The effect of HMF and furfural on the growth rate of lactic acid bacteria is dependent of the metabolic type
The effect of HMF and furfural was studied in lactic acid bacteria (LAB) displaying homo- and heterofermentative metabolism. According to the results obtained in the microplate reader cultivations, it was observed that the inhibitory compounds affected differently the two groups of LAB. The two inhibitory furan-derivative compounds had a positive effect on the growth of the heterofermentative LAB (Fig. 1), increasing its maximum specific growth rate by up to 2.4 times in comparison to the control condition (cultivation media in the absence of the inhibitory compounds). In addition, a positive effect for both compounds in the elongation of the lag phase was also observed in the heterofermentative LAB, in which a decrease in the time required to reach the exponential phase was noticed as compared to the control condition. On the other hand, in the homofermentative LAB, the complete opposite effect was observed, since their growth rates were inhibited by the furan-derivatives as compared to the control, as shown in Fig. 1. Likewise, homofermentative LAB also had a negative effect on the lag phase duration when cells were cultivated in the presence of the two inhibitors.
When comparing laboratory and industrial strains, it was observed that the growth performance (evaluated by the maximum specific growth rate and the elongation of the lag phase) of the laboratory heterofermentative LAB strains were less stimulated or partially inhibited than in the industrial LAB strains. Comparing laboratory and industrial homofermentative LAB strains, it was not possible to observe a pattern for these two parameters that could be used to differentiate them. The only exception was the fact that in all conditions tested the decrease in the growth rate of the homofermentative laboratory strain was more pronounced than the industrial strains.
Growth kinetics in the presence of HMF and furfural is only enhanced for heterofermentative lactic acid bacteria
As a follow-up, the growth kinetics was further investigated in two representative LAB strains using flask cultures with the same MBL media supplemented with the two furan derivatives separately at the highest concentration tested. For that purpose, a representative homofermentative (Lactobacillus plantarum ESALQ 4) strain, and a representative heterofermentative (Lactobacillus fermentum ESALQ 3) strain were investigated in these conditions. The measurement of growth provides reliable and sensitive information for the characterization of toxic compounds and conditions that adversely affect microbial cells (Franden et al., 2009)
As observed in the general growth screening described above in microplate cultures, the heterofermentative LAB showed faster growth kinetics and shorter lag phase in the presence of both inhibitors as compared to their absence (Fig. 2a). The exponential phase starts almost with 5 h of cultivation in the control kinetics and could be evidenced in 2 h in furfural presence. This effect is less pronounced in the presence of HMF. On the other hand, the growth kinetics in the presence of both inhibitors was inhibited as compared to the control condition (absence of furan derivatives) in homofermentative LAB. Apparently, in the concentrations tested, HMF seemed to be more detrimental to the homofermentative strain than furfural (Fig. 2b).
Sugar consumption and product formation is enhanced in the presence of furaldehydes in heterofermentative lactic acid bacteria
Heterofermentative LAB normally present slow growth kinetics on glucose that is caused by the low activity of the ethanol pathway in the reoxidation of the extra two NADH (Maicas et al, 2002). In the presence of the furan inhibitors, we observed an enhanced sugar consumption rate, and we noticed a deviation towards the formation of acetate and ethanol with a concomitant decrease in lactate production when compared to the control condition (Fig. 3). Moreover, biomass yield was lower in the presence of both furan inhibitors as compared to the control, and major conversion yields (glucose to lactate, acetate, and ethanol) showed a deviation towards acetate and ethanol formation with a concomitant decrease in lactate (Table 3). It seems this observation is caused by the fact that furfural and HMF are promoting the reoxidization NAD+ and NADP+, respectively. In addition, as they do not need to use the ethanol route to reoxidize de NADH, the acetyl-P can be used by ATP synthesis and the acetate production route becomes energetically more advantageous (Ganzle, 2015).
In cultures with the homofermentative LAB, the opposite behaviour was observed; sugar consumption rate decreased in the presence of inhibitors, and it was also possible to notice a decrease in lactate production kinetics (Fig. 4) along with a slightly decreased biomass yield (Table 4).
Detoxification of furaldehydes is very effective in heterofermentative lactic acid bacteria
Homofermentative bacteria dissimilate hexoses through glycolysis, where fermentation of 1 mol of hexose results in the formation of 2 mol of lactic acid and 2 mol of ATP. In comparison, heterofermentative bacteria present another active pathway (Kandler and Weiss, 1986) and hexoses are converted to equimolar amounts of lactic acid, ethanol or acetate, and carbon dioxide, yielding 1 mol of ATP per mol of hexose fermented (Cogan and Jordan, 1994). With the conversion of acetyl phosphate to acetate instead of ethanol, an additional ATP can be produced. Then, the regeneration of surplus NAD+ must be achieved through an alternative electron acceptor. In the heterofermentative LAB, it was possible to see the complete depletion of HMF and furfural which we hypothesized that were converted to furfuryl alcohol and 2,5- furandimethanol, respectively (Fig. 5). Previous studies indicate that yeast and bacteria strains were able to reduce furfural and HMF to their corresponding alcohols, as reported for L. reuteri (van Niel, 2012), S. cerevisiae (Liu et al., 2011), and E. coli (Jozefczuk, 2010) in which. These degradation products are of lower toxicity to microorganisms compared to their aldehyde precursors (Liu et al., 2011).
Heterofermentative L. fermentum seems to convert HMF at a slower rate as compared to furfural, which may be attributed to a lower cell membrane permeability of HMF when compared to furfural (Larsson et al., 1999). Therefore, under the oxygen-limited conditions that the experiments were performed, furan derivatives might have been reduced to their corresponding alcohols. In this way, such inhibitors seemed to be important co-substrates for heterofermentative lactobacilli, as opposed to homofermentative strains (Fig. 5).
Homofermentative bacteria is more deleterious to yeast in the context of lignocellulose-based substrates
We finally performed co-cultivations with yeast and both homo- and heterofermentative bacteria, in the presence of furfural and HMF. After 24 h co-cultivation of yeast with LAB strains, it was possible to notice that when homofermentative LAB was the only strain, the viability of yeast cells was drastically reduced (50%, in terms of the fraction of viable cells) as compared to yeast monocultures in the same medium (95%). On the other hand, in the treatment with the heterofermentative strain, viability was at an intermediate value (71%).
Regarding substrate and product kinetics (Fig. 6), glucose consumption was faster in co-cultures than in yeast monocultures. Yet, the presence of heterofermentative bacteria resulted in a faster consumption of glucose when compared to the homofermentative strain (Fig. 6A). Lactate accumulation was faster in the presence of the homofermentative strain and reached highest titters when compared to all conditions (Fig. 6B). Lactate accumulation in the presence of both bacteria (homo- and heterofermentative types) in the same fermentation flask was virtually the same as observed in cultivations with the heterofermentative strain alone. Acetate accumulation followed a similar trend as observed for lactate (Fig. 6C). Finally, ethanol accumulation was faster in the presence of the heterofermentative strain, but titters were higher when yeast was cultured alone. It seems that the higher lactic acid accumulation by homofermentative strains represents an additional source of inhibition to yeast cells, leading to decreased ethanol titters.
It was also possible to note an increase in glycerol titters in cultivations in the presence of the heterofermentative bacteria when compared to yeast monocultures. This observation is in accordance to results published by Meikle et al. (1988). On the other hand, in cultures with homofermentative and yeast cells, but without the presence heterofermentative cells, glycerol titters were much lower (Fig. 7).
Finally, furan detoxification by yeast cells is slower than by heterofermentative LAB (Fig. 8). When in the presence of the heterofermentative bacteria, both HMF and furfural concentrations displayed a significant depletion after only 7h of cultivation. On the other hand, in the presence of the homofermentative strain, complete depletion of furfural only occurred after 24h of cultivation (Fig. 8A). As for HMF, more than half of what was initially available in the culture medium remained untouched at the end of the cultivation (Fig. 8B). Taking all together, the presence of heterofermentative LAB in co-cultures with yeasts in the context of lignocellulosic ethanol processes, seems to accelerate detoxification of furan derivatives, resulting in faster kinetics of ethanol production. However, the presence of heterofermentative bacterium still reduces ethanol titters in the end of the cultivation.