Based on batch experiments conducted using media obtained to maximize biomass productivity and maximize butanol productivity, a two-stage batch fermentation strategy was developed. Comparison of butanol titer (gray line) and productivity (black circle,•) in different two-stage batch fermentations.
Background and motivation
Systems biology approaches such as static and kinetic modeling have been undertaken to understand the regulatory mechanisms involved in butanol biosynthesis (Kaushal et al., 2018). Another approach being investigated to achieve process-level improvement in ABE fermentation is media engineering (Li et al., 2014) and the use of environmental factors that increase the butanol synthesis potential of Clostridium spp.
Objectives of the present study
Process-level improvements have been achieved through approaches including the application of various alternative substrates (Gottumakkala et al., 2013; Kaushal et al., 2017), fermentation strategies to increase butanol titer, yield and productivity, and coupling of fermentation within on-site product recovery technologies. overcome the toxicity of butanol (Qureshi et al., 2008; Lu et al., 2012). Some studies examining factors such as different pH strategies (Oshiro et al., 2010); supplementation of inducers such as butyric acid/sodium butyrate, zinc, calcium carbonate (Wang et al., 2013; Wu et al., 2013; Wu et al., 2016); and limitation of trace minerals: iron, magnesium, phosphate, sulfate (Bahl et al., 1986) have resulted in improved kinetics of the butanol process.
Approach
Biochemical engineering and metabolic modeling approaches used to achieve high butanol titer and butanol productivity and to understand the regulations involved in butanol biosynthesis for Clostridium acetobutylicum MTCC 11274. Key nutrients were then optimized via a statistical optimization method to maximize maximization of achieve biomass productivity and maximization of butanol productivity.
Thesis organization
In the broader context, the present study was focused on developing an efficient strategy for butanol production through reduced fermentation time, improved butanol titer and productivity, and reduced nutrient waste and substrate costs.
These include: (i) sterilization of pretreatment biomass by autoclaving before enzymatic hydrolysis, (ii) addition of antibiotics to the hydrolysis or fermentation medium, (iii) sterilization of hydrolysis and fermentation equipment and pipelines, (iv) optimization of the products pretreatment in such a way that they inhibit contamination without affecting the producer organism and (v) engineering microorganisms to produce endolysin (Serate et al., 2015). Experiments were conducted on P2 media (Monot et al., 1982) optimized for biomass productivity and butanol productivity.
Literature Review 9
Acetone-Butanol-Ethanol (ABE) fermentation
- History
- Microorganisms
- Substrates
- Factors affecting growth and solvent production
- Solvent toxicity
- Different fermentation strategies
- Downstream processing
- Metabolic modeling
Butanol, which is the main product of the mixture, is also the most toxic (Izard et al., 1989). Butanol after permeation is fed as a vapor to the cold trap for condensation (Abdehagh et al., 2014).
Current challenges
Apart from GSM, methods for small-scale metabolic network analysis such as elementary mode analysis (EMA), flux balance analysis (FBA), and metabolic flux analysis (MFA) are widely used ( Millat et al., 2017 ). For example, Kumar et al. 2014) used elementary mode analysis as a tool to quantify metabolic fluxes inC. acetobutylicum under different external pH and in response to the addition of exogenous acids.
As described in section 5.2.3, the organism was initially grown in the medium that supported maximum biomass productivity during the first phase of the two-phase process engineering strategy. The inductors were replenished in the second phase of the two-stage fed-batch strategy. Serial enrichment of butanol tolerance was performed along with chemical mutagenesis to improve the tolerance limit of the strain.
Screening and characterization of Clostridium strains on the
Materials and methods
- Microorganisms, maintenance, and inoculum preparation
- Screening of potential butanol producing strain and selection
- Characterization of the selected strain under different carbon
- Evaluation of the strain for solvent tolerance
- Statistical medium optimization for maximization of biomass
10 mL of each of the actively growing cultures was transferred to 500 mL airtight glass bottles containing 100 mL of TGY medium and incubated under similar conditions for seed culture development. The batch fermentation was carried out in 500 ml airtight glass bottles containing 100 ml of the respective medium for 120 hours at 37°C under static conditions. Biochemical characterization of the selected strain was performed in accordance with Bergey's Manual of Determinative Bacteria (Buchanan and Gibbons, 1974) and the tests performed are as follows: motility, catalase test, oxidase, Voges Prokauer, urease utilization, indole production, lipase, growth at different initial pH and 7), Nagler, gelatin hydrolysis, hydrogen sulphide production, milk coagulation, nitrate reduction, lecithinase test and growth on 24 different carbon sources.
Results and discussion
- Screening of Clostridium strains as a potential cell factory for
- Characterization of the strain under different carbon and
- Evaluation of the strain for solvent tolerance
- Statistical optimization of media for maximization of biomass
- Characterization of the strain in optimized media in bioreactor 69
Significantly higher specific growth rate (111%) and maximum volumetric biomass productivity (75%) were achieved in the case of the growth support medium compared to the medium optimized for butanol productivity, albeit with a lower biomass titer (Table 3.7). Comparison of ABE fermentation in batch fermentation using optimized media for biomass productivity and butanol productivity with C. Statistical optimization resulted in two different media compositions for biomass productivity and butanol productivity.
A two-stage feed process engineering strategy was used to increase butanol volumetric productivity. Production of high-titer n-butanol by Clostridium acetobutylicumJB200 in batch fermentation with intermittent degassing. This titer was three times higher when compared to batch fermentation fed two stages without degassing (13.1 g L-1).
Enhancement of butanol tolerance of the selected strain via
Materials and methods
- Microorganism, inoculum, and media
- UV irradiation of cultures and induction of mutants
- Chemical mutagenesis using N-methyl-N-nitro-N-nitrosoguanidine
- Chemical mutagenesis using ethyl methane sulfonate (EMS) 81
- Characterization of mutants for butanol tolerance and butanol
- Adaptive evolution to enhance butanol tolerance
- Analytical methods
Inoculum preparation was performed in tryptone-yeast-glucose (TYG) medium as detailed in section 3.2.1. Stored glycerol stocks were revived and inoculum was prepared as explained in section 4.2.1. The mutants were also characterized for their tolerance to butanol and butanol challenge experiments were performed as detailed in section 3.2.4.
Results and discussion
- Optimization of mutagenic conditions
- Rational screening of solvent tolerant mutant(s)
- Characterization of putatively improved strain(s) for butanol
- Adaptive evolution to enhance butanol tolerance
Taking this point of view, three mutagenesis experiments, namely UV-irradiation, NTG treatment and mutagenesis using EMS, were performed according to the optimal conditions obtained in Section 4.3.1. This represented one adaptation cycle and was performed in a similar manner to cells under stress. Adjustment to 1.0% was performed for 18 cycles; however, no further improvement was achieved in terms of specific growth rate.
Conclusion
Bar chart showing the comparison of putatively improved strains after mutagenic treatment with the wild-type (WT) strain in terms of biomass (gray bar), butanol titer (back bar) and butanol productivity (red circle). Similarly, the strain was found to be adapted in the third stage of adaptation (exposure to 0.75% butanol) after seven cycles (Figure 4.7). EMS treatment yielded eight putatively improved strains, none of which showed an improved butanol titer compared to the wild type.
Production of high titer n-butanol by Clostridium acetobutylicum JB200 in fed-batch fermentation with intermittent gas stripping. Two-stage fed-batch fermentation was performed combining magnesium restriction in stage one with calcium supplementation in stage two. Furthermore, with the aim of reducing raw material costs, the two-phase fed-batch fermentation was carried out using low-cost substrates.
Studies on effect of various factors governing growth and
Materials and methods
- Microorganisms, maintenance, and inoculum preparation
- Two-stage fed-batch process engineering strategy
- Effect of limitation and starvation of metal ions in two-stage
- Effect of supplementation of metal ions in two-stage fed-batch
- Two-stage fed-batch process combined with metal ions
- Effect of calcium carbonate on butanol tolerance
- Analytical methods
- Two-stage fed-batch process engineering strategy
- Effect of limitation and starvation of metal ions in two-stage
- Effect of supplementation of metal ions in two-stage fed-batch
- Combinatorial use of limitation and supplementation of metal
After induction of solventogenic phase, the concentrations of glucose and peptone were adjusted to mimic the medium optimized for maximum butanol productivity, thereby initiating the second step of the process. With the onset of butanol production, the concentrations of glucose and peptone were adjusted (as described in section 5.2.3) and the medium was supplemented with different individual concentrations of the selected inducers. To study the effect of supplementation of the inducer on the tolerance limit, MTCC 11274 was grown, under batch mode, in the medium promoting maximum butanol productivity supplemented with optimal concentration of CaCO3.
Conclusion
Comparison of model predicted and experimentally determined specific growth rates (h-1) and butanol flux (mmol g-1h-1) for different batch and two-stage fed-batch fermentation. Distribution of carbon fluids under MgSO4-limited and CaCO3-supplemented two-stage fed-batch fermentation (A) at 10 h with maximization of biomass as objective function and (B) at 20 h with maximization of butanol as objective function. However, the titer was limited due to butanol toxicity; for this purpose, intermittent gas stripping is integrated with the two-stage fed-batch fermentation process.
Development of a mathematical model to understand the
Materials and methods
- Organism, growth, and maintenance conditions
- Flux balance analysis
- Reconstruction of metabolic network
- Biomass composition
- Measureable external fluxes
In addition, the use of a mass balance and the assumption of a pseudo-steady state resulted in a stoichiometric model (Orth et al., 2010): S.v=0. However, in the case of ferredoxin: NADPH oxidoreductase, the specific activity of the enzyme NADPH: ferredoxin oxidoreductase was found to be low (Gheshlaghi et al., 2009). Therefore, the enzyme was considered irreversible, and the main role was the production of NADPH (Jungermann et al., 1973).
Results and discussion
- Nutrient dependent modulation of biomass and product
- Flux distribution of C. acetobutylicum MTCC 11274 when
Percentage change in absolute flux values under two-stage fed-batch fermentation with respect to batch fermentation using biomass productivity medium at 10 hours and two-stage fed-batch fermentation with respect to batch fermentation using butanol productivity medium at 20 hours of growth. Percent change in absolute flux values under two-stage fed-batch fermentation with respect to MgSO4 limited two-stage fed-batch fermentation at 10 h and 20 h of growth. Percentage change in absolute flux values under CaCO3 supplemented two-stage fed-batch fermentation with respect to two-stage fed-batch fermentation at 20 hours of growth.
Conclusion
With the aim of reducing the raw material costs in experiment IV, the two-stage fed-batch fermentation was carried out using low-cost substrates. Fed-batch fermentation with intermittent gas stripping using immobilized Clostridium acetobutylicum for biobutanol production from corn stover bagasse hydrolyzate. Fed-batch fermentation for n-butanol production from cassava bagasse hydrolyzate in a fiber bed bioreactor with continuous gas stripping.
Development of a bioprocess coupled with in situ product
Materials and methods
- Organism, growth, and maintenance conditions
- Intermittent gas stripping coupled two-stage fed-batch process
- Combinatorial use of limitation and supplementation of
- Two-stage fed-batch fermentation with gas stripping using
- Analytical methods
In the setup, the condenser discharge of the bioreactor was connected to the Graham condenser inlet (Borosil, India), which was further connected to the sparger inlet via a vacuum pump (Tarsons, India). The fermenter discharges were connected to a condenser (Graham coiled still type, 62 mm internal diameter and 500 mm jacket length, Borosil, India) for condensing the solvents into the gas phase. Experiment II: Intermittent feeding of key nutrients throughout the duration of fermentation in Experiment I resulted in a significantly high amount of unused nutrients (82.9 g glucose and 49 g peptone) at the end of cultivation.
Results and discussion
- Intermittent gas stripping coupled two-stage fed-batch process
- Combinatorial use of limitation and supplementation of
Experiment III: Two-stage fed-batch strategy developed in Experiment I resulted in 26% improved butanol titer compared to control fermentation (13.1 g L-1) due to improved butanol tolerance. Integration of two-stage fed-batch process with gas stripping alleviated butanol toxicity and resulted in extended fermentation duration with a cumulative butanol titer of 40.3 g L-1 and productivity of 0.41 g L-1 h-1. Two-step fed-batch strategy with half concentration of MgSO4 compared to the original resulted in improved titer of 13.41 g L-1.
