Pretreatment of G. amansii to produce D-galactose and HMF
The feasibility of D-galactose and HMF co-production was tested with four different HCl concentrations (1%, 2%, 4%, 6% w/w) at 120 °C for 1 h. Figure 2 revealed that improving the dosage of HCl from 1 to 2% (w/w) increased the concentration of D-galactose by 23.4%, from 24.35 g/L to 30.04 g/L, and further improvement of HCl dosage almost had no impact on D-galactose yield. Conversely, the HMF concentration decreased from 5.53 to 3.13 g/L with the concomitant increase in the concentrations of levulinic acid and formic acid, suggesting a degradation process of HMF, especially improving the dosage of HCl from 2% (w/w) to much higher. Beyond that, d-glucose generated from fiber was detectable, approximately 2.40 to 3.32 g/L. The improved HCl concentration resulted in obvious levulinic acid and formic acid formation, but no other by-products could be observed, which also indicated that the physical structure of G. amansii was almost absence of lignin.
With the aim of achieving both higher yield of D-galactose and HMF, effects of temperature and time on hydrolysis of G. amansii were evaluated. Figure 3a exhibited improving the temperature from 80 °C to 160 °C gave similar trend on D-galactose and HMF production. Both the highest concentration of D-galactose (38.99 g/L) and HMF (7.56 g/L) were obtained at 120 °C. Temperature lower than 100 °C was too facile for degradation of galactan and conversion of AHG in G. amansii. In contrast, when temperature was higher than 140 °C, a gradual decrease of D-galactose and HMF was observed, suggesting a further degradation process of D-galactose to undetected compounds, and a transformation of HMF to levulinic acid and formic acid.
Compared with temperature, effect of pretreatment time on G. amansii hydrolysis was insignificant (Fig. 3b). The holding time prolonged from 20 min to 40 min brought about slight enhancement of D-galactose and HMF concentration, and further extension of time had no influence on D-galactose and negative impact on HMF production, respectively. Therefore, a relatively high titer of 39.96 g/L D-galactose and 7.48 g/L HMF, with individual yields of 82.87% and 40.71%, was achieved when holding time was 40 min.
Based on the obtained experimental results, it can be observed that the concentration of levulinic acid was much improved under higher temperatures than other conditions, from 2.56 g/L (120 °C) to 11.88 g/L (160 °C) (Fig. 3a). It should be noted that the increased production of levulinic acid could not be compensated from HMF decomposition (7.56 g/L at 120 °C vs 1.37 g/L at 160 °C). It was possible that the combination of HCl and high temperature (160 °C or higher) can drive a further dehydration of the formed D-galactose intermediate (38.99 g/L at 120 °C vs 23.93 g/L at 160 °C) during the pretreatment process to generate HMF and subsequently levulinic acid. These results were in agreement with previous studies pointing to red algae as a potential biomass for levulinic acid production, which is another versatile platform chemical with numerous application fields [4].
Oxidation of D-galactose and HMF using Pseudomonas sp. strains
In terms of oxidizing D-galactose and HMF into D-galactonic acid and HMFCA, it is desirable to explore strains that possess strong and specific activity towards the aldehyde group. As typically aerobic bacteria, the genus Pseudomonas can oxidize a wide range of aldose sugars into their corresponding aldonic acids [13, 14], but their potential capacities of HMF oxidation have not been fully investigated. In this study, five different species from Pseudomonas sp. were screened as putative candidates for both D-galactose and HMF oxidation. Additional file 1: Table S1 revealed that the D-galactose oxidation ability was universal in all tested strains. In contrast, only P. putida ATCC 47054 and P. rhodesiae CICC 21960 were capable of converting HMF into HMFCA, and the former had a little advantage. On the basis of the above results, P. putida ATCC 47054 was chosen as the biocatalyst for further investigations. Over-oxidation or further degradation reaction(s), which were readily caused by the complex enzyme systems in the cells, didn’t occur in the whole-cell biotransformation of P. putida ATCC 47054, which might be ascribed to the paucity of enzymes for assimilation of D-galactonic acid and HMFCA. Certain strains, such as P. saccharophila, Azotobacter vinelandii, Caulobacter crescentus and E. coli, possess a De Ley-Doudoroff route for dehydration of D-galactonic acid to produce 2-keto-3-deoxygalactonic acid, and the latter was further phosphorylated and cleaved to form glyceraldehyde-3-phosphate and pyruvate [15,16,17,18]. These strains can utilize D-galactose or D-galactonic acid as carbon source for growth. Nevertheless, this is not the case for P. putida ATCC 47054, which is devoid of enzymes involved in De Ley-Doudoroff pathway [19]. In the case of HMFCA, it can be converted to FDCA by full oxidation of the hydroxymethyl group on furan ring by Cupriavidus basilensis HMF14 and Raoultella ornithinolytica BF60, but the strains with such characteristic is rather rare [20, 21]. As a matter of fact, researchers have devoted many efforts to screen such strains or related enzymes for FDCA biosynthesis [20, 22,23,24]. For P. putida ATCC 47054, only the oxidation of aldehyde group on furan ring can be accomplished, which is similar to previously reported Comamonas testosterone SC1588 and Gluconobacter oxydans DSM 50049 [25, 26].
Co-oxidation of D-galactose and HMF was conducted by P. putida ATCC 47054. The concentration of D-galactose and HMF in reaction mixtures was set according to their ratio in pretreated G. amansii hydrolysates. Figure 4a, b showed the co-production process of D-galactonic acid and HMFCA. It was found that 50 g/L D-galactose was transformed completely in 5 h, while 10 g/L HMF was converted totally in 4 h, with yields of 97.7% and 99.1%, respectively. Zhang et al. reported that the catalytic activities of C. testosterone SC1588 towards HMF were enhanced markedly when they were cultivated with a low concentration of furfural and furfural alcohol as the inducers [25]. Herein, we attempted to facilitate the oxidation reactions by supplementing 4.5 mM HMF, hoping to induce the expression of the enzyme(s) responsible for the aldehyde group oxidation. Indeed, the bioconversion ability towards HMF was improved significantly when HMF was added during the cultivation period, but no positive effect was detected for D-galactose bioconversion (Fig. 4c, d). Figure 4d showed that the reaction period of HMF bioconversion decreased from 4 h to 1 h with the induced cells as biocatalyst. It was plausible that enzyme(s) responsible for oxidation the aldehyde group of HMF and D-galactose were different. To sum up, no matter the cells were induced or not, the substrates were consumed completely, and the maximal D-galactonic acid and HMFCA yield were above 97% in all cases.
Although good yields were obtained for both D-galactonic acid and HMFCA, the main enzymes in P. putida ATCC 47054 involved in D-galactose and HMF oxidation are still unknown. In our another research that focused on the role of molybdate transport system in biotransformation of furanic aldehydes, we found that deletion of modABC (encoding a molybdate ABC transporter, PP_3828–PP_3830) could prevent the oxidation of HMF to HMFCA (unpublished data, manuscript in preparation) whereas no impact on D-galactose oxidation occurred. It is likely that one or more unknown molybdate-dependent oxidoreductases can specifically accept HMF as substrate. But frustratingly, we had not identified the target enzyme(s) responsible for HMF oxidation at present. In the case of D-galactose, in literatures, the oxidoreductases from bacteria that can oxidize the C1-position of D-galactose are D-galactose dehydrogenase and L-arabinose dehydrogenase [16, 27,28,29], but none of them can be searched on the genome of P. putida ATCC 47054 (GenBank no. AE015451.2). We then turned our attention to the pyrroloquinoline quinone-dependent glucose dehydrogenase (PQQ-GCD), which can transform a diverse of aldoses into their corresponding aldonic acids [13, 30]. A gcd-disrupted mutant was constructed by homologous recombination to investigate the role of PQQ-GCD in d-glucose oxidation. The oxidative activities of P. putida ATCC 47054 and the gcd-disrupted mutant towards D-galactose were determined, respectively. Compared with the wild strain that possessed the activity of 0.13 U/mg, the gcd-disrupted mutant appeared to have lost the ability to oxidize D-galactose in a large extent, evidenced by the low activity of 8.9 × 10−4 U/mg, which verified the pivotal role PQQ-GCD in D-galactonic acid production.
Dual production of D-galactonic acid and HMFCA from G. amansii hydrolysates
Based on the obtained experimental results, the feasibility of co-production of D-galactonic acid and HMFCA from G. amansii hydrolysate was further investigated, which would contribute to the environmental protection due to the simple process for preparation of D-galactose and HMF without additional purification. Considering that the G. amansii hydrolysates might have an inhibitory effect on microbial cells, the bioconversion process was evaluated with different substrate concentrations. When G. amansii was pretreated under optimal conditions at a solid to liquid ratio of 1:10, a total of 41.24 g/L D-galactose and 7.85 g/L HMF were obtained. After mixed with wet cells of HMF-induced P. putida ATCC 47054, the initial concentration of D-galactose and HMF were diluted to 39.04 g/L and 7.29 g/L, respectively. Figure 5a depicted the change of D-galactonic acid and HMFCA yields along with reaction progress in the acid-pretreated hydrolysates of G. amansii. It revealed that the HMFCA yield increased very quickly to a maximum of 98.7% (corresponding to the overall yield of 39.2%) in 75 min, and the D-galactonic acid yield continuously rose until 7 h to a maximum of 95.2% (corresponding to the overall yield of 81.42%). We then improved the solid to liquid ratio to 1:5 in pretreatment process, in this case, 62.30 g/L D-galactose and 11.05 g/L HMF were produced. Similarly, the initial concentration of D-galactose and HMF were diluted to 56.36 g/L and 9.97 g/L individually after mixed with cells. Figure 5b showed that HMF was transformed completely in 105 min, with the maximum HMFCA yield of 98.7% (corresponding to the overall yield of 29.7%) and the highest HMFCA titer of 11.09 g/L. D-galactose was thoroughly bioconverted in 11 h, as the concentration of D-galactose was much higher than HMF in hydrolysates. The maximum D-galactonic acid yield was 91.1% (corresponding to the overall yield of 58.8%) and the highest D-galactonic acid titer was 55.30 g/L. The yields attained from G. amansii hydrolysates were slight lower than the results from commercial D-galactose and HMF.
In recent years, the exploration of macroalgae as a source for bio based chemicals and biofuels, including bioethanol [31], FDCA [32], levulinic acid [4], and hydrogen [7] etc., has attracted considerable interest. This study is the first report for D-galactonic acid and HMFCA production from macroalgae. The comparison of D-galactonic acid and HMFCA biosynthesis with previous reports was listed in Additional file 1: Table S2. Up to now, strains screened and applied for D-galactonic acid and HMFCA production are relatively few and mainly concentrated in some aerobic bacteria. Four different species of Pseudomonas were tested for their aldose-oxidizing activities and P. fragi TCCC 11892 was found to be an efficient producer of aldonic acids, including D-galactonic acid [14]. G. oxydans was another versatile species that can convert a series of biomass-derived sugars into corresponding aldonic acids [9, 33, 34]. But the enzymes responsible for D-galactose oxidation in P. fragi and G. oxydans were not unveiled. Liu et al. introduced D-galactose dehydrogenase from P. syringae and L-arabinose dehydrogenase from Azospirillum brasilense [29, 35], respectively, into E. coli to produce D-galactonate. To enhance the production of D-galactonate, inherent metabolic pathways for assimilating both D-galactose and D-galactonate must were blocked in advance [18, 29, 35]. With respect to HMFCA, Sayed et al. investigated and compared two G. oxydans strains, and one of them, G. oxydans DSM 50049, offered a much superior catalytic system for selective oxidation of HMF to HMFCA [26]. Zhang et al. isolated a new strain of C. testosterone SC1588 and proved its good biocatalytic oxidation of HMF to HMFCA [25]. They also spent much energy on identifying aldehyde dehydrogenases with ability to oxidize HMF [36]. P. putida ATCC 47054 was the first reported strain with selective biocatalytic activity towards the aldehyde group of both D-galactose and HMF, which were accomplished with utterly different enzymes. To sum up, taking advantage of the excellent efficiency and selectivity of P. putida ATCC 47054, efficient valorization of G. amansii biomass for dual production of D-galactonic acid and HMFCA at high yields was achieved for the first time, and these results further proved that algae biomass could be a promising feedstock for biochemicals production.