There are several reports on using different carbon sources with B. subtilis under aerobic conditions (among many others: Cabrera-Valladares et al., 2012; Ludwing et al., 2001; Blencke et al., 2003, Schmiedel et al., 1996). However, the reports using different sugars under anaerobic conditions are scarce (Steinmetz 1993; Nakano et al., 1997 and 1998; Cruz-Ramos et al., 2000; Stülken and Hillen, 2000; Romero et al., 2009; Härtig and Jahn, 2012). To evaluate B. subtilis 168 trp+ and ER382 (ΔalsS) performance to ferment different carbon sources we decided to use a rich medium, i.e., Luria Bertani, because it has been shown that B. subtilis has very poor growth when mineral media-glucose is used under non-aerated conditions (Nakano et al., 1997 and 1998; Cruz-Ramos et al., 2000). Under such growth conditions, B. subtilis requires the contribution of metabolic intermediates, such as pyruvate (Nakano et al., 1997 and 1998; Cruz-Ramos et al., 2000), or additional nutrients such as vitamins and/or amino acids, which are provided in a rich medium. Also, to avoid potential interferences, due to the deletion of several proteases and presence of many antibiotic markers, we decided in this study to generate the strain ΔalsS from the wild type B. subtilis 168 trp+, instead of the B. subtilis strain CHI alsS- previously developed by Romero et al., 2009.
The depletion of oxygen in B. subtilis is sensed by the transcriptional regulator FNR and the two-component regulatory system ResDE, which regulates the transcription of several genes, such as ltcE (encoding for lactate dehydrogenase) and alsSD that encodes for the acetolactate synthase and acetolactate decarboxylase, which catalyze the formation of acetoin (precursor of butanediol) (Härtig and Jahn, 2012). Under anaerobic conditions, ATP is generated at the substrate level, and NAD+ is regenerated in the conversion of pyruvate to lactate and butanediol; this maintains the redox balance and ATP generation for biosynthesis (Nakano et al., 1997 and 1998; Steinmetz, 1993; Cruz-Ramos et al., 2000; Stülken and Hillen, 2000; Härtig and Jahn, 2012). The results shown in Table 4 suggest that the metabolic balance was maintained when hexoses and disaccharides were used as a carbon source because the LA yield was higher than the theoretical. On the other hand, to conserve cellular resources, the expression of more than a hundred genes encoding for carbohydrate catabolism activities is induced only when the corresponding carbohydrate is present in the growth medium (Stülken and Hillen, 2000). In B. subtilis, this induction can occur via inducer-mediated inactivation of a repressor but can also occur by transcriptional activation or antitermination (Deutscher et al., 2002 and 2014; Fujita, 2009). The internalization of some sugars depends on the phosphoenolpyruvate system (PEP): phosphotransferase of sugars (PTS), a complex enzymatic system responsible for detecting transmembrane transport and phosphorylation of sugar substrates. While few sugars use another type of transport called ABC (Deutscher et al., 2002 and 2014; Deutscher, 2008; Fujita, 2009). The PTS is considered a bifunctional system that serves both sugar transport and signal transduction purposes. The regulatory signal senses the inducers or the levels of other important metabolites, like fructose-1,6-biphosphate (FBP) or branched amino acids, to redirect the use of carbon sources to maximize energy requirements (Sonenshein, 2007).
It has been reported that elimination of alsS does not negatively affect B. subtilis growth under anaerobic conditions (Cruz-Ramos et al., 2000; Renna et al., 1993; Frädrich et al., 2012). Accordingly, in comparison to the progenitor strain in ER382 the deletion of alsS allowed a slight increase in the amount of biomass formed and the biomass yield, the elimination of butanediol formation, the increase of the LA yield, the volumetric sugar consumption rate, and volumetric LA productivity (table 4) Overall, these results indicate that a higher fraction of carbon flux was redirected towards higher biomass and LA formation. Also, the components of the rich media contributed to the formation of LA.
B. subtilis preferentially uses glucose over other carbon sources. This study observed that B. subtilis could grow on glucose, fructose, and the capacity to metabolize these hexoses to LA by ER382 under anaerobic conditions was fast, above 1 gLA/L h (Table 4). In B. subtilis, glucose is transported and phosphorylated by a PTS-dependent glucose-specific component. The internalization, phosphorylation, and catabolism to pyruvate (glycolysis) generate a low ATP consumption, resulting in a global yield of 2 mol ATP for each mol when glucose is catabolized to LA and restores the NAD+ balance. It is also known that fructose is transported and phosphorylated via FruA (a PTS fructose transporter) at a high rate (Stülken and Hillen, 2000; Deutsher et al., 2002 and 2014; Fujita, 2009). Compared to glucose, fructose was metabolized at a lower rate because fewer cells were generated using this carbon source (Table 4); the biomass yield on sugar consumed was 33% lower with fructose. Perhaps, the fructose-specific PTS component has a lower transport capacity when compared to that for glucose.
Five genes encoding for beta-glycoside-specific PTS components and a putative 6-phospho-beta-glucosidase (licBCAH) are involved in utilizing beta-glycosidic compounds in B. subtilis. The lic operon is inducible by lichenan, lichenan hydrolysate, and cellobiose. Furthermore, the expression of the lic operon is positively controlled by the LicR regulator protein (Tobisch et al., 1999). Since cellobiose is a glucose dimer, knowing that B. subtilis has a β-glucosidase and that it requires two ATPs to phosphorylate the two glucoses obtained from cellobiose hydrolysis, it is suggested that this microorganism can use and metabolize this sugar with glucose-like efficiency. However, the rate of cellobiose consumption for both strains was 67–77% lower than the rate at which glucose was consumed. Previously, Chang et al. (1994, 1999) and Romero et al. (2009) had reported that the use of high concentration of biomass, obtained from aerobic conditions with cellobiose, to inoculate cultures under anaerobic conditions contribute to obtaining cellobiose consumption rates like those observed with glucose due to a greater induction in the genes that are required for cellobiose catabolism.
The presence of sucrose in the medium induces the transcription of genes involved in the regulation, internalization, and metabolism of this carbohydrate. B. subtilis has two systems for internalizing this disaccharide into the cell; one that is involved in catabolism is composed of a PTS-permease (sacP) and a phosphosucrase (sacA) (Débarbouillé et al., 1990; Débarbouillé et al., 1991; Crutz and Steinmetz et al., 1992; Daguer et al., 2004). Under aerobic conditions, the genes for the PTSpermease can be activated at low concentrations of sugar. The second system is mainly involved in anabolism, it is composed of the extracellular enzyme levansucrase (encoded by the sacB gene), and under aerobic conditions is more dependent on its activation at high concentrations of sucrose (Débarbouillé et al., 1990; Débarbouillé et al., 1991; Crutz and Steinmetz et al., 1992; Daguer et al., 2004). No levan or fructose formation was detected in culture supernatants; hence we suggest that internalization occurred through the SacP transport system. The lower rate of substrate consumption compared to that observed when the carbon source was glucose might have three explanations. First, the activation of the genes for the inducer (sucrose) was low. Although it is known that when only one carbon source is present, the induction of the genes encoding its own transporters must be maximum. However, PTS mediates the autoregulation of carbohydrate utilization by CcpA (a global regulator) (Gunnewijk et al., 2001; Brückner and Titgemeyer, 2002), and the phosphorylation state of the cell by HPr-Ser-P, which also contributes to this autoregulation (Saier et al., 1995; Darbon et al., 2002; Monedero et al., 2001). Therefore, depending on the phosphorylation state, the antiterminator SacT (transcriptional regulator) could activate the sacPA operon. Also, once the glycolytic phosphorylated intermediated increase (e.g., fructose-1,6-bisphosphate), the regulation mediated by CcpA occur (Sonenshein, 2007). Second, the activity of phosphosucrase (the enzyme that intracellularly hydrolyses sucrose) would be low, and therefore, the hydrolysis rate of the disaccharide is low. In addition, once the disaccharide is hydrolyzed, the glucose-6-phosphate enters the glycolytic pathway directly, while the entry of fructose to the glycolic pathway could be a limiting step because it would need two previous phosphorylation steps to be incorporated as fructose-1,6-bisphosphate into the glycolysis. Furthermore, fructose-1,6-bisphosphate is an important metabolite that stimulates HPr kinase to phosphorylate Hpr and Crh, which interact with CcpA (a global regulator) regulating glycolytic flux (Sonenshein 2007; Deutscher et al., 2002). Third, the transport of sucrose is less active than glucose. On the other hand, as with glucose or fructose, with disaccharides, a yield higher than the theoretical was obtained (between 20 and 30% more, Table 4), indicating that the strain metabolizes carbon contained in the Luria medium to lactate.
B. subtilis can import arabinose under anaerobiosis by an ABC-type transporter protein. After entering the cell, arabinose is sequentially converted to ribulose, ribulose-5-phosphate, and xylulose-5-phosphate by the action of the arabinose isomerase ribulokinase and ribulose-5-phosphate epimerase, respectively. The regulation of the biosynthesis of these enzymes is transcriptional and is negatively controlled by the transcriptional factor araR and by the presence of the inducer (arabinose). Once the inducer is present, AraR releases the operator sites in the promoter regions of araABDLMNPQ-abfA, araE, and araR operons (Rodionov et al., 2001). In our study, we observed low biomass formation with arabinose as compared to glucose and almost one order of magnitude lower volumetric rate of arabinose consumption compared to glucose. This may be due to two factors, one energetic and the other at the transcriptional level. The energy balance indicates that the catabolism of arabinose to pyruvate yields 0.67 mol ATP/mol arabinose (Nakano et al., 1998; Cruz-Ramos et al., 2000), while 2 mol ATP is obtained from the catabolism of 1 mol of glucose to pyruvate; hence the lower ATP yield with arabinose could hinder B. subtilis growth. On the other hand, it is possible that there was a low expression of genes that code for proteins that take up arabinose, as well as those genes encoding for the enzymes of the pentose phosphate pathway. Also, the metabolism of non-PTS carbohydrates can trigger PTS-mediated mechanisms to autoregulate the rate of sugar metabolism (Gunnewijk et al., 2001; Brückner and Titgemeyer, 2002). Besides, it has been reported that AraE, an unspecific transporter, is the most relevant transporter at a low concentration of arabinose (Krispin and Allmansberger, 1998). The expression of araE depends on the negative transcriptional regulator AraR. Therefore, a low expression of araE or a low affinity of the AraE transporter for the substrate could cause a slow transport of arabinose and, therefore, a slow expression of the genes involved in arabinose catabolism (Sá-Nogueira and Ramos, 1997; Krispin and Allmansberger, 1998). Moreover, there is evidence that the metabolism of non-PTS carbohydrates influences the intracellular [PEP]/[pyruvate] ratio (Gunnewijk et al., 2001; Brückner and Titgemeyer, 2002), and this, in turn, influences PTS autoregulation mediated by CcpA and HPr-Ser-P. To our knowledge, there are no previous reports about the utilization of arabinose by B. subtilis under anaerobic conditions.