Identification of the Toxic Mechanisms Employed by Lignin Derivatives and Its Application to Improve n-Butanol Production within Clostridium beijerinckii

Department of Biological SciencesThe main focus of this study is the transcriptomic analysis of bacterial stains exposed to lignin hydrolysate-related compounds to determine the effects of them on global gene expression thereby evaluating their stresses, and the use of the results from the analysis to make novel strains. To achieve this, some works were performed: transcriptomic analysis with a model organism and its use for biosensor construction, and another transcriptomic analysis using a fermentative strain and construction of lignin-derived phenolic compound tolerant strain. Initially, global transcriptional response of E. coli BL21 (DE3) exposed to ferulic acid was investigated by microarray. When E. coli BL21 (DE3) was cultured in presence of 0.25 or 0.5 g/L ferulic acid, several gene sets showing significant change were identified. It was also shown that several individual genes were strongly up-regulated, including marA, inaA, aaeA and aaeB. Thus, these 4 genes, along with htpG, were selected as biomarker genes for phenolics toxicity. Five biomarkers were applied to monitor the response of E. coli exposed to various phenolic acid and aldehyde, which can be generated from lignin, and spruce hydrolysate, where the potential of those five genes was demonstrated as biomarkers to determine toxicity of lignin-derived chemicals. Afterwards, based upon the analysis, transcriptional fusions with the lux operon were constructed using the promoter of inaA, aaeXAB, and yhcN, giving pSP4, pDMA3, pDMY1, respectively, in collaboration with Ajay Kalanjana Monnappa. First, pSP4 was characterized within E. coli BL21 (DE3) with several compounds, suggesting that phenolics were major inducers other than acetic acid and furfural. The Plasmid pSP4 inside different hosts was also tested, including mar regulon mutants, and compared with pDMA3 and pDMY1. Furthermore, these bioreporter strains were applied to monitor the degradation of several aromatic compounds by T. aromatica or A. baylyi, and to analyze spruce and rice straw hydrolysates, which demonstrates that the bioreporters constructed are possible to analyze lignocellulosic biomass hydrolysates for the presence of inhibitory phenolic compounds. In addition, a novel technology for improving response of bioreporter strains was investigated by using serum complement. The effect of this method was proven using genotoxin detectable bioreporter, pRec3. Finally, the effects of ferulic acid on growth, solvent production, and transcriptome of C. beijerinckii NCIMB 8052 were investigated. C. beijerinckii growth rate and butanol production was decreased by ferulic acid at 0.5 g/L. Through microarray analysis with 3 different exposure duration, several groups of the genes were found to be highly up-regulated or down-regulated, including efflux system, heat shock proteins, and two-component system. Among the genes showing significant induction responding to ferulic acid, groE operon genes was overexpressed within C. beijerinckii to have this strain tolerant to lignin hydrolysate-related compounds, which was characterized in this study.ope

Biological activities of lignin hydrolysate-related compounds

Lignin hydrolysates contain many different chemical species, including ferulic acid, coumaric acid, vanillic acid, vanillin, syringaldehyde and furfural. From the perspective of biofuels, these compounds are problematic and can cause downstream loss of product if not removed prior to beginning the fermentative process. In contrast, a search for these compounds within the literature turns up many papers where the same compounds have beneficial properties pertaining to human health, including as antioxidants and in cancer prevention, or are involved in bacterial cell-to-cell signaling. Consequently, this article reviews the dual nature of these and other compounds found in lignin hydrolysates, highlighting both their detrimental and beneficial activities.close7

Improved Sugar Production by Optimizing Planetary Mill Pretreatment and Enzyme Hydrolysis Process

This paper describes an optimization of planetary mill pretreatment and saccharification processes for improving biosugar production. Pitch pine (Pinus rigida) wood sawdust waste was used as biomass feedstock and the process parameters optimized in this study were the buffering media, the milling time, the enzyme quantity, and the incubation time. Glucose yields were improved when acetate buffer was used rather than citrate buffer. Initially, with each process variable tests, the optimal values were 100 minutes of milling, an enzyme concentration of 16 FPU/g-biomass, and a 12-hour enzymatic hydrolysis. Typically, interactions between these experimental conditions and their effects on glucose production were next investigated using RSM. Glucose yields from the Pinus rigida waste exceeded 80% with several of the conditions tested, demonstrating that milling can be used to obtain high levels of glucose bioconversion from woody biomass for biorefinery purposesopen

Analysis of Clostridium beijerinckii NCIMB 8052's transcriptional response to ferulic acid and its application to enhance the strain tolerance

Background: Plant-based cellulose presents the best source of renewable sugars for biofuel production. However, the lignin associated with plant cellulose presents a hurdle as hydrolysis of this component leads to the production of inhibitory compounds, such as ferulic acid. Results: The impacts of ferulic acid, a phenolic compound commonly found in lignin hydrolysates, on the growth, solvent production, and transcriptional responses of Clostridium beijerinckii NCIMB 8052 were determined. Addition of ferulic acid to growing cultures resulted in a decrease in the growth and solvent production by 30% and 25%, respectively, when compared to the control cultures. To better understand the toxicity of this compound, microarray analyses were performed using samples taken from these cultures at three different growth states. Several gene ontology terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were identified showing significant change at each status, including ATP-binding cassette (ABC) transporters, two component system, and oxidoreductase activity. Moreover, genes related with efflux systems and heat shock proteins were also strongly up-regulated. Among these, expression of the groESL operon was induced by more than fourfold and was consequently selected to improve C. beijerinckii tolerance to ferulic acid. Real-time quantitative PCR (RT-qPCR) analysis confirmed that C. beijerinckii harboring the plasmid, pSAAT-ptb_Gro, had a two-to fivefold increased groESL operon expression during growth of these cultures. Moreover, this strain was more tolerant to ferulic acid as the growth of this recombinant strain and its bioconversion of glucose into solvents were both improved. Conclusions: Using transcriptomics, we identified numerous genes that are differentially expressed when C. beijerinckii cultures were exposed to ferulic acid for varying amounts of time. The operon expressing groESL was consistently up-regulated, suggesting that this gene cluster may contribute to strain tolerance. This was confirmed as recombinant cultures showed both an enhanced growth and solvent yield in the presence of 0.5 g/L ferulic acidopen00

Detection of toxic lignin hydrolysate-related compounds using an inaA::luxCDABE fusion strain

Real-time quantitative PCR analyses of Escherichia coli str. BL21(DE3) exposed to 0.5 g/L ferulic and coumaric acid showed that the inaA gene was significantly induced (7.7- and 3.6-fold higher, respectively). Consequently, a transcriptional fusion of the inaA promoter with the luxCDABE operon was constructed and characterized with several compounds identified within hydrolysates. Tests demonstrated that the phenolics were major inducers, while acetic acid and furfural had only a minor or no effect on the inaA expression respectively. Additional tests with mutant E. coli strains found that a marA partially abolished the response while a marB knock-out led to a 2-3-fold higher basal level expression as evidenced by the bioluminescent levels of the cultures. However, a significant induction was seen even in the marA mutant, suggesting some other control mechanism is involved in regulating inaA expression during an exposure to the hydrolysate compounds. Finally, E. coli str. BL21(DE3)/pSP4 was used to analyze a spruce hydrolysate sample. Real-time quantitative PCR showed a 2.8-fold induction of the inaA expression level while the bioluminescence from the exposed culture was 22-fold higher than the control, demonstrating the possible application of this reporter strain to analyze hydrolysates for the presence of fermentation-inhibiting phenolics.close6

The production of biofuels from carbonated beverages

Commonly sold carbonated beverages typically contain 110 g/L of sugar. Consequently, this study evaluated the use of four products, including two colas, one diet cola and one lemon-lime flavored beverage from two large distributors world-wide and one local distributor, as fermentative sugar sources. Zymomonas mobilis ZM4 cultures were able to ferment the sugars present, which included sucrose, fructose and glucose, consuming all three completely within about 10 h after addition. The results from each of the beverages tested were similar, except for the diet beverage, with a final ethanol concentrations of around 25 g/L, an average productivity of approximately 2 g*L(-1)h(-1) and average yields of 0.43 g ethanol/g glucose. Using a diet beverage with a fructose-glucose mixture added extraneously, we demonstrated that the beverages were not inhibitory or toxic to the cultures. Subsequent experiments with three clostridial strains showed that these beverages can also be used within fermentative processes to generate butanol and butyric acid. As with Z. mobilis ZM4, Clostridium acetobutylicum ATCC 824 and Clostridium beijerinckii NCIMB 8052 were capable of fermenting all three sugars and had final butanol concentrations of 5.3 and 8.7 g/L, respectively. In contrast, Clostridium tyrobutyricum ATCC 25755 is unable to utilize sucrose as a carbon source. Although limited to only the monosaccharides fructose and glucose, the butyric acid concentration was 15.4 g/L and had a yield of 0.43 g butyrate/g glucose, which is only slightly below the theoretical maximum of 0.47. This study illustrates the potential of using wasted or expired carbonated beverages in the biofuels industry.close

Environmentally friendly pretreatment of plant biomass by planetary and attrition milling

This study evaluated the use of planetary and attrition milling as pretreatment processes for lignocellulosic biomass using rice straw. Planetary milling reduced the rice straw crystallinity from 0.48 to 0.11. Since the samples could be milled and enzymatically treated using the same media, loss of the biomass due to washing was effectively eliminated. In contrast, conventional sodium hydroxide and soaking in aqueous ammonia (SAA) processes showed a loss of 34.2% and 14.8%, respectively. Furthermore, milling produced significantly lower concentrations of soluble phenolics than the alkali treatments. Using a bioluminescent bioreporter strain that is sensitive to these phenolics, neither of the milled samples elicited a response while the sodium hydroxide and SAA samples led to a 25.8 and 4.7 -fold induction, respectively. Although planetary milling produced more reducing sugars than attrition milling before saccharification, both had similar monosaccharide yields, i.e., 0.38 and 0.34. g/g-biomass, respectively, when 40. g/l rice straw was treated.close3