Global Advanced Research Journal of Microbiology (ISSN: 2315-5116) Vol. 2(3) pp.

054-064, March, 2013 Available online http://garj.org/garjm/index.htm Copyright 2013 Global Advanced Research Journals

Full Length Research Paper

Microbial production of polyhydroxybutyrate, a biodegradable plastic using agro-industrial waste products.

Alyaa Hamieh*, Zakia Olama and Hanafi Holail
BAU, Debbiye, Lebanon
Accepted 24 February, 2013

Plastics produced from petrochemical sources and known as polypropylene are now accumulating in our environment at rates of millions of tons per year creating severe problems. The present study aims to the production and isolation of PHB (polyhydroxy-buty rate ), a biodegradable plastic, from agro-industrial waste products (whey and date molasses) due to its high economic and industrial importance , taking into consideration many points that lead to produce PHB on large scale. The methodology of this study includes screening study for the isolation of a promising microbial producer of PHB , and optimization experiments to evaluate the best environmental and physiological factors that lead to maximum PHB production. Under the optimized conditions, Lactobacillus acidophilus has shown maximum production when grown for 4 days on date molasses supplemented with NB yielding 0.412g/50ml of PHB, followed by Bacillus thuringiensis (0.367g/50ml) grown for 4 days on the same medium, and Bacillus subtilis (0.337g/50ml) grown for 6 days on whey supplemented with glucose, yeast extract, and peptone. Eleven nutritional factors were examined for their significance on PHB production using a statistical design known as Plackette-Burman. Maximum PHB output of 43.1 g/l produced by Lactobacillus acidophilus was revealed by the statistical design, which represents about 7.04 fold increase in PHB production. Fedbatch fermentation was carried out using the optimized fermentation medium and PHB production has been increased to 27.5% as compared with batch closed process. PHB was detected by transmission electron microscopy and monitoring UV spectra of the sample by scanning the samples between 220 and 300nm compared with standard PHB. Lactobacillus acidophilus can be used for PHB production on large industrial scale, solving by this one of the problems of solid waste management that results from the accumulation of plastics and saving the environment from additional air pollution caused by its recycling. Keywords: Microbial production, polyhydroxybutyrate, agro-industrial waste products. INTRODUCTION Plastics known as polypropylene are the essential ingredients that enhance the quality of our life. Its manufacturing has increased significantly since the 1940s and has successfully replaced wood, mud, metals, glass and other materials.

*Corresponding Authors E-mail: zakia.olama@bau.edu.lb

Hamieh et al. 055 The low cost, stability, durability, good mechanical and thermal properties of plastic make it the best choice for widespread applications (Amara, 2008). However, the extensive use of materials made from plastics causes a worldwide problem because they are non-degradable (Muller et al., 2001). Another problem is that traditional plastics are produced from petrochemical sources that may be depleted and takes millions of years to be renewed. Recently issues concerning the global environment and solid waste management have created much interest in the development of biodegredable plastics that should be produced on an industrial scale to be commonly used (Anderson & Dawes, 1990). Polyhydroxy-butyrate (PHB) is a linear polyester of D(-)-3- hydroxyl-butyrate , and the best known of the polymers of related polyhydroxyalkanoates, that is produced by several microorganisms as an energy source(Page, 1995 and Lee, 1996). It is a biodegradable plastic that can be degraded aerobically and anaerobically by soil microorganisms. It has similar physical properties as polypropylene so it can be used as an alternative source of plastics (Bryom, 1987). Eventhough there are more than 250 different microorganisms synthesizing PHAs, only several of these, such as Alcaligenes eutrophus (Kim et al., 1994), Alcaligenes latus (Yamane et al., 1996), Azotobacter vinelandii (Page & Knosb, 1989), methylotrophs (Kim et al., 1996), Pseudomonas oleovorans (Brandl et al., 1988) and recombinant Escherichia coli (Lee & Chang, 1994 and Lee et al., 1997) are suitable for the production of PHAs to a high concentration with high productivity. Various researchers have explained that soil bacteria generally produce PHB (Hanzlikova et al., 1985). A large proportion (80%) of the tested soil streptomyces was able to produce PHB. However the actual amounts of PHB accumulation were generally lower for industrial application than for other bacterial groups which are known to produce PHB (UGUR et al., 2002). Biodegradable plastics are seeing some use and they have been available for many years on the market. However, their high cost has meant they have not replaced the traditional non-degradable plastics. The present study aims to the optimization of PHB production by microorganisms through evaluating the physical and environmental factors that lead to the maximization of PHB production using agro-industrial waste products as cheap carbon sources, and trials for scaling up the product as a step to be used in large scale industry for its high economic and commercial value and its value as a product that is safe for the environment. MATERIALS AND METHODS Microorganisms Five different bacterial isolates were used throughout the screening experiments namely: Bacillus subtilis and Bacillus thuringiensis were isolated from the garden soil of the faculty of science, Alexandria University, the former was identified by IMI (International Mycological Institute, UK); Bacillus thuringiensis was identified using 16S rDNA; Lactobacillus acidophilus was isolated from whey and identified using 16S rDNA; Escherichia coli and Staphylococcus aureus were kindly provided by the Microbiology Research lab, Faculty of Science, Beirut Arab University, they were isolated from urine samples and identified using phenotypic characterizations. Fermentation media Unless otherwise indicated, media were prepared with distilled water, sterilized by autoclaving for 20 min at pressure 15 lb/inch2 to raise the temperature to 121C. Fermentation medium (I) containing (g/l): whey supplemented with peptone, 3 and yeast extract, 3. Fermentation medium( II) containing (g/l): whey supplemented with peptone, 3; yeast extract, 3 and sucrose,5. Fermentation medium (III) containing (g/l): whey supplemented with peptone, 3; yeast extract, 3 and glucose, 5. Fermentation medium (IV) containing (g/l): treated date molasses, 50 dissolved in 1L nutrient broth . Fermentation medium (V) containing (g/l): treated date molasses, 50 dissolved in 1L nutrient broth, supplemented with glucose,5. Maintenance of the microorganisms Bacterial strains were maintained on nutrient agar slants and culture is renewed at monthly intervals. For long preservation, the bacteria were folded with 25% glycerol. Preparation of seed culture Transfers from single slant bacterial cultures (24 hours old) into 250 Erlenmeyer flasks containing 50 ml seed culture medium were incubated to initiate growth for 18 hrs at 37C to reach an OD1 at 600 nm. Aliquotes of 3 ml were taken from the latter liquid culture as a standard inoculum unless otherwise indicated. Fermentation Techniques Using Free Cells Submerged Fermentation Technique Cultivation was achieved in 250ml Erlenmeyer flasks each containing 50ml aliquots of the specified fermentation medium. The media were sterilized by autoclaving for 20 min at 121C. Each flask received 3 ml of seed culture as a standard inoculum and was incubated at 37C for 4 days under shaken conditions (160 rpm). Each treatment was carried out in triplicates and the results obtained

056. Glo. Adv. Res. J. Microbiol. Throughout this work were the mean of at least two experiments. Fed-batch Fermentation Technique In a trial to test the effect of the fermentation technique on PHB production as a step for scaling up the product a continuous open system using fermentor (Biotron Liflus GR Fermentor, 3L) was evaluated. The fermentor was fortified with 2.5 L optimized fermentation medium. The aeration was 2 L/min; the pH of the medium was 5.5. The cultivation time of each batch was 4 days at 37C on 160 rpm. At the end of each batch, 500ml fermentation medium was taken out and 500ml fresh fermentation medium was added into the fermentor. Screening experiments for microbial production of PHB The five bacterial isolates used throughout the screening experiment were screened for their potentiality for PHB production under shaken conditions using five different fermentation media for different time intervals (2-8 days) at 37C. Physiological and environmental factors affecting PHB production by the bacterial isolates under test Different environmental factors such as pH, inoculum size, culure volume and incubation temperature were tested and screened for maximium PHB production. Optimization of nutritional factors affecting PHB production using multifactorial statistical design (Plackett-Burman). Application of a complete factorial design would require 2n experiments if n factor have to be investigated. In the present case, eleven variables would lead to 1024 trials, which is a very large number. Using a fraction of the factorial design without losing information about the main effects of variables (Ooijkas et al., 1998) can reduce the number of experiments. The Plackett-Burman experimental design, a fractional design, (Plackett & Burman, 1946 and Yu et al., 1997) was used in this research to reflect the relative importance of various environmental factors on PHB production by the promising bacterial strain. The design is recommended when more than five factors are under investigation (Lavilla et al., 1998 and Abdel-Fattah et al., 2002). Eleven independent variables were screened in twelve combinations organized according to the PlackettBurman design matrix described in the results section (table 1). For each variable, a high (+) and low (-) level was tested. All trials were performed in duplicates and the averages of obtained PHB was treated as the response for each trial. The main effect of each variable was determined with the following equation: Exi= (Mi+ - Mi-) / N Where; Exi is the variable main effect, Mi+ and Mi- are PHB amounts in trials where the independent variable (xi) was present in high and low levels, respectively, and N is the number of trials divided by 2. A main effect figure (figure 6) with positive sign indicates that the high level of this variable is nearer to optimum and a negative sign indicates that the low level of this variable is nearer to optimum. Using Microsoft Excel, statistical t-values for equal unpaired samples were calculated for determination of variable significance. Extraction of PHB PHB was extracted following the method adapted by (UGUR et al., 2002) after some modifications, and includes extraction of the product from the cells after they are harvested by centrifugation, washed by distilled water and then lysed in 30 ml of sodium hypochlorite for 24 hours. The solution was subjected to double extraction by chloroform in which PHB is highly dissolved , preceded with extraction with ethanol acetone solution (1:1 v/v) to remove cell lipids. Chloroform extract was evaporated to dryness at 70C in a water bath, then dry PHB crystals were weighed. Detection of PHB PHB granules were detected using TEM microscopy, and by monitoring UV spectra of PHB samples (after treatment with concentrated sulfuric acid) by scanning between 220 and 300nm, and compared with standard PHB which has highest absorbance at 235nm. Statistical analyses The values were subjected to linear regression analysis for the determination of R (regression coefficient) using SPSS16 (statistical package software) (Kutner et al., 2004). RESULTS AND DISCUSSION Screening Experiments for Microbial Production of PHB Data revealed that the highest PHB output (0.412g/50ml) was achieved with Lactobacillus acidophilus when grown on medium IV for 4 days under shaken conditions, followed by Bacillus thuringiensis (0.367g/50ml) when grown under the same conditions, and Bacillus subtilis (0.337g/50ml)

Hamieh et al. 057

Figure 1. PHB production by different bacterial isolates grown on different media at different time intervals.

when grown on medium III for 6 days under shaken conditions (figure 1). Physiological and Environmental Factors Affecting PHB Production For maximum PHB production, it is important to evaluate the physiological and environmental factors of the bacterial

isolates under investigation for the PHB production process. Medium IV was selected as the fermentation medium for further experimentation to evaluate the effect of environmental and physiological factors affecting the PHB production by the selected bacteria.

058. Glo. Adv. Res. J. Microbiol.

Table 1. Randomized Plackett-Burman experimental design for evaluating factors influencing PHB production by Lactobacillus acidophilus.

Variable s Trials 1 2 3 4 5 6 7 8 9 10 11 12 13

Date molasse s
+

(NH4)2S O4

Na2HP O4

KH2PO
4

Oliv e oil

NH4C l

MgSO 4. 7H2O + + + + + + 0

Malt Extrac t
+ +

FeSO4.7H2 O

Cystein e

Glycin e

PHB output (g/50m l) 0.25 0.399 2.188 0.786 1.381 0.451 0.489 0.614 1.235 0.212 0.27 0.141 0.306

+ + + + + 0

+ + + + + + 0

+ + + + + + 0

+ + + + + + 0

+ + + + + + 0

+ + + + + + 0

+ + + + 0

+ + + + + + 0

+ + + + + + 0

+ + + + + + 0

pH Relations Maximum PHB output (0.412 and 0.367g/50ml) was achieved by Lactobacillus acidophilus and Bacillus thuringiensis respectively at pH 5.5 (figure 2). Therefore pH 5.5 was selected for further experimentation. Higher or lower pH values showed inferior results. Metabolic processes are highly susceptible to even slight changes in pH (Wei et al., 2011), and drastic changes in PHB production seems to be due to the effect of initial pH on the bioavailability of trace elements (Ramadas et al., 2009). Flora et al. (2010) revealed that the maximum PHB

production (25%) by Bacillus sphaericus was at pH range from 6.5-7.5, and the reduction of polymer accumulation at higher pH values is due to the effect on the degradative enzymes of polymer breakdown, so that PHB is utilized at a rate almost equal to the rate of its synthesis. Effect of Inoculum Size Results revealed that maximum PHB output (0.412 and 0.367g/50ml) were achieved by Lactobacillus acidophilus and Bacillus thuringiensis respectively, with 3ml inoculum level/flask; however, minimal PHB contents (0.064 and

Hamieh et al. 059

0.139g/50ml) were achieved by Lactobacillus acidophilus and Bacillus thuringiensis respectively, with 7ml inoculum level/flask (figure 3). Accordingly, 3ml/50ml inoculum level was selected to carry out the next part of the research. Low inoculum size required longer time for cells to multiply and produce the desired product (Jiff et al., 1998). A small amount of inoculum can lead to insufficient number of microbial cells and a reduced amount of the secreted enzymes while a much higher inoculum could lead to or cause a lack of oxygen and depletion of nutrients in the culture media (Abusham et al., 2009). Effect of Culture Volume Both Lactobacillus acidophilus and Bacillus thurnigiensis has attained maximum PHB production (0.412 and 0.367g) respectively with 50 ml culture volume (figure 4) that was

utilized as the optimum culture volume in the next experiments. The finite volume of the culture medium means the limitation of the nutrients for the microorganism. The consumption of the nutrients is largely dependent on the bacterial population. To ensure a high PHB output in limited culture volume, the inoculum size should therefore be controlled (Abusham et al., 2009). Effect of Incubation Temperature Results indicated that maximum PHB production was achieved at 37C incubation temperature. Higher or lower temperatures showed inferior results (figure5). This result coincides with that represented by Aslim et al. (2002), who reported that optimum incubation temperature for PHB production by Bacillus thuringiensis, Bacillus

060. Glo. Adv. Res. J. Microbiol.

Figure 6. Main effect of variables on PHB production by Lactobacillus acidophilus.

subtilis, and Bacillus pumilis was at 37C. Tamodgan & Sidal (2011) reported that higher and lower temperatures than 30C lead to decrease in PHB synthesis by Bacillus subtilis ATCC 6633, as well as cell mass, probably due to the low enzymes activity. Optimization of the best nutritional factors affecting Lactobacillus acidophilus PHB production using multifactorial experiments (Plackett- Burman Design) Sequential optimization approaches were applied in the present part of the study. The first approach dealt with screening for the environmental factors affecting PHB production by the bacteria under investigation. The second approach was to optimize the nutritional factors that control PHB production process. The best culture conditions such as, incubation time for 4 days; initial pH at 5.5; 3 ml inoculum level; 50ml culture volume and 37C incubation temperature were used for the optimization of the

nutritional factors using the Plackett-Burman statistical design. Evaluation of the Factors Affecting PHB Production In screening and optimizing the factors affecting PHB production, it is very important to test as much factors as possible and to identify the significance of each of them. Plackett-Burman design offers good and fast screening procedure and mathematically computes the significance of large number of factors in one experiment, which is time saving and maintain convincing information on each component (Srinivas et al., 1994). The design is recommended when more than five factors are under investigation (Abdel-Fattah et al., 2002). The influence of eleven factors including carbon, nitrogen, amino acids and metal ions on PHB production were tested. A wide variation was shown in Lactobacillus acidophilus that was chosen rather than Bacillus

Hamieh et al. 061

Figure 7. Effect of initial glycine and olive oil amounts on PHB production by Lactobacillus acidophilus

thuringiensis as the promising microbial producer of PHB (that is safe to be used on large industrial scale) throughout the different trials. The variation in PHB production was ranging from 0.141 2.188g/50ml. This revealed that these factors have a strong influence on PHB production. It was shown that glycine and olive oil had a significant effect on PHB production (figure 7), whereas the other factors affected slightly the PHB production process. The main effect that was estimated as a difference between both average of measurements made at the high level (+1) and at the low level (-1) of the factor) of the examined factors affecting PHB production was calculated and presented graphically (figure 6). On the analysis of the regression coefficients of the eleven variables, (NH4)2SO4, Na2HPO4, olive oil, KH2PO4 , FeSO4.7H2O, glycine, and cysteine showed a positive effect, whereas date molasses, malt extract, NH4Cl,and MgSO4.7H2O showed a negative effect. Data in the present investigation showed that PHB production increased with high levels of glycine (0.05g/50ml), and olive oil (1g/50ml). PHB production was best achieved at high concentration of glycine, it was a positively significant factor. Maximum PHB output (7.6% PHB of cell dry weight) was produced by Streptomyces MU 117 when glycine was used as nitrogen source. Similarly high amount of PHB was accumulated (61.43%) when glycine was used as nitrogen source by Rhizobium sp. 2426 (Mercan et al., 2002), and by Bacillus subtilis ATCC 6633 (7.683%) (Tamdogan & Sidal, 2011). Olive oil used as a carbon source was also a positively significant factor, it affects PHB production in its high level (1g/50ml). Fukui & Doi (1998) reported that plant oils such as olive, corn, and palm oils were good carbon substrates for PHB production by A. eutrophus. Kahar et al. (2004).

Thakor et al. (2005) used the vegetable oils for PHB production by Ralstonia eutropha and Commomonas testosterone respectively. Oleic acid was good for the growth of Pseudomonas putida and it was used as carbon source instead of alkanes for production of PHAs since it exhibits less toxicity (Lee et al., 2000). Schizosaccharomyces pombe, achieved high PHB content on using oleic acid and glucose as carbon sources (Abdulhamid et al., 2007). Malt extract and date molasses used as carbon sources had negative effect on PHB production when used in high levels. Maximum Bacillus sp JMA5 growth and PHB production were noticed with 10% (w/v) molasses and higher concentrations lead to negative effect on both growth and PHB output (Younes et al., 2010). Data of the present study revealed that (NH4)2SO4 in its higher level had a positive effect on PHB production, whereas NH4Cl had a negative effect on PHB production in its higher level and it must be maintained with lower levels. It was reported that (NH4)2SO4 was the optimal nitrogen source for PHA production compared to NH4Cl as well as urea used by: Alcaligenes eutrophus (Grothe et al., 1999 & Koutinas et al., 2007), Methylobacterium sp. (Kim et al., 2006), Sinorhizobium fredii (Liangqi et al., 2006), and Rhodobacter sphaeroides (Sangkharak & Prasertan 2007). Forty times lower amount of (NH4)2SO4 (from 0.8 to 0.02g/l) in the optimum medium could tremendously reduce the medium cost, hence, gave higher potential for large scale production of PHB by Rhodobacter sphaeroides (Sangkharak & Prasertan 2007). In the present study both KH2PO4, Na2HPO4 supplied as phosphorus sources seemed to have positive effect on PHB production in their high levels at 0.15g/50ml and 0.05g/50ml respectively. Phosphorous limiting condition in

062. Glo. Adv. Res. J. Microbiol.

Figure 8. Fed batch fermentation for PHB production by Lactobacillus acidophilus.

the presence of KH2PO4 and K2HPO4 was important factor for PHB production (Sangkharak & Prasertan, 2007). However, addition of phosphate was also required for cell growth, and loss of buffer capacity led to high pH of approximately 10-11 which might be growth inhibiting level. On the contrary KH2PO4 was used with higher concentration of 2g/l with Na2HPO4 for PHB production (0.6g/l) by Bacillus sphaericus 5149 (Ramadas et al., 2009), and 3.75g/l of Na2HPO4 was used for PHB production by Hydrogenophaga pseudoflava (Mahmoudi et al., 2010). On the other hand Fe2+ ions induced PHB production by Lactobacillus acidophilus whereas Mg2+ ions negatively affected PHB production in its higher level, and it must be maintained in its lower level. PHB production (7g/l) by C. taiwanensis 184 was noticed with limited not lacking 2+ magnesium ions (Wei et al., 2011). Mg ions were used in higher amounts (0.6g/l) for PHB production by Bacillus sphearicus (Ramadas et al., 2009); 2g/l with Hydrogenophaga pseudoflava (Mahmoudi et al., 2010); 1.14g/l with Glycogen accumulating organisms (Dai et al., 2007) and 0.2g/l with Rhizobia spp. (Mercan et al., 2002). Fe2+ ions were used in trace amounts for PHB production by Azotobacter vinelandii (Yan et al., 2000), and by R. eutropha (Khanna & Srivastava, 2006). However, it was used in larger amounts by Mahmoudi et al. (2010) for PHB production by Hydrogenophaga pseudoflava. By the end of the present study, the conditions achieved nearer to optimum one for PHB production by Lactobacillus acidophilus were (g/l): date molasses, 50 ; (NH4)2SO4, 0.8 ; Na2HPO4, 1 ; olive oil, 20 (with few drops of tween 80) ; KH2PO4, 3 ; NH4Cl, 1 ; MgSO4.7H2O, 1 ; malt extract,2 ; glycine,1 ; cysteine,1 ; and 40 ml/l of 1 mm/l FeSO4.7H2O solution. Flasks with 50 ml culture volume were inoculated

with 3ml of 18 hrs seed culture and incubated for 4 days at 37C. Verification of Plackett-Burman A verification experiment was applied to examine model validation and to evaluate the basal versus the optimized conditions. Verification result revealed 98.4% validity and showed 43.1 g/l which represents about 7.04 fold increase in PHB production when compared to the control medium. Fed-batch fermentation technique Five batches of fermentation are done within 20 days with a 4 day time interval between every single batch. The amount of PHB at the end of each of the five batches was as following: 2.18, 2.3, 2.45, 2.54, 2.78g/50ml respectively. The PHB production of the 5 batches are shown in figure 8. During the five batches of the continuous fermentation, PHB production has been increased to 27.5% as compared with batch closed process. The greater cost of PHB production on large industrial scale is largely due to low productivity. Therefore, improvement of fermentation efficiency is a key factor in making a microbial PHB process viable (Khanna & Srivastava, 2005). Fedbatch cultivation is often employed to achieve high product concentrations (Sun et al., 2007). PHB detection PHB granules were detected using TEM microscopy (figure 9) and by monitoring UV spectra of PHB samples (after

Hamieh et al. 063

Figure 9. Transmission electron micrograph of Lactobacillus acidophilus grown on nutrient broth supplemented with date molasses showing PHB granules inside the cell.

Figure 10. UV spectra of PHB extracted from Lactobacillus acidophilus.

treatment with concentrated sulfuric acid) by scanning between 220 and 300nm. Maximum absorbance was at 235nm (figure 10). PHB was detected by transmission electron microscopy (Luengo et al., 2003 and Sangkharak & Prasertan, 2007). Ugur et al. (2002) and Abdulhamid et al. (2007) analyzed PHB after conversion into crotonic acid and absorbance spectra were determined by scanning the samples between 220 and 300 nm according to standard PHB that has maximum absorbance at 235nm. The number and size of granules vary depending on the producer microorganism (Anderson & Dawes, 1990 and Murray et al., 1994). CONCLUSION The results of the present investigation provides basis for assessing a potential for using Lactobacillus acidophilus for PHB (a biodegradable plastic) production, which is an economically and environmentally important product, on

large industrial scale, solving by this one of the problems of solid waste management that results from the accumulation of plastics and saving the environment from additional air pollution caused by its recycling.
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