Chapter 3. Screening and characterization of Clostridium strains on the
3.3 Results and discussion
3.3.5 Characterization of the strain in optimized media in bioreactor 69
effects of all the parameters along with interaction between glucose and peptone were found to be significant. Maximum biomass productivity of 0.28 g L-1h-1was predicted by the RSM for concentrations of glucose, peptone, and trace elements of 33.12 g L-1, 21.12 g L-1and 18.1 mL L-1, respectively. Similarly, maximum butanol productivity of 0.36 g L-1h-1was predicted for 82.04 g L-1of glucose, 49.66 g L-1of peptone and 23.2 mL L-1 of trace elements. Concentrations of glucose, peptone, and trace elements were found to be significantly different in the medium optimized for butanol and biomass productivity implying mutually exclusive nature of these two parameters.
The RSM predicted media composition was validated by corresponding experimental values. CCD-RSM based media optimization resulted in 103.5% increment in biomass productivity and 63.6% increment in butanol productivity as compared to the biomass (0.135 g L-1 h-1) and butanol productivity (0.22 g L-1 h-1) obtained from the un- optimized media composition (Table 3.7).
Various studies have been targeted towards increasing butanol titer (Kaushal et al., 2017; Kao et al., 2013; Yadav et al., 2014). However, limited reports are available with respect to improved butanol productivity for Clostridium spp. via media optimization. One such study was carried out by Moon et al. (2011), where maximization of butanol and 1, 3-PDO was achieved during active growth phase of C. pasteurianumDSM 525 through medium optimization.
Fig. 3.6. Dynamic profile for growth (•), pH (Æ, glucose (È), butanol (Î), acetone (×), ethanol (), acetic acid (◦) and butyric acid () whenC. acetobutylicum MTCC 11274 was grown in optimized medium for (A) maximum biomass productivity and (B) maximum butanol productivity.
When grown on the medium optimized for biomass productivity, cells entered into the exponential phase of growth without any distinct lag phase (Fig. 3.6A).
This dynamics of growth was concomitant with the rapid utilization of glucose from extracellular medium. However, in case of the medium optimized for butanol productivity, a distinct lag phase of 8 h was observed characterized by insignificant change in the extracellular glucose concentration (Fig. 3.6B). This distinct lag phase may be attributed to higher initial concentration of glucose in the medium resulting in substrate inhibition for growth (Qureshi and Blaschek, 2001). From the Fig. 3.6B, it is also evident that the rate of glucose utilization increased with the onset of the exponential phase of growth. Significantly higher specific growth rate (111%) and maximum volumetric biomass productivity (75%) was achieved in case of growth supporting medium as compared to the medium optimized for butanol productivity albeit with a lower biomass titer (Table 3.7). In both the media, butanol production was triggered as soon as the extracellular medium pH reached a critical value of 4.5 though the time required for initiation of butanol synthesis differed significantly depending on the type of medium used. In case of growth supporting medium, butanol production was initiated at 10 h, whereas the medium supporting butanol productivity required an additional 8 h for butanol induction. However, a two-fold higher maximum butanol titer of 12.56 g L-1 with a productivity of 0.31 g L-1 h-1 was achieved in case of butanol supporting medium as compared to the medium favoring growth (Table 3.8).
In case of growth supporting medium, cessation of butanol synthesis at 26 h Page|70 Department of Biosciences & Bioengineering| TH-2103_126106021
Table 3.8. Comparison of ABE fermentation in batch fermentation using optimized media for biomass productivity and butanol productivity by C. aceto- butylicumMTCC 11274
Parameters Medium optimized for biomass productivity
Medium optimized for butanol Productivity
Biomass (g L-1) 2.92 4.48
Butanol (g L-1) 6.07 12.56
Acetone (g L-1) 1.4 5.9
Ethanol (g L-1) 0 2.5
ABE (g L-1) 7.47 20.96
Lag time (h) NL 8
Max specific growth rate
(h-1) 0.38 0.18
Maximum biomass
productivity (g L-1h-1) 0.28 0.16
Overall butanol productivity
(g L-1h-1) 0.23 0.31
Butanol yield (g g-1glucose) 0.18 0.23
Batch time (h) 26 40
Residual acetic acid (g L-1) 1.2 1.8
Residual butyric acid (g L-1) 0.2 0.3
Residual glucose (g L-1) 0 26.9
NL-No lag phase
of fermentation may be attributed to lower concentration of glucose below a critical value of 5 g L-1(Long et al., 1984). It is important to note that when grown on the optimized medium for butanol productivity, the growth and butanol production was terminated followed by a decrease in the biomass concentration at around 40 h, in spite of presence of sufficient amount of residual glucose in the extracellular medium (Fig. 3.6B). This may be attributed to the possible butanol toxicity on the cells which corroborates with the findings of butanol tolerance experiments (Fig. 3.3). Similar results were reported forC. acetobutylicumATCC 824, where accumulation of butanol around 12-14 g L-1 resulted in onset of sporulation and cessation of fermentation (Maddox et al., 1989).
The organism exhibited a marked difference with reference to total solvent production in two different media. When the organism was cultivated in the medium optimized for butanol productivity approximately three-fold higher total ABE titer (20.96 g L-1) was obtained as compared to the growth supporting medium (7.47 g L-1). This lower titer of total solvent in case of the medium optimized for biomass productivity was attributed to non-production of ethanol coupled with lower production of acetone and butanol. ABE titer together with the distribution of individual solvent was reported to vary significantly over a range of 5.76 g L-1 to 19.9 g L-1 depending on the medium composition and the strain used (Chen et
al., 2014, Wu et al., 2013, Ezeji et al., 2004; Monot et al., 1982). Termination of fermentation due to glucose depletion in batch fermentation using medium optimized for biomass productivity gave the lead for development of a fed-batch strategy for improvement of butanol titer coupled with high biomass productivity. On the other hand, Termination of fermentation due to butanol toxicity in batch fermentation using medium optimized for maximization of butanol productivity gave the lead for development of fermentation strategy coupled with continuous product recovery.
3.4 Conclusion
Six strains were screened for maximum butanol production in two media compositions. Clostridium acetobutylicumMTCC 11274 was identified as the best butanol producer strain among the strains screened and selected for further studies.
The tryptone-yeast extract-glucose media (TYG) with P2 salt solution was found to be the best growth promoting and butanol producing media. Glucose and peptone were selected as the carbon and nitrogen source, respectively, on the basis of maximum butanol production. Butanol tolerance limit of C. acetobutylicum MTCC 11274 was found to be 14 g L-1. Statistical optimization resulted in two different media composition each for biomass productivity and butanol productivity. In case of media optimized with biomass productivity objective function an increase of 103.5%
was observed, whereas 63.6% increase in butanol productivity in case of butanol productivity as objective function. Glucose depletion in batch fermentation using medium optimized for biomass productivity gave the lead for development of a fed- batch strategy for improved butanol titer, while butanol toxicity in batch fermentation using medium optimized for maximization of butanol productivity actuated the need for integration of fermentation within situproduct recovery.