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PHB production in Azomonas, Acinteobacter and Bacillus species: Isolation, screening and identication

Noha Salah Elsayed, Mohammad Aboulwafa*, Khaled Aboshanab, Nadia Hassouna

Department of Microbiology & Immunology, Faculty of Pharmacy, Ain Shams University, Cairo, Egypt.
Correspondencia: maboulwafa@yahoo.com

* Address: Department of Microbiology and Immunology, Faculty of Pharmacy, Ain Shams University, Al Khalifa Al Maamoun St., Abbassia, Cairo, Egypt. Tel:  (202) 22628017. Fax: (202) 24051107.

Abstract
Background: Biopolymers (polyhydroxyalkanoates; PHA) are the hope for solving problems of synthetic polymers. They have a lot of applications in medicine and industry. However their production cost is still a problem. Our study aimed to solve part of the problems of PHA production. First we chose polyhydroxybutyrate (PHB) as an abundant and very well characterized member in PHA family. Then isolating easy growing bacterial isolates from soil capable of industrial production of polyhydroxybutyrate and estimating minimum time of incubation needed for its maximum production.

Materials and ndings: A total of 251 bacterial isolates were recovered from
various soil samples. Screening for PHA production was done by viable colony staining method using Nile red. A total of 66 isolates appeared to produce PHA. Screening for PHB was done by spectrophotometric analysis where three promising bacterial isolates were obtained. These isolates were fully identied using microscopical examination, culture characteristics, biochemical reactions and sequencing of 16S ribosomal RNA gene. They were identied as Acinteobacter baumannii isolate P39, Bacillus cereus isolate P83 and Azomonas macrocytogenes P173. The nal 16S ribosomal RNA sequences of the respective isolates were assembled, analyzed and submitted into Genbank. Acinteobacter isolate P39 produced 32 % PHB per dry weight after 30 hours of incubation. After 48 hours of incubation Bacillus isolate P83 and Azomonas isolate P173 produced 13 % and 24 % PHB per dry weight, respectively.

Conclusion: Since PHB production had not been extensively studied in both
Acinteobacter and Azomonas, this study valorizes usage of both isolates in PHB production. Further optimization studies will be conducted in the future for maximum PHB production from these three promising isolates.

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Keywords: Polyhydroxybutyrate, Acinteobacter baumannii, Azomonas macrocytogenes, Bacillus cereus, biopolymers.

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Introduction
Plastics are considered to be an invaluable gift of modern sciences and technology to mankind [1]; they are used in many applications such as packaging. They are widespread not only due to their uses but also due to their stability [2-4]. On the other hand, plastics have some problems. First they are persistent in the environment to the extent that communities are now more sensitive to the impact of discarded plastic on the environment. Second plastics originate from nite resource (petroleum product). Solutions to plastic waste management include source reduction, incineration, recycling and bio- or photo-degradation. However, these solutions added more problems to the existing grave of plastics [4]. Since there is a need for plastics in our daily life, so there has been an urge for development of biopolymer (biodegradable polymers) materials which must still retain the desired physical and chemical properties of conventional synthetic plastics [3]. Among the candidates for biodegradable plastics, polyhydroxyalkanoates (PHAs) have been drawing much attention because of their similar material properties to conventional plastics and complete biodegradability. Famous example of PHA is 3-polyhydroxybutyrate (PHB). PHB was the rst PHA to be discovered and is also the most widely studied and best characterized PHA [5]. PHB has a lot of medical applications as developing therapeutic devices for example temporary prostheses, three-dimensional porous structures as scaffolds for tissue engineering and as controlled/sustained release drug delivery vehicles [1]. Moreover, according to Lee (1995) the degradation product of PHB, D (-)-3-hydroxybutyrate has been detected in relatively large amount in human blood plasma. Therefore, it is highly plausible that implanting PHB in mammalian tissues would not be toxic. The two most notable features of PHB in medical applications are that it is very biocompatible producing an exceptionally mild foreign body response, and that the biodegradation rate is slow [6]. PHB has a lot of useful properties such as moisture resistance, water insolubility, oxygen impermeability and optical purity; this makes PHB a unique member in PHA family [7]. PHB are synthesized by both Gram positive and Gram negative bacteria. It is accumulated under unfavorable growth conditions such as limitation of nitrogen, phosphorus, magnesium, or oxygen in the presence of excess carbon. Therefore, a lot of research had adopted several cultivation strategies that can

simulate these conditions for the efcient production of PHB [8]. Among examples of bacteria studied for PHB production, Alcaligenes eutrophus, it produced 80 % PHB (w/w) of dry cell mass after 60 hours of incubation. Also Protomonas extorquens used methanol as a carbon source and produced 50-60 % of dry weight [8]. It might be noteworthy that PHA has been found both in Eubacteria and Archaebacteria [9]. The aim of our study was screening of different soil samples to get a promising bacterial isolate (s) capable of industrial production of PHB from soil and identify them. We aimed that these isolates will pave a way to decrease PHB production cost by its productivity.

Materials and methods

Bacterial isolates: A total of 251 isolates were recovered from different soil samples collected from different localities in Egypt.

Chemicals and culture media

Different chemicals used in the present study were of high est quality available and obtained mainly from Sigma-Aldrich (Munich, Germany), El-Nasr chemical Co. (Adwic, Cairo, Egypt) and other local suppliers. While ready-made culture media and media ingredients were obtained from Lab M (Topley house, England), Oxoid (USA) and Difco (Detroit, USA). Mineral salt medium (MSM) was used for PHB production, it is composed of (g/liter): Na2HPO4 12H2O (10.2); NaCl (10); KH2PO4 (1.5); NH4Cl (0.1); MgSO4 7H2O (0.2); CaCl2 (0.01); ferrous ammonium citrate (0.06); and 1 ml trace elements solutions [10]. Collection of soil samples: Twenty soil samples were collected from a depth of approximately 10 cm below surface (as microbial ora is affected by U.V of sunlight) of agricultural elds and gardens and stored at 4C till use. Recovery of bacterial isolates from soil samples: Bacterial isolates were recovered from soil samples using the method developed by Yilmaz et al. (2005) and Berlanga et al. (2006) with minor modications [11, 12]. Three grams of each soil sample were homogenized in 30 ml of MSM broth and incubated for 24 hours at 37 C in shaker at 200 rpm and dilution series were made in saline and aliquots of 0.1 ml were taken and surface inoculated using bent glass rod onto R2A agar. The plates were incubated for 24-48 hours at 37C. Morphologically different colonies were picked up from plates showing separate colonies to be puried on nutrient agar plate by streaking method.

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Maintenance of recovered isolates: All the recovered isolates were maintained onto nutrient agar slants at 4 C and sub-cultured every month. After screening for PHB production, the tested isolates showed high production of PHB were maintained in L.B medium supplemented with 20% glycerol [13]. Screening for polyhydroxyalkanoates (PHA) production: The screening was done by the viable-colony staining method using Nile red [11, 14]. All isolates were cultured on MSM agar medium supplemented with Nile red for 4-5 days at 37 C .The agar plates were exposed to UV light to detect accumulation of PHA. The positive isolates gave pinkish uorescence under UV. Screening for PHB production: The screening was done on positive isolates obtained from primary screening. Secondary screening was done by spectrophotometric assay [15, 16]. This method depends on conversion of extracted PHB with concentrated sulphuric acid to crotonic acid which was estimated spectrophotometerically at 235 nm. This was done by transferring a loopful from culture grown on nutrient agar slant to test tube containing 5 ml L.B medium. The tube was incubated in shaking water bath at 37 C for 20 hours at 160 rpm. The production was carried out in 100 ml Erlenmeyer ask containing 20 ml MSM. The asks were inoculated with 5 % v/v pre-culture and incubated in shaking incubator at 200 rpm and 37 C for 48 hours. At the end of incubation, volume of 1 ml was taken for PHB determination and 5 ml sample was taken for dry weight determination.

PHB determination: The withdrawn sample contained in eppindorff tubes (1.5 ml) previously washed with alcohol followed by hot chloroform (to remove plasticizers) [16].The cells were harvested by centrifugation at 12,000 rpm for 5 min. The cell pellets were digested with a sodium hypochlorite solution (equivalent to active chlorine 4-6 %; density at 20 C of 1.12) at 37 C for 1 hour with stirring at 160 rpm. The insoluble materials containing PHB were collected by centrifugation at 12,000 rpm for 10 min. The pellets were washed with 1ml aliquot each of water, ethanol, and acetone, respectively. About 1 ml hot chloroform was added to extract PHB and After that 10 ml concentrated sulphuric acid (98%) were added to residue and the tubes were kept in a boiling water bath for 15 min. The solution was left for cooling then it was measured spectrophotometery at 235 nm. Construction of standard calibration curve: A standard curve was constructed between crotonic acid concentrations and absorbance at 235 nm. A linear relationship was observed (Figure 1). This was done by dissolving 50 mg crotonic acid in 50 ml concentrated sulphuric acid (98 %) and then appropriate dilutions were made with distilled water and the absorbance was measured for the obtained dilutions [15]. The amount of crotonic acid per ml of sample solution was calculated from the standard curve equation: Y=0.017 X+0.0419 where y equals to absorbance of measured sample and X represents the crotonic acid concentrations (g/ml) in the sample solution.

Figure 1. Calibration curve of crotonic acid concentration versus absorbance at 235 nm.

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Dry weight determination: 5 ml aliquot contained in preweighed centrifuge tubes was centrifuged and supernatant was carefully removed by aspiration. The cell pellets were dried at 37 C for 24 hours. Then each tube was weighed. The dry weight of cells was calculated from subtracting the initial weight from nal weight of each centrifuge tube from which the cell dry weight of 1 ml culture was determined. The percentage of PHB in terms of crotonic acid per dry weight was calculated as follows: Amount of crotonic acid of 1ml culture Dry weight of 1ml culture X100

these tubes containing dried cell pellets. The values obtained were averaged and the dry cell weights at different dilution levels were calculated. In parallel serial dilutions of bacterial suspension (similar to those used for dry weight calculations) were done and their optical densities were measured at 640 nm (Figure 2).

Identication of the selected isolates with promising polyhydroxybutyrate productivity: By microscopical examination was done using gram stain and Gram positive isolates were grown on nutrient agar and mannitol salt agar while gram negative isolates were grown on nutrient agar, MacConky and EMB agar. Biochemical reactions were done to promising isolates according to method designed in Bergeys manual [17]. Sequencing of 16S rRNA gene: Isolates P39 and isolate P173 were sent to Microbiological Resources Center (MIRCEN), Faculty of Agriculture, Ain Shams Universty, Cairo, Egypt. At the center DNA extraction and PCR amplication of 16S rRNA gene were carried out for isolates P39 and P173. Through MIRCEN, the PCR products of both isolates were sent and sequenced at Bioneer Company, Germany. For isolate P83, organism culture and DNA extraction were carried out according to Pospiech and Neumann 1955 [18] and the extracted DNA was sent to Sigma scientic company for PCR amplication of 16S rRNA gene. Through Sigma, the PCR products of isolate P83 was sent and sequenced at GATC Company, Germany. Sequence analysis: The sequence obtained from company was analyzed to identify bacterial species. The sequence assembly and DNA analysis was carried out using Staden package program (http://staden.sourceforge.net/) and the nal sequence was aligned against sequences on Genbank using Blast (http://blast.ncbi.nlm.nih.gov/Blast.cgi) [19, 20]. Biomass determination: A calibration curve was constructed between optical density at 640 nm and dry weight. To establish this calibration curve, test organisms were cultured as in secondary screening. At the end of incubation period aliquots of 5ml each were taken in pre-weighed centrifuge tubes, centrifuged at 12,000 rpm for 5 min and the supernatants were removed carefully by aspiration and the cell pellets were washed once with physiological saline. Then the tubes were dried at 37 C for 24 hours (pre-determined time for constant weight). The dry cell weight was calculated by subtracting the weight of empty centrifuge tubes from nal weight of

Figure 2. Calibration curves between dry cell weight and optical density of the selected test isolates (P39, P83 and P173).

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Studying the time course of PHB production: The three isolates were pre-cultured in LB broth at 37 C for 20 hours then inoculation in main medium (MSM) was done as in screening for PHB. PHB concentration and dry weight were measured after 6, 24, 30, 48 and 72 hours of incubation in the main culture.

Results
Recovery of bacterial isolates from soil samples: A total of 251 different isolates were recovered from 20 different soil samples. Screening for polyhydroxyalkanoates (PHA) production: A total of 66 isolates accumulate HA. This was evidenced by pinkish uorescence of colonies on MSM-Nile red agar medium under U.V (Figure 3). Screening for PHB production: A total of 52 producing isolates (20 were Gram positive and 32 were Gram negative) gave positive results for PHB production. The percentages of PHB production per dry weight (specic productivity) by the test isolates. One Gram positive isolate coded P83 and two gram negative isolates coded P39 and P173 produced maximum percentage of PHB production per dry weight when compared with other tested isolates within the same Gram category. Therefore these isolates were selected for further studies. Identication of bacterial species: Preliminary identication was carried out using culture media and biochemical reactions. The obtained nal 16S ribosomal RNA sequences

showed 100 % homology of P39 to Acinetobacter baumannii, 99 % homology of P83 to Bacillus cereus and 90 % homology of P173 to Azomonas macrocytogenes. For isolate P83, the obtained microscopical, culture and biochemical reactions are similar to those of Bacillus cereus according to Bergyes manual of systematic bacteriology except for the two tests citrate utilization and tyrosine degradation. Taken together (microscopical, culture and biochemical results plus sequence analysis) the test isolate P83 could be identied as Bacillus cereus. For the other two selected isolates, the obtained microscopical, culture and biochemical reactions were found to be correlated with the results obtained for 16S rRNA sequence. Therefore the isolate P39 was identied as Acinetobacter baumannii isolate P39 and isolate P173 was identied as Azomonas macrocytogenes isolate P173. The full assembled 16S ribosomal RNA sequences of the isolates P39, P83 and P173 were submitted to the nucleotide Genbank database (http://www.ncbi.nlm.nih.gov/genbank/) and the submitted sequences were accepted with accession codes KC685000 for Azomonas macrocytogenes isolate P173, KC876036 for Acinetobacter baumannii isolate P39 and KC876035 for Bacillus cereus isolate P83. Studying time course: The proles of both PHB production and the growth for the three test isolates (P39, P83 and P173) at different time intervals are shown (Figure 4). The PHB production paralleled growth for isolate P39 while in case of isolate P173 maximum growth preceded maximum PHB productivity (obtained after 48 hours). For isolate P83 the increase in growth over time was at slow rate while the increase in PHB production was at higher rate reaching its maximum after 48 hours.

Discussion
In this study, we were able to isolate 3 promising bacterial strains out of 251 isolates capable of producing relatively high concentration of PHB. These species were identied by microscopical examination, culture characteristics, biochemical reactions and 16S rRNA gene sequencing. Their identity was P39 as Acinetobacter baumannii, P83 as Bacillus cereus and P173 as Azomonas macrocytogenes. Acinetobacter P39 produces 32 % per dry weight PHB after 30 hours, while bacillus produces PHB up to 13 % per dry weight and Azomonas produces 24 % PHB per dry weight after 48 hours of incubation. Isolation was carried according to Berlanga et al. (2006) but with some modication in the medium used for isolation of microorganism. First soil was homogenized in MSM broth to provide bacteria with minerals essential for growth. After dilution R2A medium was used as Reasoner et al. 1985 stated
Figure 3. Fluorescence of PHA producing isolates on MSM.

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Figure 4. Time course of PHB production and biomass in the three selected isolates ( Acinteobacter isolate P39 (a), Bacillus isolate P83 (b) and Azomonas isolate P173 (c). This experiment was done in triplicate.

that R2A medium allow colonies to develop slowly and large enough to be counted easily so it is best used for isolation of bacteria based on morphological differences [21]. The screening for PHA was done according to Spiekermann et al. (1999). This method is based on the direct inclusion of the lipophilic dyes Nile red in the agar medium such that the growth of the cells is not affected and the occurrence of PHAs in the colonies can be directly monitored. This method gives a powerful discrimination between positive isolates accumulating PHA and negative isolates. We chose PHB as an example of PHA. It is abundant in nature and has a lot of good properties distinguishing it from other PHA [7]. Since PHB are synthesized and accumulated under unfavorable growth condition in the presence of ex-

cess carbon source [22]. It is important to develop cultivation strategies that simulate these conditions and allow efcient production of PHB. The medium used in production of PHB is MSM. It is designed to stimulate conditions of PHB production. Generally bacteria able to synthesize PHA can be divided into two groups. The rst group, accumulating PHA during the stationary phase, requires limitation of N, P, Mg and oxygen, for example, and an excess of the carbon sources. The second group accumulates PHA during the growth phase. At the end of incubation in MSM, screening for PHB was done by spectrophotometric assay. Crotonic acid was used as standard instead of PHB. Law and Slepecky (1960) mentioned that PHB cannot be used as standard because they are contaminated with esters and probably with water and it necessities purication. Karr et al. 1983 stated that crotonic acid better used as a standard as purity of PHB differs with Copyright iMedPub

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the purication method used so crotonic acid was used as standard. Dilution of samples with distilled water occurred to decrease viscosity of sulphuric acid [15]. For efcient recovery of polymer, sodium hypochlorite was used to digest cells at 37 C. The residue of hypochlorite digestion was washed with distilled water to remove soluble salts and with acetone and alcohol to remove non-PHB lipid. After screening, we picked up three isolates; isolate P83 among Gram positive isolates and isolates P39 and P173 among Gram negative isolates. They produced a relatively high concentration of PHB in comparison to their morphological group. Identication of the selected isolated were done by culturing them on different media after knowing their Gram behavior. Biochemical reactions were done afterwards to assure identication of bacterial species. 16S rRNA gene sequencing was done as ribosomal 16S exists universally among bacteria and includes regions with species specic variability, which makes it possible to identify bacteria to the genus or species level by comparison with databases in the public domain [23]. Time course of PHB production in the three selected isolates was studied. Mostly the isolates produced our polymer in their stationary phase. It started its production in growth but maximum production in stationary phase. Afterwards the polymer concentration decreases due to the bacterial consumption. It makes use of its carbon and energy reserve for drastic conditions.

PHB production was studied deeply over the last years. Being biodegradable and from renewable sources attracted a lot of scientists. According to our study we concluded PHB production in the three species Acinetobacter baumannii, Bacillus cereus and Azomonas macrocytogenes. Acinetobacter was not studied extensively in literature; Gavin et al (1992) pointed 7 % PHB production in Acinteobacter after 24 hours [24]. However PHB production in Bacillus was studied to a great extent. Yuksekdag et al. (2003) stated that B.subtilius produces 18 % PHB after 45 hours and B. megatiurum produces 14.7 % after 45 hours [25]. Nazan et al. (2011) stated that Bacillus subtilius produces 23 g/ml after 24 hours [26]. Borah et al. (2002) stated that B.mycoides 69 % PHB after 24 hours [27]. Gram positive isolates lack lipolysaccarides so its PHB is used in biomedical applications so Bacillus is the best candidate for industrial production [28]. This eliminates the need for purication as an extra step. According to Azomonas species, PHB accumulation was stated in other nitrogen xing bacteria as Azotobacter. Azotobacter species are well known for industrial production of PHB [29]. Further studies will be conducted to optimize PHB production by the three selected isolates. This will include both physiological and environmental factors that inuence maximum PHB production by the respective isolates.

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