Korean Society for Biotechnology and Bioengineering Journal 2022; 37(2): 49-57
Published online June 30, 2022 https://doi.org/10.7841/ksbbj.2022.37.2.49
Copyright © Korean Society for Biotechnology and Bioengineering.
Jueun Jang&dagger,, In Jung Kim&dagger,, Suhyeung Kim, Jamin Shin, Sejin Geum, and Soo Rin Kim*
School of Food Science and Biotechnology, Kyungpook National University, Daegu 41566, Korea
Research Institute of Tailored Food Technology, Kyungpook National University, Daegu 41566, Korea
Correspondence to:†These two authors contributed equally.
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Sargassum horneri is a sea-drifting brown macroalga often found along the coast of East Asian countries. It was recently found to be drifting from China toward Jeju Island in South Korea, causing damage to fisheries and vessels. Being considered as a marine waste, a huge amount of S. horneri was collected in the past 5 years, but an efficient and proper way to treat it has still not been found. Therefore, it is required to develop technologies to tackle this issue. Here, we conducted bioconversion of S. horneri by the yeast Saccharomyces cerevisiae in order to utilize it as a biomass source for producing ethanol. First, S. cerevisiae was engineered to extend its substrate range to mannitol, which is one of the major components of brown algae. Activation of the native HXT17 and MAN2 genes enabled the yeast to metabolize mannitol as the sole carbon source. Impact of pretreatment conditions, the type of hydrolytic enzymes, and biomass solid loadings on the ethanol production by the yeast were evaluated. The highest ethanol productivity was obtained when the biomass was pretreated at 121oC and ethanol concentration was the highest when the biomass loading was 24% (w/v), giving the maximum concentrations of monosaccharides and ethanol of 47.29 g/ L and 22.94 g/L, respectively. The results obtained from this study suggest possible utilization of S. horneri as a raw material for cellulosic bioethanol production.
Keywords: Sargassum horneri, marine waste, mannitol, bioethanol, Saccharomyces cerevisiae, brown algae
Global warming and consequent climate change caused by fossil fuel-based economy call for sustainable bioenergy production in the modern society [1,2]. Biofuel production based on land plants as a biomass source requires several factors such as land and water for cultivation and thus cannot provide a suitable solution [3,4]. In particular, bioconversion from an edible feedstock such as corn (
Mannitol, a six-carbon sugar alcohol widely distributed in nature, is known as one of the main carbohydrate components of brown algae such as
In this study, ethanol production from
To develop mannitol-fermentable
Table 1 Strains, plasmids, and primers used in the present study
|SR8||D452-2 with an optimized xylose-metabolic pathway||[45, 46]|
|JE 01||D452-2 PTDH3-||This study|
|JE 02||SR8 PCCW12-MAN2||This study|
|JE 03||SR8 PCCW12-MAN2, PTDH3-||This study|
|pRS41N-Cas9||A single-copy plasmid containing Cas9 and |||
|pRS42H-INT#1||A multicopy plasmid containing an |||
|pRS42K-||A multicopy plasmid containing the ||This study|
The obtained yeast colonies were precultured in YPD medium (1% yeast extract, 2% peptone, and 2% D-glucose) for 24 h at 30°C and 250 rpm. Cell pellet of the preculture was subsequently collected by centrifugation, washed with sterilized water, and resuspended to the optical density of either 0.1 or 0.5 at 600 nm (OD600), as the measure of cell density. Finally, the cell suspension was inoculated into YPD medium, YPM medium (1% yeast extract, 2% peptone, and 2% D-mannitol) or a pretreated algal biomass solution and further cultivated at 30°C at either 130 or 250 rpm. All experiments were performed in triplicates.
h. The dried sample was then milled in a grinder with a pore size of 180−245 μm and stored at −80°C until use. The sample powder was soaked in 4% (v/v) H2SO4 with different solid loadings (16%, 20%, and 24%, w/v) and pretreated at 90°C, 105°C, and 121°C for 30 min using an autoclave. The pretreated samples were neutralized to pH 5.5−6.0 by adding 1.5 g CaCO3.
For the enzymatic hydrolysis of the pretreated sample, Cellic CTec2 (40 filter paper unit/g biomass) and Viscozyme L (100 fungal β-glucanase unit/g pretreated algae) were added (Novozymes, Krogshoejvej, Denmark). SSF was carried out using the
For determination and quantification of glucose, mannitol, and ethanol in both YPM medium and algal hydrolysate, a high-performance liquid chromatography system was employed (Agilent 1260 series; Agilent, Santa Clara, CA, USA) equipped with a Rezex-ROA Organic Acid H+ column (8%, 150 mm × 4.6 mm; Phenomenex, Torrance, CA, USA). The column was eluded with 0.005 N H2SO4 at 50°C and the flow rate was set to 0.6 mL/min. Statistical analysis of differences among mean values was performed by either Student’s t test using Microsoft Excel or one-way ANOVA test using IBM SPSS (Armonk, New York, USA).
A previous study reported that native
To confirm the activation of the mannitol metabolic pathway in the engineered
Bioconversion of biomass into ethanol normally requires four steps: pretreatment, saccharification, fermentation, and ethanol extraction (
Pretreatment is an important step in biomass conversion into a fuel or other chemicals that facilitates both enzymatic saccharification and microbial fermentation. Up to date, a variety of pretreatment methods have been developed for seaweed based on
Brown algae contain cellulose and laminarin as the major polysaccharides . Cellulose, a linear glucan with β-1,4 linkages, is the most abundant carbon source on the planet and its enzymatic hydrolysis kinetics is well-known [2,37]. In many cases, cellulase mixtures composed of lytic polysaccharide monooxygenase, cellobiohydrolase (CBH), endoglucanase (EG), and β-glucosidase (BG) (
The pretreated algal samples were hydrolyzed either with only Cellic CTec2 or a mix of Cellic CTec2 and Viscozyme L (Fig. 5), and the released glucose and mannitol concentrations were monitored. During a 24-h incubation, when Cellic CTec2 only was used, 22.21 g/L glucose was obtained with a marginal amount of mannitol (< 3 g/L). When using the two-enzyme mixture, however, significantly higher concentrations of glucose and mannitol were obtained, 34.29 g/L and 19.56 g/L, respectively. The result suggested that Viscozyme L is able to hydrolyze laminarin of the brown algae, resulting in the release of glucose and mannitol.
To increase the final ethanol concentration, the solid loading of algal biomass for the pretreatment was increased from 16%, to 20% and 24% (w/v). A mixture of Cellic CTec2 and Viscozyme L was used for saccharification and the JE 03 strain was used for fermentation (Table 2 and Fig. 6). The final ethanol concentrations were 16.31 g/L, 20.46 g/L, and 22.95 g/L at 16%, 20%, and 24% (w/v) solid loadings, respectively. However, ethanol productivity (0.680 g/L/h) was the highest at the lowest solid loading tested (16%). Ethanol yield from 16% biomass was 10.19% (w/w, based on biomass), which was higher than those
Table 2 Effect of biomass solid loading on ethanol production
|Solid loading (%, w/v)||Ethanol (g/L)||Ethanol productivity (g/L/h)|
|16||16.3 ± 3.4||0.680 ± 0.140|
|20||20.4 ± 1.0||0.568 ± 0.027|
|24||22.9 ± 2.7||0.479 ± 0.056|
Table 3 Comparison of ethanol yields from various brown algae
|Brown algae||Ethanol yield (%, w/w)||Reference|
|Sargassum horneri||10.19||This study|
Glucose repression is the phenomenon widely observed in microorganisms including
Performing SSF with a high solid loading is important to achieve an economically effective ethanol recovery process . Ethanol productivity in our study decreased as the solid loading increased. A SSF process at a high solid loading is known to suffer from low ethanol yield and slow fermentation due to a poor access of microbial cells and hydrolytic enzymes to a substrate (
This work was supported by Center for Women in Science, Engineering and Technology (WISET) grant funded by the Ministry of Science and ICT (MSIT), Korea under the team research program for female engineering students.
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