production of approximately 300 g kg)1 TS pea-shells. Although pea shell slurry still need a supplementation of CEH and GM2 medium, which adds to process cost, further optimization is needed for improving the process economics. Acknowledgements We are thankful to Acting Director, Institute of Genomics and Integrative Biology, CSIR, Acting Director, National Environmental Engineering Research Institute, CSIR and CSIR Task Force project for providing the necessary funds, facilities and moral support. References
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