Victor Ujor

Use of Proteomic Analysis To Elucidate the Role of Calcium in Acetone-Butanol-Ethanol Fermentation by Clostridium beijerinckii NCIMB 8052

ABSTRACT Calcium carbonate increases growth, substrate utilization, and acetone-butanol-ethanol (ABE) fermentation by Clostridium beijerinckii NCIMB 8052. Toward an understanding of the basis for these pleiotropic effects, we profiled changes in the C. beijerinckii NCIMB 8052 proteome that occur in response to the addition of CaCO3. We observed increases in the levels of different heat shock proteins (GrpE and DnaK), sugar transporters, and proteins involved in DNA synthesis, repair, recombination, and replication. We also noted significant decreases in the levels of proteins involved in metabolism, nucleic acid stabilization, sporulation, oxidative and antibiotic stress responses, and signal transduction. We determined that CaCO3 enhances ABE fermentation due to both its buffering effects and its ability to influence key cellular processes, such as sugar transport, butanol tolerance, and solventogenesis. Moreover, activity assays in vitro for select solventogenic enzymes revealed that part of the underpinning for the CaCO3-mediated increase in the level of ABE fermentation stems from the enhanced activity of these catalysts in the presence of Ca2+. Collectively, these proteomic and biochemical studies provide new insights into the multifactorial basis for the stimulation of ABE fermentation and butanol tolerance in the presence of CaCO3.

Butanol production from hydrothermolysis-pretreated switchgrass: Quantification of inhibitors and detoxification of hydrolyzate

The present study evaluated butanol production from switchgrass based on hydrothermolysis pretreatment. The inhibitors present in the hydrolyzates were measured. Results showed poor butanol production (1g/L) with non-detoxified hydrolyzate. However, adjusting the pH of the non-detoxified hydrolyzate to 6 and adding 4 g/L CaCO3 increased butanol formation to about 6g/L. There was about 1g/L soluble lignin content (SLC), and various levels of furanic and phenolic compounds found in the non-detoxified hydrolyzate. Detoxification of hydrolyzates with activated carbon increased the butanol titer to 11 g/L with a total acetone, butanol and ethanol (ABE) concentration of 17 g/L. These results show the potential of butanol production from hydrothermolysis pretreated switchgrass.

Glycerol supplementation of the growth medium enhances in situ detoxification of furfural by Clostridium beijerinckii during butanol fermentation.

Lignocellulose-derived microbial inhibitors such as furfural and 5-hydroxymethyl furfural adversely affect fermentation of lignocellulosic biomass hydrolysates to fuels and chemicals due to their toxicity on fermenting microbes. To harness the potential of lignocellulose as a cheap source of fermentable sugars, in situ detoxification of furfural and other lignocellulose-derived microbial inhibitors is essential. To enhance in situ detoxification and tolerance of furfural by Clostridium beijerinckii NCIMB 8052 during acetone-butanol-ethanol (ABE) fermentation, the effect of glycerol on NADH/NADPH generation and ABE production by furfural (4, 5, and 6 g/L)-challenged cultures was investigated in this study. In all instances, beneficial outcomes were observed. For example, the fermentation medium supplemented with glycerol and subjected to 5 g/L furfural elicited up to 1.8- and 3-fold increases, respectively, in NADH and NADPH levels in C. beijerinckii 8052 relative to the control culture. These critical changes are the likely underpinnings for the glycerol-mediated 2.3-fold increase in the rate of detoxification of 5 g/L furfural, substrate consumption, and ABE production compared to the unsupplemented medium. Collectively, these results demonstrate that increased intracellular NADH/NADPH in C. beijerinckii 8052 due to glycerol utilization engenders favorable effects on many aspects of cellular metabolism, including enhanced furfural reduction and increased ABE production.

Evaluation of industrial dairy waste (milk dust powder) for acetone-butanol-ethanol production by solventogenic Clostridium species.

Readily available inexpensive substrate with high product yield is the key to restoring acetone-butanol-ethanol (ABE) fermentation to economic competitiveness. Lactose-replete cheese whey tends to favor the production of butanol over acetone. In the current study, we investigated the fermentability of milk dust powder with high lactose content, for ABE production by Clostridium acetobutylicum and Clostridium beijerinckii. Both microorganisms produced 7.3 and 5.8 g/L of butanol respectively, with total ABE concentrations of 10.3 and 8.2 g/L, respectively. Compared to fermentation with glucose, fermentation of milk dust powder increased butanol to acetone ratio by 16% and 36% for C. acetobutylicum and C. beijerinckii, respectively. While these results demonstrate the fermentability of milk dust powder, the physico-chemical properties of milk dust powder appeared to limit sugar utilization, growth and ABE production. Further work aimed at improving the texture of milk dust powder-based medium would likely improve lactose utilization and ABE production.

Process development for biological production of butanol from Eastern redcedar

Eastern redcedar is an invasive softwood species in Oklahoma and across grasslands in the Central Plains of the United States and potential feedstock for butanol production. Butanol has higher energy content than ethanol and can be upgraded to jet and diesel fuels. The objective of this study was to develop a process for production of butanol from redcedar. Results showed that Clostridium acetobutylicum ATCC 824 and Clostridium beijerinckii NCIMB 8052 did not grow in fermentation medium with citrate buffer. However, both strains grew in the medium with acetate buffer, resulting in 3-4g/L greater butanol than without acetate. Detoxification of redcedar hydrolyzate was required to increase butanol concentration from 1 to 13g/L. Hydrolyzate was detoxified by activated carbon to remove inhibitors. Fermentations in detoxified redcedar hydrolyzate reached 13g/L butanol and 19g/L total ABE, comparable to glucose control. This shows the potential for redcedar use in butanol production.

Allopurinol-mediated lignocellulose-derived microbial inhibitor tolerance by Clostridium beijerinckii during acetone-butanol-ethanol (ABE) fermentation.

In addition to glucans, xylans, and arabinans, lignocellulosic biomass hydrolysates contain significant levels of nonsugar components that are toxic to the microbes that are typically used to convert biomass to biofuels and chemicals. To enhance the tolerance of acetone–butanol–ethanol (ABE)-generating Clostridium beijerinckii NCIMB 8052 to these lignocellulose-derived microbial inhibitory compounds (LDMICs; e.g., furfural), we have been examining different metabolic perturbation strategies to increase the cellular reductant pools and thereby facilitate detoxification of LDMICs. As part of these efforts, we evaluated the effect of allopurinol, an inhibitor of NAD(P)H-generating xanthine dehydrogenase (XDH), on C. beijerinckii grown in furfural-supplemented medium and found that it unexpectedly increased the rate of detoxification of furfural by 1.4-fold and promoted growth, butanol, and ABE production by 1.2-, 2.5-, and 2-fold, respectively. Since NAD(P)H/NAD(P)+ levels in C. beijerinckii were largely unchanged upon allopurinol treatment, we postulated and validated a possible basis in DNA repair to account for the solventogenic gains with allopurinol. Following the observation that supplementation of allopurinol in the C. beijerinckii growth media mitigates the toxic effects of nalidixic acid, a DNA-damaging antibiotic, we found that allopurinol elicited 2.4- and 6.7-fold increase in the messenger RNA (mRNA) levels of xanthine and hypoxanthine phosphoribosyltransferases, key purine-salvage enzymes. Consistent with this finding, addition of inosine (a precursor of hypoxanthine) and xanthine led to 1.4- and 1.7-fold increase in butanol production in furfural-challenged cultures of C. beijerinckii. Taken together, our results provide a purine salvage-based rationale for the unanticipated effect of allopurinol in improving furfural tolerance of the ABE-fermenting C. beijerinckii.

The mycelial response of the white-rot fungus, Schizophyllum commune to the biocontrol agent, Trichoderma viride.

In this study, agar plate interaction between Schizophyllum commune and Trichoderma viride was investigated to characterise the physiological responses occurring during interspecific mycelial combat. The metabolite profiles and morphological changes in both fungi paired on agar were studied relative to the modulation of phenoloxidase activity in S. commune. The calcium ionophore A23187 was incorporated in self-paired cultures of S. commune to explore possible involvement of calcium influx in the response of S. commune to T. viride. The levels of lipid peroxides and protein carbonyls in the confronted mycelia of S. commune were also measured. Contact with T. viride induced pigmentation and cell wall hydrolysis in S. commune with concomitant increase in phenoloxidase activity, rise in the levels of oxidative stress indicators and increased levels of phenolic compounds, antioxidant γ-amino butyric acid, and pyridoxine and osmo-protective sugar alcohols. Calcium ionophore mimicked the pigmentation in the T. viride-confronted mycelia of S. commune, implicating calcium influx in the response to T. viride. The changes in S. commune are indicative of targeted responses to osmotic and oxidative stresses and phenoloxidase-mediated detoxification of noxious compounds in the contact interface with T. viride, which may confer resistance in natural environments.

Quantitative proteomic analysis of the response of the wood-rot fungus, Schizophyllum commune, to the biocontrol fungus, Trichoderma viride

Aims:  Investigation of changes in the protein profile of the wood‐rot fungus, Schizophyllum commune, when paired against the biocontrol fungus, Trichoderma viride, for 48 h.

Global Climate Change: Enteric Methane Reduction Strategies in Livestock

The greenhouse gas (GHG) emission from the agricultural sector is considered to be a key contributor to the climate change, accounting for about 25.5% of total global anthropogenic emission. While carbon dioxide (CO2) receives the most attention as a factor, which causes global warming, methane (CH4), nitrous oxide (N2O), and chlorofluorocarbons (CFCs), also cause significant radiative forcing. With the relative global warming potential of 25 compared with CO2, CH4 is one of the most important GHGs. This chapter reviews the prediction models, estimation methodologies, and strategies for reducing enteric CH4 emissions. Emission of CH4 in ruminants differs between developed and developing countries, depending on factors like animal species, breed, pH of rumen fluid, ratio of acetate: propionate, methanogen population, composition of diet, and amount of concentrate fed. Among ruminants, cattle contribute the most toward greenhouse effect through methane emission, followed by sheep, goat and buffalo, respectively. The estimated CH4 emission rate per cattle, buffalo, sheep, and goat in developed countries are 150.7, 137, 21.9, and 13.7 (g/animal/day) respectively. However, the estimated rates in developing countries are significantly lower, at 95.9 and 13.7 (g/animal/day) per cattle and sheep, respectively. There is a strong interest in developing new and improving existing CH4 prediction models that are effective in identifying mitigation strategies for reducing the overall CH4 emissions. A careful examination of the literature suggests that the mechanistic models are superior to empirical models in accurately predicting the CH4 emission from dairy farms. The latest development in prediction model is the integrated farm system model, which is a process-based whole farm simulation technique. Several techniques are used to quantify enteric CH4 emissions starting from whole animal chambers to sulfur hexafluoride (SF6) tracer techniques. The latest technology developed to estimate CH4 more accurately is the micrometeorological mass difference technique. Understanding this basic information about enteric methane is very vital for formulating suitable mitigation strategies to curtail methane production. There are varieties of mitigation strategies available, which can be grouped under managemental, nutritional, and advanced molecular technologies. Strategies that are cost-effective, improve productivity, with potentially limited negative effects on livestock production are likely to be adopted by producers.

Unorthodox methods for enhancing solvent production in solventogenic Clostridium species

While production of biofuels from renewable resources is currently receiving increased attention globally, concerns on availability and sustainability of cheap substrates for their production are growing as well. Lignocellulose-derived sugars (LDS) remain underutilized and merit consideration as a key feedstock. Among other obstacles such as low yield and low solvent titer, mitigation of stresses stemming from lignocellulose-derived microbial inhibitory compounds (LDMICs) that severely impair cell growth and solvent production is a major area of research interest. In addition to attempts at developing LDMIC-tolerant strains via metabolic engineering to enhance utilization of LDS, unconventional approaches that elicit different metabolic perturbations in microorganisms to relieve solvent- and LDMIC-mediated stresses have been explored to increase solvent production from LDS. In this review, the impacts of metabolic perturbations including medium supplementation with glycerol; furfural and 5-hydroxymethyl furfural; allopurinol, an inhibitor of xanthine dehydrogenase; calcium (Ca2+) and zinc (Zn2+) ions); and artificial electron carriers, methyl viologen and neutral red, on butanol production are discussed. Although these approaches have brought about considerable increases in butanol production, both from LDS and defined glucose-based media, the modes of action for most of these perturbations have yet to be fully characterized. Better understanding of these mechanisms would likely inform development of LDMIC-tolerant, butanol-overproducing strains, as well as possible combinatorial application of these approaches for enhanced butanol production. Hence, delineating the underlying mechanisms of these perturbations deserves further attention.