While inhibitory properties of degradation products of lignocellulosic biomass are widely recognized as a major limitation to bioconversion of biomass to biofuels and chemicals , the gap in knowledge with respect to detoxification of these lignocellulose-derived inhibitory compounds by fermenting microorganisms continues to impede the development of inhibitor-tolerant strains vis–à–vis commercialization of biofuels. This study presents the physiological changes and transcriptional responses of C. beijerinckii NCIMB 8052 to furfural challenge at different growth and fermentation stages, highlighting a systematic pattern of gene regulations and revealing potential target genes for strain improvements by genetic engineering.
Genome-wide microarray analysis demonstrated a clear perspective on the effect of the lignocellulosic biomass-derived inhibitor furfural on the transcriptional profile of C. beijerinckii 8052. This study revealed for the first time that changes in physiological activities of furfural-challenged cultures of C. beijerinckii are coordinated with transcriptional variations during ABE fermentation. Validation of microarray data by Q-RT-PCR using samples from independent biological treatment showed high degrees of correlation coefficient (R) at both acidogenic and solventogenic phases (0.87 and 0.84, respectively), which fall into the upper values of the reported range (−0.48 to +0.93) , thus, confirming strong reliability of data obtained by microarray analysis. This result is significant because the correlation coefficient is the generally accepted criterion for assessing reliability of microarray data . Ramifications of obtained results are discussed below under different attributes.
Redox and cofactor genes are crucial for detoxification of furfural by C. beijerinckii 8052
Furfural challenge increases the expression of genes encoding redox proteins in C. beijerinckii 8052 (Figure 1A and Additional file 1: Table S4A and S4C). Differential expression of redox genes involved in antioxidant activity suggests that furfural causes oxidative stress in C. beijerinckii 8052. In yeast, furfural induces the accumulation of reactive oxygen species (ROS), that are known to damage DNA, lipids and proteins and that subsequently induce programmed cell death . Glutathione peroxidases and thioredoxin peroxidases, which were differentially induced in furfural-challenged C. beijerinckii 8052 (Figure 1A and Additional file 1: Table S4A and S4C), can reduce H2O2 to H2O via oxidation of thiol groups. The reduction of oxidized glutathione and thioredoxin is catalyzed by glutathione reductase or thioredoxin reductase, respectively, using NADPH as the electron donor . Thioredoxin and glutathione can also function as oxygen quenchers and hydroxyl radical scavengers [13–15]. Given that this study was conducted in an anaerobic chamber with less than 1 ppm of molecular oxygen (monitored by an oxygen detector), the origin of ROS is not clear. However, production of ROS, hydroperoxide or other radical species by anaerobes has been hypothesized and elucidated previously [16, 17]. Organic hydroperoxide generated under anaerobic condition, therefore, may induce the expression of alkyl hydroperoxide reductase gene in E. coli, and glutathione peroxidase and thioredoxin peroxidase genes in C. beijerinckii 8052. Another gene encoding a thioredoxin family protein dsbA oxidoreductase, a periplasmic oxidoreductase that facilitates disulfide bond formation in proteins, was found in this study to be induced by furfural. Overexpression of dsbA in E. coli increased soluble protein level in the periplasm and improved enzyme secretion and activity . Elevated levels of antioxidant activity due to furfural challenge indicate increased oxidative stress, thus, accentuating innate detoxification capabilities of C. beijerinckii under the influence of furfural stress. Additionally, thioredoxin and thioredoxin reductase work in tandem with ribonucleotide reductase during reduction of ribonucleoside diphosphates to deoxyribonucleoside diphosphates . The induced expression of these redox enzymes (Figure 1A and Additional file 1: Table S4A) involved in purine and pyrimidine metabolism in C. beijerinckii 8052 (Additional file 3: Table S3) suggests greater demand of nucleotides due to furfural stress. This premise is supported by the fact that DNA molecules are prone to damage in the presence of furfural , and in this case, DNA repair or biosynthesis is activated leading to induced expression of redox enzymes.
The differentially induced genes encoding oxidoreductases such as aldo/keto reductase (AKR) and short-chain dehydrogenase/reductase (SDR) in C. beijerinckii, which are involved in the reduction of furfural to furfuryl alcohol, have been reported elsewhere as scavengers of furfural in Escherichia coli[20, 21], Saccharomyces cerevisiae, and Zymomonas mobilis fermentations. Direct reduction of furfural to the less toxic furfuryl alcohol  is another strategy C. beijerinckii 8052 uses to mitigate toxic effects of furfural.
Moreover, differential expression of genes encoding the iron-sulfur cluster and cobalamin- and riboflavin-associated proteins, was observed in furfural-challenged C. beijerinckii 8052 (Figure 1A and Additional file 1: Table S4A), thus, accentuating cellular responses to furfural stress by redox balancing because these proteins require cofactors such as NADH and NADPH to facilitate catalysis. Notably, the iron-sulfur cluster plays important roles in electron transfer by redox enzymes, disulfide reduction by ferredoxin:thioredoxin reductase, regulation of gene expressions associated with Ferredoxin-NADP+ reductase, and iron and sulfur storage in ferredoxins . Similar to iron-sulfur clusters, cobalamin, known as vitamin B12, can also function as redox enzyme cofactors ; a typical example is the cobalamin-mediated biodegradation of chloroform by the methanogenic consortium obtained from an anaerobic distillery waste water treatment plant . Riboflavin, the redox active moiety of flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN), has been shown to be differentially induced in response to furfural challenge during ethanol production by S. cerevisiae. Broadly, these results support the idea that induction of genes encoding redox proteins and cofactors and transformation of furfural to less toxic furfuryl alcohol are important mechanisms that C. beijerinckii 8052 uses to restore redox balance under furfural stress and mitigation of toxic effects of furfural on the cell.
Membrane transporter genes play active role in furfural tolerance and detoxification by C. beijerinckii 8052
Fluctuations in differential expressions of the membrane transport system, including ATP-binding cassette transporters (ABC-transporter) and phosphotransferase system (PTS), signify possible physiological adaptations in C. beijerinckii 8052 in response to furfural stress (Figure 1B). The increased expression of sulfate ABC transporter genes (Figure 1B) in C. beijerinckii 8052 may be interpreted following a previously proposed model in E. coli in which furfural depresses sulfur assimilation with concomitant inhibition of cell growth, but supplementing the fermentation medium with sulfur-containing amino acids (cysteine and methionine) reversed cell growth and increased cell tolerance to furfural. It is plausible that under furfural stress, C. beijerinckii 8052 may sense sulfur limitation and consequently elevate expression of sulfate ABC transporter genes in preparation for potential increased absorption of sulfur from the fermentation medium. It is important to note that there are sulfates (MgSO4, MnSO4, and FeSO4) in the P2 growth medium.
The expression of the phosphate-specific transport (Pst) system in C. beijerinckii 8052 was significantly enhanced under furfural stress (Figure 1B and Additional file 1: Table S4A). Phosphate is an essential component of nucleotides; hence, it plays a central role in chemical energy and DNA/RNA synthesis. The elevated expression of the Pst system may indicate shortage of intracellular phosphates, thus, the need for increased absorption of phosphorus from the environment. Elsewhere, while Pst in E. coli was demonstrated to have decreased expression in the presence of excessive inorganic phosphate, phosphate limitation induces the expression of Pst. Similarly, elevated expression of multiple operons encoding ABC transporters for branched-chain amino acid transportation was observed (Figure 1B and Additional file 1: Table S4A and S4C). It is conceivable that the biosynthesis of branched-chain amino acids (leucine, isoleucine and valine) in C. beijerinckii 8052 is perturbed when furfural is present in the medium, hence, the induction of genes encoding a related membrane transportation system to mitigate the perturbation. This line of reasoning agrees with the fact that furfural induces the accumulation of reactive oxygen species and superoxide anions, which may damage the synthesis of amino acids, especially the branched chain amino acids . Therefore, it is reasonable that C. beijerinckii 8052 increases the expression of these ABC transporters to facilitate enhanced absorption of exogenous amino acids.
In contrast to ABC transporters, the phosphotransferase system (PTS) reveals decreased expression in furfural-challenged C. beijerinckii 8052 (Figure 1B and Additional file 1: Table S4B and S4D). Since the bacterial PTS plays crucial roles in sugar reception, transport and phosphorylation in addition to regulation of catabolic pathways , the expression level of PTS may reflect the physiological state of cell metabolism and, consequently, could rationalize the low ABE production and premature termination of the C. beijerinckii 8052 fermentation process after furfural challenge at the solventogenic growth phase (Figures 5, 6, 7).
Furfural influences the adaptation machinery of C. beijerinckii 8052
The two-component signal transduction system (TCS) is a stimulus–response coupling signal transduction machinery that allows bacteria to respond and adapt to changes in a wide range of environmental conditions, such as nutrient assimilation , cellular redox state  and bacterial virulence regulation . Chemotaxis, controlled by TCS, is the cells’ response to stressful environments. In chemotaxis, signals are first sensed by transmembrane receptors known as methyl-accepting chemotaxis proteins (MCPs), which control the autophosphorylation of a kinase protein and then a regulator protein. The regulator protein interacts directly with flagellar proteins that act as motor switches and, thus, controls the swimming pattern of the bacterial cell . Exposure of C. beijerinckii 8052 to furfural stress elicits repression of genes that code for MCPs, CheA, CheY, and flagellar proteins (Figure 1C), plausibly causing temporary (at the acidogenic phase) and permanent (at the solventogenic phase) defects in the adaptation machinery of C. beijerinckii 8052.
In bacteria, the carbon storage regulator (CsrA) is recognized as an activator of glycolysis, acetate metabolism, and flagellum biosynthesis  and as a global regulator of bacterial virulence and stress response . E. coli csrA
− (csrA deficient) strains are known to have severe growth problems due to central carbon stress , and the csrA
− strain of Helicobacter pylori significantly attenuates its virulence . In the presence of furfural, the global regulator CsrA in C. beijerinckii 8052 was significantly repressed (Figure 1C and Additional file 1: Table S4B and S4D), which may result in the repression of glycolysis and consequently, may trigger repertoires of transformations in stationary-phase physiology . This could rationalize the low ABE production and premature termination of the C. beijerinckii 8052 fermentation process following furfural challenge at the solventogenic growth phase (Figures 5, 6, 7).
Furthermore, repression of glutamine synthetase (GS) in C. beijerinckii 8052 during ABE fermentation in the presence of furfural may decrease the production of glutamine, which may have undesirable effects with respect to nutrient assimilation and cellular redox balance. The GS strain (glutamine-requiring strain) of Bacillus subtilis was found to cause pleiotropic effects on glucose catabolite repression . Moreover, glutamine is a precursor of glutamate, which may be used to synthesize glutathione, an important cellular antioxidant (albeit in reduced form) that mitigates stresses . Since the product of GS plays an important role in cellular redox balance, the repression of GS may impair the tolerance of C. beijerinckii 8052 to furfural.
Basis for both stimulatory and inhibitory effects of furfural on C. beijerinckii 8052
Addition of furfural (<3 g/L) to the fermentation medium inhibits ABE production by C. beijerinckii 8052 to various degrees regardless of the growth stage (acidogenic or solventogenic) of the culture (Figures 3 and 5). While C. beijerinckii 8052 challenged with furfural during the acidogenic phase experienced short-term ABE production inhibition (Figure 3), rapid depletion of furfural in the fermentation medium (Figure 3G), full recovery even with elevated cell growth (data not shown), and increase in ABE production following exhaustion of furfural in the growth medium (Figure 4), C. beijerinckii 8052 challenged with furfural at the solventogenic growth phase resulted in immediate termination of ABE fermentation (Figures 5 and 6). This finding partly agrees with previous investigations [2, 7], which reported that furfural could stimulate growth and ABE production when added at the beginning of fermentation, but it also expands knowledge in the field by uncovering the fact that furfural is most toxic to C. beijerinckii 8052 during the solventogenic growth phase.
C. beijerinckii 8052 did not recover from the toxic effect of furfural when it was challenged with it at the solventogenic growth phase, yet why does furfural enhance growth of C. beijerinckii 8052 when it is added either at the beginning of fermentation or during the acidogenic growth phase? The answer may be found in the genes. Genes GrpE, DnaK and DnaJ in DnaK operon encoding GrpE, DnaK and DnaJ proteins are induced under furfural stress (Additional file 1: Table S4A), and they play an important role in mitigating harmful effects of environmental stresses such as UV irradiation , ethanol , and butanol  on microorganisms, and stresses on the cellular chaperone machinery . While GroES and GroEL in the groE operon [which are also highly conserved molecular chaperons and are known to be induced by the presence of butanol  were differentially induced by more than threefold when C. beijerinckii 8052 was challenged with furfural during the solventogenic growth phase (Additional file 1: Table S4C), the operon was differentially induced by less than threefold when C. beijerinckii 8052 was challenged with furfural during the acidogenic growth phase (Additional file 1: Table S4A). Notably, overexpression of groES and groEL increases production of ABE, tolerance to toxic products, and metabolism in solventogenic Clostridium species , but their increased expression during the solventogenic phase was perhaps to overcome (albeit increased expression was not enough to overcome furfural toxicity) the high toxicity of furfural to C. beijerinckii 8052 at this physiological growth phase.
Then, why is furfural more toxic to C. beijerinckii 8052 during the solventogenic phase than during the acidogenic phase? Three hypotheses are proposed. First, the presence of ABE enhances cell membrane fluidity and inhibits cell metabolism , which leads to significant loss of cell functions and weakening of the cellular defense system to furfural. This was underscored by the fact that C. beijerinckii 8052 was unable to completely reduce 2 g/L furfural in the presence of ABE during acidogenic growth phase, unlike the control without ABE, which reduced the entire amount of furfural (Figure 8). Second, the biotransformation of furfural, which is catalyzed by NAD(P)H-dependent oxidoreductase , competes with NAD(P)H-dependent dehydrogenase (that catalyzes alcohol production) for NAD(P)H coenzymes . The need for NAD(P)H by NAD(P)H-dependent oxidoreductase to boost cellular defense against furfural is a high priority, which leads to a decrease in the NAD(P)H pool and subsequently impedes alcohol production by NAD(P)H-dependent dehydrogenase. This hypothesis is supported by a previous finding in ethanologenic E. coli[20, 48], wherein silence of an oxidoreductase involved in furfural conversion relieves the diversion of NAD(P)H away from other important biosynthetic processes, thus, increasing cell growth and furfural tolerance. Competition between oxidoreductase and alcohol dehydrogenase for NAD(P)H is severe at the solventogenic growth phase, during which NAD(P)H is needed for the conversion of butyryl-CoA to butyrylaldehyde and subsequently to butanol, unlike the acidogenic growth phase, during which acids are produced in tandem with NAD(P)H production . Third, while furfural repressed the expression of only two genes involved in cell motility by more than threefold when C. beijerinckii 8052 was challenged with furfural during the acidogenic phase, more than forty genes were differentially repressed by up to 18-fold when C. beijerinckii 8052 was challenged with furfural at the solventogenic phase (Figure 1C and Additional file 1: Table S4B and S4D). Notably, the non-motile strain of C. acetobutylicum has been shown to produce lower ABE than the motile parent strain .