The optimization of fermentation conditions particularly physical and chemical

Transcription

1 4.1 Preamble The optimization of fermentation conditions particularly physical and chemical parameters are of primary importance in the development of any fermentation process owing to their impact on the economy and practicability of the process. Similarly purification is another important step. Hence in the present study these steps were considered in the PHB production. 4.2 Materials and Methods Process of optimization Optimization of culture media 1% of the 18-24hrs young seed culture was inoculated in to 5 different culture media. Growth and PHB accumulation was monitered at every 6hrs from shak flaske batch culture. Production of PHB was estimated from the total cell dry mass (CDM) obtained and compared with other media tested. Table. 3 Culture media tested for the optimization process Media tested Composition(g/L) Reference PHB medium 1 (NH 4 ) 2 HPo 4.4H 2 O : 3.5g K 2 HPO 4. 3H 2 O : 7.5g KH 2 PO 4 : 3.7g MgSO 4 : 0.17g Rawte and Mavinkurve, Glucose: 2.0g Yeast extract: 0.04g Microelement solution:1ml Agar: 1.8g ph: 7±0.2 52

2 (Micro element stock solution/1000ml FeSO 4. 7H 2 O: 2.7 mg MnCl 4. 4H 2 O: 1.98 mg CoSO 4. 7H 2 O: 2.8 mg CaCl 2. 2H 2 O: 0.17 mg ZnSO 4. 7H 2 O: 0.29 mg PHB medium 2 Na 2 HPO 4. 7H 2 O: 6.7g KH 2 PO 4 : 1.5g (NH 4 ) 2 SO 4 : 2.5g MgSO 4. 7H2O: 0.2g FeS: 60 mg CaCl 2 : 10 mg Trace mineral solution: 5 ml. (The trace mineral solution/l) Na 2 EDTA, 6.0 g FeCl 3.6H 2 O:0.29 g H 3 BO 3 :6.84 g MnCl 2. 4H2O: 0.86 g ZnCl 2 : 0.06 g CoCl 2.6H 2 O:0.026 g and CuSO 4. 5H 2 O, g PHB medium 3 Sucrose :20g (NH 4 ) 2 SO 4 :1.0g KH 2 PO 4 :4.4g Na 2 HPO 4 :4.8g MgSO 4 7H 2 O :0.5g ph: 7.0 Trace element solution: 1ml (Trace element solution Ammonium) Fe(III)citrate:50mg CaCl 2. 2H 2 O: 5mg Yu et al., 2005 Grothe and Chisti,

3 PHB medium 4 sesame oil:10g Na 2 H 2 PO4 12H 2 O: 5g (NH 4 ) 2 SO 4: 2 lg MgSO4 7H 2 O: 0.4lg Tween-80: 0.1% Wong et al., 2004 ph:7.0 PHB medium 5 NaNH 4 HPO 4. 4H 2 O: 3.5g 2006 K 2 HPO 4 : 10g MgSO4 7H2O: 0.2g Citric acid: 2.0g Acetate: 5.0g Agar: 15g ph:7.0 54

4 4.2.2 Effect of inoculum size on PHB accumulation The volume of 18-24hrs old lag phase seed culture was used in different volume to find the influence of inoculums size on growth and PHB accumulation. 1-5% of the of the culture was tested in the shake flasks. Samples were analyzed at every 6hrs of incubation. Cells were harvested when the biomass started to decline. % of PHB yield was estimated form the total cell dry mass obtained. 150ml of media was used in 250ml conical flasks and each time 3ml sample was used for analysis Effect of ph on biomass and PHB accumulation The batch culture was started with an initial of ph 3,5,7,9 and11. But no further adjustment in the ph level was done till the end of culture processes. Estimation of biomass and PHB production were done Effect of temperature on biomass and PHB accumulation The bacterial isolate was grown on culture media in a rotary shaker at 30, 35, 40, 45 and 50 0 C to find the influence of temperature on biomass and PHB production. Samples were analyzed at every 6 hrs as before Effect of salinity on biomass and PHB accumulation Finding the optimal salt concentration is also essential for strains isolated from marine environment. Hence 1-5% of salt concentrations were tested to check their impact on biomass and PHB production. Samples were analyzed at every 6 hrs. 55

5 4.2.6 Effect of DO concentration on biomass and PHB accumulation 5, 5.5, 6.0, 6.5 and 7.0 ranges of DO concentrations were tested in a laboratory scale fermentor by supplementing the air. Continuous monitoring on DO was done. Biomass and PHB production were estimated at every 6hrs Effect of different carbon sources on PHB production The selected bacterial isolate was grown in 250 ml conical flasks containing 100 ml E 2 mineral media broth with different carbon sources like glucose, fructose, molasses, wheat bran and diluted sewage (9:1). The flasks were incubated at 30 0 C Effect of substrate concentration on PHB production After finding the suitable substrate, its concentration on biomass and PHB production was found with various levels starting from 1-5%. As per the previous experiments done, samples were analyzed for every 6hrs Effect of different nitrogen sources on PHB production Different nitrogen sources like (NH 4 ) 2 HPO 4, (NH 4 )SO 4, (NH 4 )Cl, Na NH 4 HPO 4 and peptone (1.5%) were tested for their influence on biomass and PHB production. The flasks were incubated at 30 0 C Effect of nitrogen source on PHB production Different concentrations like 0.01, 0.02, 0.03, 0.04 and 0.05% of the suitable nitrogen source were tested to find their impact on biomass and PHB production Mass scale culture of the most potential strain under optimized conditions Mass scale culture of the most potential strain under optimized conditions was done with a laboratory fermentor with ph-7, temperature 30 o C, Saliniy 3.5%, D.O 6.5. molasses as Carbon source (NH 4 ) 2 HPO 4 as nitrogen source. 56

6 4.3 Results and Discussion Fig.18 Comparison of PHB production in different culture media Based on the optimization processes, E 2 Mineral medium (medium1) of Rawte and Mavinkurve (2002) was selected as the most suitable medium as it resulted in 60% of yield where as medium 2 was with 35% followedby media 3 and 4 with 20% yield. Grothe et al., 1999 suggested that the basal medium was suitable for the optimization experiments, among 5 differrent media tested by them. According to their results, medium 1 and 3 produced 61.5% of cell mass Panda et al, 2006 used Bg-11 medium supplemented with citric acid and their PHB production was 29% of the total cell dry weight. However the present study showed, A. eutrophus was the most potential strain for the maximum production of PHB with the E 2 mineral medium. 57

7 Fig.19 Impact of inoculum size on biomass production Fig.20 Impact of inoculum size on PHB production Maximum of biomass was observed at 72hrs of incubation and it started decline afterwards.. PHB accumulation was started from the 18 hrs of incubation onwards. Hence the cells were harvested at 72 of incubation. Maximum of PHB production also was occurred at 72hrs of 58

8 incubation i.e where the cell density also was high. 1% of inoculum was resulted 2.71g/L of PHB obtained from 7.2g/L of cell dry mass. When the size of the inoculum increased, the PHB accumulation was reduced. Fig. 21 Effec of ph on biomass production Fig.22 Effec of ph on PHB production 59

9 Different ph was maintained in the medium and its effect on PHB production was evaluated. Out of the different ph of the medium tested, ph 7.0 was found to be the optimum for the maximum PHB production which resulted in 9.7 g/l of cell dry mass where as the PHB yield was 3.6 g/l. ph 3 was the least preferred and it resulted in 2.1g/L of PHB yield. Results of the present study on ph was matched with that of Grothe et al., 1999 where they found that the initial ph value of 6.5 gave the best PHB yield whereas the ph values that changed even slightly from the optimum reduced the culture performance in accumulation of PHA to a greater extent. The initial ph value may have affected the bioavailability of some of the trace elements. It is the investigation of Sharma and Mallick (2005) that PHB accumulation was found to be the maximum at ph 8.5 (8.9%, w/w of dry cells) followed by ph 9.5 (7.8%) and 10.5 (7.2%) on 21 st day of incubation. Acidic ph was not found to support PHB accumulation. Alkaline ph did not support the PHB accumulation. This was in parallel with the result obtained by Khatipov et al., 1998, who observed that, PHB accumulation in the bacterium, Rhodobacter sphaeroides was significantly enhanced when ph of the culture medium was increased to 7.5 from 6.8. Quillaguam et al., 2006 observed that the ph of the medium was increased during cultivation from 7.5 to about 9.7±0.2. PHB accumulation was found to be the maximum at ph 8.5 followed by ph 7.5. Acidic ph was not found suitable for PHB accumulation (Panda et al., 2006). Aslim et al., 2002 who observed the PHB in Rhizobium strain grown on yeast extract mannitol broth adjusted to ph 7.0 found that the the amount of PHB produced by the strain was 0.01 to 0.5 g/l and the percentage of PHB in these cells was between 1.38 and 40 per cent of total cell dry weight. Tavernler et al., 1997 also investigated the effect of different sources of nitrogen, carbon and different ph levels on exopolysaccharide and PHB production in two 60

10 different strains of Rhizobium meliloti. They observed higher PHB content in both strains at ph 7.0. Fig.23 Effect of temperature on biomass production Fig. Effect of temperature on PHB production 61

11 In the present study, it was found that 35 0 C was the optimum temperature and 3.24g/L of PHB was obtained from 8.8g/L of cell dry mass which was followed by 40 0 C at which the yield was 2.92g/L. Extremely lower and higher level of temperature ranges gave led to poor PHB accumuation. Flora et al., tried PHB production in different media and varying temperature and found the maximum at 30 0 C in glucose medium (63 %)followed by 30 o C. 33 C appeared as the optimal temperature for growth and PHB synthesis; however, over the C range, the temperature effect was negligible in view of the reproducibility. It was clear that the temperature range of C was more suitable for PHB production. Fig.24 Effect of salinity on biomass production 62

12 Fig.25 Effect of salinity on PHB production In the present study, 3% of the NaCl concentration resulted in 40.49% yield (3.928g/L of PHB) from 9.7g/L of total cell dry mass. The 4% of the salt concentration resulted in 2.9g/L of total cell dry mass and 2% of the NaCl concentration resulted in 2.6% of PHB of total cell dry mass. Quillaguam et al., 2006 found that the sodium chloride concentration of % (w/v) provided the highest cell densities and also the PHB accumulation of about 54%. 63

13 Fig.26 Effect of DO on biomass production Fig. 27 Effect of DO on PHB production In the present study 3.0g/L of PHB was produced with 6.5mg/L of DO concentration that is 36.66% from 8.4g/l of total cell dry mass. Next to that DO 8mg/l provided 3.01g/l of PHB. 64

14 Level of 5% of DO gave only 2.39% of PHB yield. Third et al., reported that the production of biomass requires energy; thus, one expects that if biomass increases, then oxygen consumption should also increase. Fig.28 Effect of Carbon source on biomass production Fig. 29 Effect of Carbon source on PHB production 65

15 Fig.30 Effect of substrate concentration on biomass production Fig.31 Effect of substrate concentration on PHB production Among the different carbon sources tested molasses was found to be the best source. The yield of PHB was 3.81g/L with a cell mass concentration of 8.9g/L. This was followed by 66

16 glucose with 3.80g/L of PHB. Among the different concentration tested 3% of glucose produced the maximum (i.e) 3.78g/L of PHB from 9.24g/L of cell dry mass. In another study molasses, lactose and table sugar (Rohini et al., 2006) resulted respectively in a biomass of 3.11, 2.67 and 2.01g/l and PHB accumulation was 28.23%, 23.06% and % (0.57; 0.72 and 0.34g/l) respectively). Compared to their study the percentage of PHB produced in the present investigation on the higher side (i.e.) by two folds. Rohini et al., 2006 produced 0.13g/L of PHB when glucose was used as the substrate. In a different study the maximum PHB yield obtained was 60% of the dry cell mass after 93 hrs of incubation using sucrose as the carbon source (Grothe et al., 1999). Lillo and Valera (1990) stated that cellobiose, lactose, and sucrose were unsuitable substrates for PHB production. But the ability to use starch, a cheaper and abundant by available substrate, as the carbon source was appreciated as advantages in economic point of view. potential substrate. Wong et al., 2000 made an experiment with the shake flask culture using the commercial carbohydrates viz., sucrose, fructose, mannose, xylose, arabinose, galactose, lactose, and maltose as the sole carbon source. Higher cell mass and higher polymer content was obtained when using malt wastes as nutrients. Hence, they suggest that the malt wastes were considered as best inducer of bacteria to grow and produce PHB. In the present study, fructose produced only 2.8 g/l of PHB at 72hrs of incubation from 5.1 g/l of total cell dry mass and this was the least amount of yield when compared to other substrates tested g/l of PHB was produced when wheat bran used as the substrate. Diluted sewage also used as the sole carbon source and the yield was 3.79 g/l. There is an experiment with the mixture of carbon sources to test their influence on PHB production. Sharma and mallick (2005) found that the stimulation of PHB accumulation under carbon sources was 0.4% acetate + 0.4% glucose-supplemented 67

17 cultures (35%, w/w of dry cells), 0.2% acetate + 0.2% glucose (32%), 0.2% acetate (28%), 0.4% glucose (26%), 0.2% ethanol (20%), 0.4% maltose (18%) and 0.4% fructose (18%) on 21st day of incubation. No further rise in PHB pool was observed at increasing concentrations of the above carbon compounds. Cultures incubated with a combination of carbon sources depicted a significant rise in PHB pool rather than a single carbohydrates. In a different study done by Dawes (1992) found that the positive impact of acetate on PHB accumulation was observed and acetate was directly utilized for the synthesis of polyester. Lee et al., 1995 found that the glucose utilization in cyanobacteria occurs via pentose phosphate pathway and the stimulation of PHB accumulation in glucose-supplemented cultures could be due to the production of reduced cofactor NADPH. Similar explanation could be valid for fructose- and maltose-supplemented cultures (Sharma and mallick, 2005). But Panda et al., 2006 found a rise in PHB pool up to 11.2% (w/w of dry cells) in 0.4% acetate-supplemented cultures. Contrary to this, supplementation of glucose, fructose, ethanol and maltose were not found to stimulate PHB accumulation on dry weight basis. The superiority of the glucose in increasing PHB was observed in the present study, which was in line with Choi et al.,1994. They studied the biosynthesis of PHB by Hydrogenophaga pseudoflava using various carbon substrates. D-glucose (1%) yielded the highest PHB content of per cent.working with different carbon sources in MSM broth, Khanna and Srivastava (2005) observed higher PHB yield on fructose by A. eutrophus. They reported that glucose andfructose, being monosaccharides were readily utilized by bacteria and, hence, havesupported growth and subsequently PHB production. The complex molecules like starch andlactose were not utilized. Godabole et al., 2000 studied PHB production using whey as the substrate for biomass production in the first stage and lactic acid as the substrate for 68

18 accumulation of PHBin the second stage through which the maximum PHB content of 74 per cent of cell dry weight was obtained. Fig.32 Effect of different nitrogen source on biomass production Fig.33 Effect of different nitrogen source on PHB production Among different nitrogen sources tested, Di-ammonium hydrogen orthophosphate was found to be the best nitrogen source as it resulted in PHB of 3.59 g/l. The next promising 69

19 nitrogen source was peptone with 2.79 g/l and sodium ammonium hydrogen orthophosphate ((NH 4 ) 2 HPO 4 ) with 2.59 g/l PHB yield. Lillo and Valera (1990) found that ammonium chloride was found to be the least supporter of PHB production. It was evident from the results obtained from the present study that di-ammonium hydrogen orthophosphate was the best supporter of PHB production (3.59 g/l), followed by peptone (2.79g/L). These results were in agreement with the results obtained by Khanna and Srivastava 2005 who also observed the highest PHB production (2.2 %) by R. eutropha on MSM medium supplemented with ammonium sulphate. Mulchandani et al.,1989 and Raje and Srivastava (1989) also worked on the accumulation of PHB by A. eutrophus with different salts of ammonium. The highest PHB yield was obtained in ammonium sulphate followed by ammonium chloride. Thus the present study showed that the ph 7, 35 0 C, 3% NaCl, 6.5% DO, molasses as carbon source and di-ammonium hydrogen orthophosphate. Mass scale fermentation was done with theoptimized condiotns 70