Synergistic enzymatic saccharification and fermentation of agar for biohydrogen production
Introduction
Currently, the depleting fossil fuel reserves and the continuous growing fuel demand has led to the exploration of the safe and environment-friendly alternatives (e.g., biofuels). Owing to its abundant production and non-polluting characteristics, biohydrogen is one of the ideal and potentially sustainable biofuels with bright future prospects (Mathews and Wang, 2009). Marine biomass, such as algal biomass, is considered as another renewable source for bioenergy conversion due to several advantages, including the high growth rate, the high carbohydrate content (Falter et al., 2015), and the low concentration or lack of lignin, which can strongly inhibit the attack from the enzymes or microorganisms to hydrolyze the polysaccharides within the biomass (Yanagisawa et al., 2013).
Agar, composed of agarose and agaropectin, is the main component in the cell walls of red algal biomass, and agarose is a linear polysaccharide, containing d-galactose and 3,6-anhydro-l-galactose (AHG) via the alternative α-1,3 and β-1,4 linkage (Yun et al., 2015). In the practice of converting such algal biomass into biofuels or other valuable biochemicals, the critical step is to decompose the substrate into fermentable oligo sugars or monosugars through the chemical liquefaction or enzymatic saccharification process (Naik et al., 2010). Kim et al. (2012) combined the enzymatic hydrolysis with acetic acid treatment to obtain monosugars from agarose for ethanol fermentation by Saccharomyces cerevisiae. Seo et al. (2016) also demonstrated that a two-stage enzymatic process without acid pretreatment was involved in converting the agarose into ethanol by S. cerevisiae KL17. Enterobacter species has the potential in the biohydrogen production using various fermentable sugars, such as glucose, xylose, sucrose, etc. (Lu et al., 2011, Mohanraj et al., 2014, Subudhi et al., 2013). However, there have been no reports on the H2 fermentation by Enterobacter sp. using galactose, the major ingredients in agar polysaccharides, as the substrate so far. Moreover, the research on the biofuels production from agar biomass has been very limited, and even there has been no systematical investigation, including the galactose utilization enhancement, the agarase performance analysis as well as the synergistic process of saccharification and fermentation, on the biohydrogen production directly using agar as the substrate by Enterobacter species.
By simultaneously applying the agarase AgaXa and neoagarobiose hydrolase NH852, the agar can be decomposed into galactose and AHG, further converted to biohydrogen with the addition of a galactose-utilizing bacterial strain Enterobacter sp. CN1. Therefore, the objective of this study is to develop a feasible process on the synergy of the agarase saccharification and fermentation by strain CN1.
Section snippets
Bacterial strains and culture conditions
Enterobacter sp. CN1 was isolated from the previous study (Long et al., 2010) and maintained in the laboratory. The fermentation medium for biohydrogen generation by strain CN1 was prepared according to the method of Xin et al. (2014), and the fermentation process was carried out in 100 mL serum bottles sealed with butyl rubber stopper and aluminum seal caps, containing 50 mL of the fermentation medium with 2% of inoculum (v/v). The recombinant agarase (AgaXa) and neoagarobiose hydrolase (NH852)
Production of biohydrogen from galactose by Enterobacter sp. CN1
The statistical analysis of biohydrogen fermentation from galactose was performed via the Box-Behnken design using RSM. The effect of individual factors and their interaction on the biohydrogen yield was determined, and the model was established to obtain the optimal fermentation conditions (Table S2). A quadratic polynomial equation for identifying the relationship between the hydrogen yield and the selected variables was described as follows:
Conclusions
The maximal production of biohydrogen by Enterobacter sp. CN1 using galactose was optimized to be 1780 mL/L with a yield of 303.2 mL/g. Within the optimal fermentation conditions, agar hydrolysate was obtained through the synergistic process of agarases AgaXa and NH852, and further utilized for the biohydrogen production to achieve the maximal production of 5047 ± 228 mL/L from 50 g/L agar, resulting in 3.86-fold higher than the control without pretreatment. This work for the first time demonstrates
Acknowledgement
This work was supported by the National Natural Science Foundation of China (No. 41476150), the Guangdong Natural Science Foundation (No. S2012040006279), the Start-up Funding of Shantou University (No. NTF15007), the “Sail Plan” Program for the Introduction of Outstanding Talents of Guangdong Province of China (No. 14600601) and the Major University Research Foundation of Guangdong Province of China (Nos. 2015KQNCX041 and 2016KZDXM011).
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These authors contributed equally to this work.