Response Surface Methodology for Understanding Glucose and Xylose Utilization by Clostridium beijerinckii NCIMB 8052

We applied response surface methodology to understand the effect and extend of carbon catabolite repression (CCR) on growth of Clostridium beijerinckii NCIMB 8052 using xylose and glucose as representative lignocellulosic sugars. We performed batch growth experiments based on the central composite design with different concentrations of glucose and xylose, and estimated the respective growth rates as the response. Fitting the quadratic model with interaction coefficient to experimental data gave a good quality of fit (R-squared=0.939). We found that glucose is the most significant factor affecting the growth rate. Interaction between glucose and xylose is another highly significant factor. Response surface illustrated that increasing or decreasing both sugar concentrations at the same time results in a decreasing growth rate, and increasing either sugar concentration while decreasing the other sugar increases the growth rate. It is an important finding as it suggests that CCR can be not only from glucose on xylose but also from xylose on glucose. A transcriptional study will be necessary to understand the repression mechanism and to improve the utilization of sugars in mixed form, thus lignocellulosic fermentation processes.publishedVersio

The Effect of Feeding Strategy on Butanol Production by Clostridium beijerinckii NCIMB 8052 Using Glucose and Xylose

We performed fed-batch fermentations of glucose and xylose mixtures producing butanol. Our aim was to develop a feeding strategy for coping with carbon catabolite repression (CCR) and sequential utilization problems as well as understanding the effect of feeding strategy on fermentation kinetics. Experimental results showed that fermenter 1 with only xylose as the initial carbon source could co-utilize sugars for all mixed sugar feeds. On the other hand, fermenter 2 with only glucose as the initial sugar showed sequential utilization. Xylose in fermenter 2 accumulated while glucose was present; it was only utilized after the glucose was completely exhausted. Besides the sugar utilization profile, the feeding strategy had an impact on the fermentation kinetics. Maximum specific growth rates were 0.68 h-1 and 0.94 h-1, for fermenter 1 and 2,respectively. Fermenter 1 produced 4.98 g/l butanol and yield was 0.28 g/g, while fermenter 2 produced 0.5 g/l butanol with a yield value of 0.05 g/g. Total sugar utilization was also higher for fermenter 1, 81 % and 46 % for fermenter 2. The feeding strategy we proposed showed that wild type Clostridium beijerinckii NCIMB 8052 can co-utilize glucose and xylose, and produce butanol. Our observation suggests that we can tackle sequential utilization problem and enhance fermentation process with the proposed feeding strategy without having to manipulate the strain.publishedVersio

Modeling the Growth of Clostridium beijerinckii NCIMB 8052 on Lignocellulosic Sugars

To our knowledge, this is the first growth model of Clostridium beijerinckii NCIMB 8052 on glucose and xylose as representative lignocellulosic sugars, which considers the synergistic effects of sugars on the growth rate. We fitted models with different types of interactions between the substrates to the growth rate data obtained with varying sugar concentrations. Noncompetitive binary substrate growth model gave the best fit with the smallest mean standard errors (MSE), and sum of squares error (SSE), 0.0778 and 0.0071, respectively. Confidence intervals for the parameter estimates showed that the substrate affinity constant for xylose, KsX (g/l) had the largest uncertainty, while the maximum specific growth rate on xylose, µmaxX (h-1) had the smallest. The correlation matrix showed that the model parameters were highly correlated. Carbon cataboliterepression (CCR) effect on the growth rate was of the noncompetitive type. Validation with other sugar concentration values is necessary to evaluate the prediction capability of the proposed model. A transcriptional study will be beneficial to understand global gene regulation mechanisms as guidance for improving the efficiency of lignocellulosic fermentation processes.publishedVersio

Continuous fermentation and kinetic experiments for the conversion of crude glycerol derived from second-generation biodiesel into 1,3 propanediol and butyric acid

This study investigated the performance of different mixed microbial cultures (MMC) able to ferment crude glycerol generated from animal fat-based biodiesel to produce 1,3 propanediol (1,3 PDO) and butyric acid, under non-sterile conditions. Eight different continuous flow stirred-tank reactors (CSTR) were set up with different inoculum types and growth media. The distribution of metabolic products under variable operating conditions was determined. All MMC were characterized from a kinetic point of view and overall stoichiometric reactions were constructed. Changes in the microbial communities were monitored by means of Next Generation Sequencing (NGS). Maximum substrate degradation rate reached approximately 110 g/L/d of glycerol (with a productivity of 38 g/L/d and 11 g/L/d for 1,3 PDO and butyric acid, respectively), obtained with an hydraulic retention time of 12 h and 60 g/L feed. The maximum feed concentration reached almost 90 g/L, leading though to an incomplete substrate degradation.acceptedVersio

Butanol production from lignocellulosic sugars by Clostridium beijerinckii in microbioreactors

Butanol (n-butanol) has been gaining attention as a renewable energy carrier and an alternative biofuel with superior properties to the most widely used ethanol. We performed 48 anaerobic fermentations simultaneously with glucose and xylose as representative lignocellulosic sugars by Clostridium beijerinckii NCIMB 8052 in BioLector® microbioreactors to understand the effect of different sugar mixtures on fermentation and to demonstrate the applicability of the micro-cultivation system for high-throughput anaerobic cultivation studies. We then compared the results to those of similar cultures in serum flasks to provide insight into different setups and measurement methods

Butanol production from lignocellulosic sugars by Clostridium beijerinckii in microbioreactors

Butanol (n-butanol) has been gaining attention as a renewable energy carrier and an alternative biofuel with superior properties to the most widely used ethanol. We performed 48 anaerobic fermentations simultaneously with glucose and xylose as representative lignocellulosic sugars by Clostridium beijerinckii NCIMB 8052 in BioLector® microbioreactors to understand the effect of different sugar mixtures on fermentation and to demonstrate the applicability of the micro-cultivation system for high-throughput anaerobic cultivation studies. We then compared the results to those of similar cultures in serum flasks to provide insight into different setups and measurement methods

Efficacy of a novel sequential enzymatic hydrolysis of lignocellulosic biomass and inhibition characteristics of monosugars

Efficient production of sugar monomers from lignocellulose is often hampered by serious bottle-necks in biomass hydrolysis. The present study reveals that ultra-sonication assisted pretreatment following autoclaving, termed as combined pretreatment, can lead to more efficient delignification of lignocellulosic biomass and an open, deformed polysaccharide matrix, found favorable for subsequent enzymatic hydrolysis, is formed. The pattern of inhibition for the enzymatic hydrolysis reaction on combined-pretreated saw dust is identified. Two main inhibition models (competitive and noncompetitive) are proposed and a better fit of experimental values with the theoretical values for the competitive inhibition model validates the proposition that in the present experiment, glucose inhibits the enzymes competitively. Additionally, accuracy of the inhibitory kinetics based models is estimated over a series of enzyme and substrate concentrations.acceptedVersio

Valorization of Lignocellulosic Waste (Crotalaria <i>juncea</i>) Using Alkaline Peroxide Pretreatment under Different Process Conditions: An Optimization Study on Separation of Lignin, Cellulose, and Hemicellulose

<p>Lignocellulose materials, essentially consisting of lignin, cellulose and hemicelluloses, are abundant sources of fermentable sugars. The bast fiber of Crotalaria <i>juncea</i> (<i>Sunn hemp</i>), a native cover crop of India, was used as feedstock for this study. The primary objective of this study was to add value to the waste C. <i>juncea</i> bast fiber. The same was achieved by pretreating the waste fiber using alkaline peroxide solution at various process conditions. The optimal process condition for alkaline peroxide pretreatment was identified for the fiber to pretreatment solution (S/S) ratio of 1/40 at 50°C for 5h with respect to all response variables (lignin removal, hemicellulose recovery, recovery of solid pretreated material, and crystallinity of lignocellulose). Three-way ANOVA results revealed that S/S ratio had no significant effect; whereas, both temperature and time, and the combination of parameters, exhibited significant effect on response variables. The characteristic peaks associated with lignin and cellulose demonstrated a higher amount of lignin removal and increased cellulose content with elevated treatment time. Autoclave assisted pretreatment proved to be inefficient due to removal of lower amount of lignin in addition to higher hemicellulose degradation. On the other hand, pretreatment using ultrasound was found to be most effective in removing lignin, liberating hemicelluloses along with diminition in cellulose crystallinity.</p

Studies on delignification and inhibitory enzyme kinetics of alkaline peroxide pre-treated pine and deodar saw dust

Delignification of lignocellulosic biomass by alkaline peroxide pre-treatment is a preliminary important step for an overall biomass fractionation process. In the present work, saw dusts are pre-treated by aqueous alkaline peroxide solution under different temperatures over a predetermined time. It is seen that Combined Pre-treatment (CP) removes a substantially higher quantity of lignin from biomass under a particular temperature. At elevated temperatures, the extent of delignification is observed much better. The % removal is: [PR: 19.35%(30 °C):25.26%(50 °C):33.30%(100 °C)]; [CD:14.64%(30 °C):23.64%(50 °C): 28.83%(100 °C)]. Batch kinetics is investigated with certain models and corresponding parameters are estimated. As pre-treatment severity is strongly correlated to the pre-treatment temperature, increased value of “potential degree of delignification” is observed at escalated temperatures. Kinetics of enzymatic hydrolysis of delignified biomass shows decreased product inhibition with increased substrate concentration under a particular enzyme loading. Starting with a combination of 50 g/L substrate concentration with an enzyme loading of 13.23 g/L, an optimum concentration of 17.2 g/L and 21.19 g/L of glucose are produced from Pinus roxburghii and Cedrus deodara respectively. Experimental data fit quite well with the competitive inhibition kinetics based theoretical models with r2 ≥0.95. It is inferred that enzymes are competitively inhibited by glucose

Towards a low CO2 emission building material employing bacterial metabolism (1/2): The bacterial system and prototype production

The production of concrete for construction purposes is a major source of anthropogenic CO2 emissions. One promising avenue towards a more sustainable construction industry is to make use of naturally occurring mineral-microbe interactions, such as microbial-induced carbonate precipitation (MICP), to produce solid materials. In this paper, we present a new process where calcium carbonate in the form of powdered limestone is transformed to a binder material (termed BioZEment) through microbial dissolution and recrystallization. For the dissolution step, a suitable bacterial strain, closely related to Bacillus pumilus, was isolated from soil near a limestone quarry. We show that this strain produces organic acids from glucose, inducing the dissolution of calcium carbonate in an aqueous slurry of powdered limestone. In the second step, the dissolved limestone solution is used as the calcium source for MICP in sand packed syringe moulds. The amounts of acid produced and calcium carbonate dissolved are shown to depend on the amount of available oxygen as well as the degree of mixing. Precipitation is induced through the pH increase caused by the hydrolysis of urea, mediated by the enzyme urease, which is produced in situ by the bacterium Sporosarcina pasteurii DSM33. The degree of successful consolidation of sand by BioZEment was found to depend on both the amount of urea and the amount of glucose available in the dissolution reaction