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Xylo-oligosaccharides enriched yeast protein feed production from reed sawdust
Accepted Manuscript Short Communication Xylo-oligosaccharides enriched yeast protein feed production from reed sawdust Meixia Chen, Qiang Li, Ya Zhang, Haiming Li, Jie Lu, Yi Cheng, Haisong Wang PII: DOI: Reference:
S0960-8524(18)31380-4 https://doi.org/10.1016/j.biortech.2018.09.127 BITE 20543
To appear in:
Bioresource Technology
Received Date: Revised Date: Accepted Date:
27 July 2018 22 September 2018 25 September 2018
Please cite this article as: Chen, M., Li, Q., Zhang, Y., Li, H., Lu, J., Cheng, Y., Wang, H., Xylo-oligosaccharides enriched yeast protein feed production from reed sawdust, Bioresource Technology (2018), doi: https://doi.org/ 10.1016/j.biortech.2018.09.127
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Xylo-oligosaccharides enriched yeast protein feed production from reed sawdust Meixia Chena #, Qiang Lib #, Ya Zhanga, Haiming Lia, Jie Lua, Yi Chenga and Haisong Wanga* aLiaoning
Key Laboratory of Pulp and Papermaking Engineering, Dalian Polytechnic
University, Dalian 116034, Liaoning, China. bDepartment
of Plant Pathology and Microbiology, Texas A&M University, College
Station, TX 77840, USA. #These
authors contributed equally in this work.
*Corresponding
authors: Haisong Wang. Tel: +86 532 80662725. E-mail address:
[email protected] Abstract The aim of this study was to convert the cellulose and hemicellulose, in reed sawdust from the pulp mills, into yeast protein and Xylo-oligosaccharide, then functionalize xylooligosaccharide as yeast feed. Both synchronous saccharification and fermentation and separate hydrolysis and fermentation of cellulase and Candida utilis were investigated to produce protein feed. By optimizing the fermentation conditions, 6.1 g/L of protein with 76.1 % (7.1 g/L) Xylo-oligosaccharide as the sugar was obtained. The final glucan and xylan utilization efficiencies in reed sawdust were 85.45% and 91.03%, respectively. Xylo-oligosaccharide enriched yeast protein feed from reed sawdust was thus realized by pretreatment, enzymatic hydrolysis and synchronous saccharification and fermentation. Keywords: Reed sawdust; synchronous saccharification and fermentation; xylooligosaccharide; protein feed.
1. Introduction Reed, as a rhizomatous perennial graminoid plant, can absorb toxic pollutants in water and maintain wetland ecology, which brings in high adaptability to extremely harsh and cruel conditions such as saline land and heavy metal soil (Chu, et al., 2017). In the past decades, reed was mainly used for construction and making daily products like thatching and mats. Besides these applications, reed has recently become an sought after energy crop for papermaking due to its high content of celluloses (40.52%) and hemicelluloses (25.86%) (Ge, et al., 2016; Lu, et al., 2013). In China, pulp and paper industries could annually consume 2.5×106~3×106 tons of reeds, generating about 2×105 tons reed sawdust (RS) as a byproduct. RS thereby represented a waste stream to be upgraded. Nutritional yeast could be used as feed protein, since it has high contents of protein and vitamin B. In addition, many researchers have reported that nutritional yeast could prevent important diseases from animals, regulate metabolic balance and improve the feed efficiency to animals (Shurson, et al., 2018; Dias, et al., 2018). All these functions make nutrition yeast a promising nutritional ingredient to be utilized in feed (Dias, et al., 2018). All current yeast proteins were produced from starch and sucrose, which competed with food supplies (Shurson, et al., 2018). Lignocellulosic biomass has high polysaccharides content, which could be an alternative of traditional starch and sucrose for yeast protein production. Moreover, antibiotics in animal feed has been world-widely restricted, which provides biomass-based alternatives opportunity to replace the traditional yeast protein (Carvalho, et al., 2013). Xylo-oligosaccharides (XOS), a functional sugar, has great potential in various applications such as food and health care
products. XOS has the functions of immune, antimicrobial and other health benefits (Moure et al., 2006). XOS could not be digested or absorbed in the gastrointestinal tract (Zhao, et al., 2017; Boonchuay, et al., 2018), which means it can reach colon and promote the growth of beneficial bacteria, and further to enhance short chain fatty acid production and improve the immunity of animals (Carvalho, et al., 2013). Nevertheless, the commercialization of XOS is still impractical due to the single feedstock and the high cost of the purification process (Wang, et al., 2017). This paper thus exploited the production of yeast protein from XOS. Previous study has shown that RS is a promising feedstock for XOS production. 0.136 g XOS, 0.274 g glucose and other pentose yielded were 0.105 g /g of RS, respectively. The impurities in the mixture liquor were mainly monosaccharides at 73.59%. However, the produced XOS accounted for only 26.41% of the total sugars, which was significant below the usual purity of commercial XOS＞ 50% as required by the yeast feed. The reason of this low purity could be attributed to the similar structure characteristics between xylose and glucose, and makes XOS difficult to be separated from the glucose in the subsequent purification process (Li, et al., 2015; Chang, et al., 2017). To address the problem, the commercial Candida utilis was used to convert the fermentable sugars into animal nutrient feed proteins and improve the purity of XOS in this study. Utilization of XOS as the animal protein feed could thus add into the traditional animal feed and boost the application of functional XOS in feed field. 2. Materials and methods 2.1 Materials and pretreatment
RS in this study was provided by a reed pulp mill (Jinzhou in Liaoning Kim Paper Co, Ltd, Liaoning province, China). The moisture content of the RS after air-dried at room temperature was 9.80%. Liquid hot water (LHW) pretreatment was conducted in a 15 L digester with four small tanks. About 100 g oven-dried RS and 800 mL working volume were loaded in each (RSto-liquid ratio was 1:8). The cooking mixture was heated to 170ºC and hold for 30 min, then separated by 300 mesh nylon gauze. The processed solids were air-dried for chemical composition analysis and enzymatic hydrolysis. The pre-hydrolysate was detoxificated with sodium hydroxide at 30°C after neutralized by adjusting the pH to above 7.0, then stored at 4ºC before use. 2.3 Microorganism and growth conditions The yeast strain (Candida utilis), a commercial strain cocktails, procured from Cangzhou Xingye Biotechnology Co, Ltd. Hebei province, China. The activated culture medium used was YPD (contained 20 g of glucose, 20 g of peptone, 10 g of yeast extract in 1 L of deionized water) culture medium. Seeds were cultured at 32 ± 2ºC and in a shaking incubator at 200 rpm / min (ZWY-240, Shanghai, China) for 20 h. 2.4 Procedures of separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) for consuming glucose. For SSF process, enzymatic hydrolysis experiments were carried out in 250 mL flask loaded with 4g processed solid, 66 mL sodium citrate buffer (PH 4.8) and 0.25 mL xylanase (1000 IU/ mL). The experiments were performed at 50ºC and 120 rpm / min for 8h, then followed by boiling water bath to inactivate enzyme, after that, adding the pre-
hydrolysate at a ratio of 5:6 (v/v) to xylanase solution, then the mixture was concentrated to 50 mL before the pH was adjusted to 5.0 with solid citric acid. 2.5 mL activated yeast seed culture, 0.3 mL cellulase (80 FPU/ mL) and the formulated nutrient (8 g/L of yeast extract powder, 1.5 g/L of MgSO4, and 1 g/L KH2PO4) were inoculated to start the fermentation. Another parallel experiment was without pre-hydrolysate. The process of SHF was like SSF except for the cellulase hydrolysis and fermentation was operated respectively. The xylanase enzymatic residue was washed and further hydrolyzed by cellulase for 30h. The two stages hydrolysate were followed by inactivate enzyme, then adding pre-hydrolysate as SSF. The pH of fermentation was adjusted to 5.5. The fermentation temperature was maintained at 32°C, agitation at 200 rpm. The samples were withdrawn every 12h to determine residual sugars by harvesting entire flasks. 2.5 Analytical methods 2.5.1 Compositional analysis of processed solid The chemical components in processed solid was determined by the NREL/TP-51042618 standard analytical procedure (Sluiter et al, 2008). 2.5.2 Compositional analysis of the residual sugars and by-products in fermented broth Fermented broth samples were filtered by 0.2-μm syringe filters. The residual glucose and xylose were identified using the SBA-40D Biological Sensing Analyzer (Biology Institute of the Shandong Academy of Sciences, Jinan, China). XOS content was measured by ion chromatography with the CarboPacTMPA1 column (Dionex-300, Dionex Corporation, USA). The eluent was a mixture of NaOH and CH3COONa, and the
flow rate was 1 mL / min. 0.5 mL fermented broth was collected and washed with 0.9% normal saline for 3 times, and then the precipitate was diluted 5 times for measuring the activity of the yeast (colony forming units, cfu) and dry cell weight (DCW, g/L). The viable cell density of different periods was calculated accordingly by the values of optical density (OD600), and the XOS ratio was determined by the data of produced XOS accounted for total sugars. All the experiments were done in triplicate and the data shown were the average ± standard deviation. 3. Results and Discussion 3.1 Compositional analysis of processed solid and pre-hydrolysate In a previous work, the optimal condition for the LHW pretreatment was at the temperature of 170ºC have been found. About 0.24 g acetic acid was obtained, meanwhile, the hydrolysis of hemicellulose mainly produced 10.56 g XOS and about 0.95 g xylose / 100g of RS, the furfural was only 0.07 g/100g of RS. All these degraded productions corresponded to 64.4% of initial xylan (based on the xylan in RS). Moreover, by degradation of xylan into XOS, xylose and furfural, the glucan content in processed solid was enriched (49.88%). The components in processed solid will be further hydrolyzed and utilized with the enzymatic hydrolysis and the followed fermentation. 3.2 Compositional analysis of fermented broth 3.2.1 Comparison of the SHF process with and without pre-hydrolysate SHF was carried out in a 250 mL flask. As shown in Fig. 1a and 1b, glucose was rapidly
consumed and the data of OD600 was increased during 24h, due to that the glucose was assimilated prior to xylose (Boonchuay, et al., 2018). By applying the pre-hydrolysate, the logarithmic growth phase was delayed 12~24h, and the data of the OD600 was about 0.43 at 36h (Fig. 1b), which was lower than that of the process without pre-hydrolysate (0.71). The furfural content we detected in this medium was 0.10 g / L. Moreover, extractions such as pigments, lipids, formic acid, etc (data not shown), which were dissolved into pre-hydrolysate during pretreatment could be the inhibitors of fermentation (Li, et al., 2018). The existence of these solubilized inhibitors could explain the reasons that Candida utilis cannot grow well in a medium of containing pre-hydrolysate. However, Candida utilis could assimilate pentose and XOS (Koyama, et al., 2014), leading to rapid decrement of the XOS after 36h incubation. Therefore, the optimized terminal fermentation period was defined at 36h. Interestingly, XOS have an increased point at 24h (Fig. 1a and 1b), which was probably due to that some xylose chain with higher degree of polymerization can be hydrolyzed by Candida utilis metabolites. As a result, for the process of SHF with 36h incubation, 94% of glucose can be assimilated and 5.60 g/L of yeast protein obtained, and XOS ratio with pre-hydrolysate (76.1%) was much higher than that without pre-hydrolysate (67.7%). 3.2.2 Comparison of the SSF process with and without pre-hydrolysate Besides SHF, SSF was further used to produce the yeast feed. As shown in Fig. 1c, the glucose content was increased to 17 g/L at 12h, due to that the glucose release rate during SSF was faster than the assimilate of Candida utilis, while the increament of glucose was
not obvious in the medium with pre-hydrolysate (Fig. 1d), although the data of OD600 was even lower. For example, the residual of glucose and xylose was only about 1.0 g/L at 36h, and the data of OD600 was only 0.35, which was 2-fold lower than that of without pre-hydrolysate (0.75). However, significant difference was not found in final DCW between with and without hydrolysate, which could be explained as the sugar metabolic was mainly used to provide enough Adenosine-triphosphate (ATP) for survival with the cumulative of metabolites led to the yeast cells were dying more rapidly (Yin, et al., 2018). Similar to SHF, the logarithmic growth phase of SSF with pre-hydrolysate was delayed 12~24h, which could also be rendered by the soluble XOS as an inhibitor to cellulase (Limayem, et al., 2012). The cellulase enzymatic hydrolysis could be lowered by the XOS inhibition. Furthermore, XOS could also be the substrate of the cellulase (Zhang, et al., 2014). For example, in SSF, the XOS was decreased from 10.4 to 7.1 g/L and the xylose was increased from 5.5 to 6.4 g/L at 24h (Fig. 1d). At last, the consumption of glucose urged the assimilation of xylose and XOS, leading to a rapid decrement in XOS from 7.0 to 2.6 g/L after 36h to 60h incubation (Fig. 1d). Meanwhile, the data of OD600 (from 0.37 to 0.45) was slightly increased, however, the assimilation and reduction of XOS is not expected. Based on these results, 36h of incubation was defined as the terminal time. Overall, in the process of SSF, XOS ratio with pre-hydrolysate (76.30%) was relatively higher than that without pre-hydrolysate (37.61%). 3.3 Comparison of the composition in ultimate fermented broth of both SHF and SSF. The effect of feedback inhibition on cellulase by glucose and cellobiose could be
elucidated from the DCW data of SSF and SHF. For both with and without the prehydrolysate, the DCW data of SSF was higher than that of SHF process. The higher DCW of SSF could be attributed to that the assimilation of glucose decreased the inhibition of glucose on cellulase (Qi, et al., 2018) and improved the utilization of the components in RS. The improved fermentation could also be observed from the relatively higher DCW. As a result, 76.3% (7.1 g/L) XOS ratio was also the highest in this condition. More importantly, SSF with pre-hydrolysate can omit purification process of XOS and could thereby significantly reduce the processing cost. Overall, the data of DCW and XOS ratio in the process of SSF with pre-hydrolysate were 6.1 g/L and 76.3% (7.1 g/L), respectively, the recovery of XOS based on the initial XOS before fermentation was 67.3%, the Candida utilis viability was about 9.38 log cfu / mL, and the final glucan and xylan utilization efficiencies were 85.45%, 91.03%, respectively. In another word, when 100g RS (35.90g glucan, 21.21g xylan and 20.76g lignin) was pretreated with LHW for 30min and followed by xylanase enzymatic hydrolysis and 36h SSF of cellulase and fermentation, 6.21g XOS, 5.50g protein and 1.05g xylose can be obtained. Overall, the research paved a new way that using lignocellulosic biomass as material for the yeast protein and XOS enriched feed production. Such utilization could overcome the main challenging in using XOS as the feed additive, which have a high-cost in the purification process (Wang, et al., 2017), and finally render it luxurious to use in feed field. XOSderived yeast protein and XOS enriched feed thus could be an alternative to nutritional yeast and has great potential to transform both RS pulping industry and nutritional yeast
industry. 4. Conclusion RS as the waste from reed pulping mills was pretreated with LHW and then fermented for yeast feed production. Processes of SSF and SHF with or without pre-hydrolysate were investigated. SSF process with pre-hydrolysate for 36h incubation was optimized, and 6.1 g/L of protein with 76.3% XOS as the sugar was obtained. In addition, the recovery of XOS based on the initial XOS before fermentation was 67.3%, and the final glucan and xylan utilization efficiencies were 85.45% and 91.03%, respectively. By producing RS into XOS enriched yeast feed, both XOS and yeast proteins were enriched in the same product. Acknowledgments The authors are grateful for the financial support from the Natural Science Foundation of China (No. 31370582 and No. 31770624), the Natural Science Foundation of Liaoning (No. 20170540069) and Liaoning Provincial Department of Education science and technology research projects (No. LZ2015005 and 2017J002). Appendix A. Supplementary data E-supplementary data for this work can be found in e-version of this paper online References 1. Boonchuay, P., Techapun, C., Leksawasdi, N., Seesuriyachan, P., Hanmoungjai P., Watanabe, M., Takenaka, S., Chaiyaso, T., 2018. An integrated process for xylooligosaccharide and bioethanol production from corncob. Bioresour Technol. 256, 399-407.
2. Benkun, Qi., Jianquan, Luo., Yinhua, Wan., 2018. Immobilization of cellulase on a core-shell structured metal-organic framework composites: better inhibitors tolerance and easier recycling. Bioresour Technol. 268, 577-582. 3. Brancoli, Pedro., Ferreira, Jorge A., Bolton, Kim., Taherzadeh, Mohammad J., 2018. Changes in carbon footprint when integrating production of filamentous fungi in 1st generation ethanol plants. Bioresour Technol. 249, 1069-1073. 4. Carvalho, A., Neto, P., Silva, D., Pastore, G., 2013. Xylo-oligosaccharides from lignocellulosic materials: Chemical structure, health benefits and production by chemical and enzymatic hydrolysis. Food Research International. 51:75-85. 5. Chang, S., J. Chu., Y. Guo., H. Li., B. Wu and B. He., 2017. An efficient production of high-pure xylooligosaccharides from corncob with affinity adsorptionenzymatic reaction integrated approach. Bioresour Technol. 241, 1043-1049. 6. Chen, T., Wen, J., Wang, B., Wang, H., Liu, C., Sun, R., 2017. Assessment of integrated process based on autohydrolysis and robust delignification process for enzymatic saccharification of bamboo. Bioresour. Technol. 244 (Part 1), 717-725. 7. Chu, X., Yang, L., Wang, S., Liu, J., Yang, H., 2017. Physiological and metabolic profiles of common reed provide insights into plant adaptation to low nitrogen conditions. Biochemical Systematics and Ecology. 73, 3-10. 8. Dias, JDL., Silva, RB., Fernandes, T., Barbosa, EF., Graas, LEC., Araujo, RC., Pereira, RAN., Pereira, MN., 2018. Yeast culture increased plasma niacin concentration, evaporative heat loss, and feed efficiency of dairy cows in a hot environment.
Journal of Dairy Science. 101, 5924-5936. 9. Feng-Wei, Yin., Dong-Sheng, Guo., Lu-Jing, Ren., Xiao-Jun, Ji., He, Huang., 2018. Development of a method for the valorization of fermentation wastewater and algal-residue extract in docosahexaenoic acid production by Schizochytrium sp. Bioresour Technol. 266, 482-487. 10. Li, Q., Koda, K., Yoshinaga, A., Takabe, K., Shimomura, M., Hirai, Y., Tamai, Y., Uraki, Y, 2015. Dehydrogenative Polymerization of Coniferyl Alcohol in Artificial Polysaccharides Matrices: Effects of Xylan on the Polymerization. J. Agric. Food Chem. 63, 4613-4620. 11. Li, Y., Z. Zhang, S. Zhu, H. Zhang, Y. Zhang, T. Zhang and Q. Zhang., 2017. Comparison of bio-hydrogen production yield capacity between asynchronous and simultaneous saccharification and fermentation processes from agricultural residue by mixed anaerobic cultures. Bioresour Technol, 247, 1210-1214. 12. Limayem, A., and S.C, Ricke., 2012. Lignocellulosic biomass for bioethanol
production: Current perspectives, potential issues and future prospects. Progress in Energy and Combustion Science. 38(4), 449-467. 13. Lu, J., Li, X., Yang, R., Yang, L., Zhao, J., Liu, Y., Qu, Y., 2013. Fed-batch semisimultaneous saccharification and fermentation of reed pretreated with liquid hot water for bio-ethanol production using Saccharomyces cerevisiae. Bioresour Technol. 144, 539-547. 14. Moure, A., Gullo′n, P., Dom′nguez, H., 2006. Advances in the manufacture,
purification and applications of xylo-oligosaccharides as food additives and nutraceuticals. Process Biochemistry. 41(9), 1913–1923 15. Ren, J., L. Liu, J. Zhou, X. Li and J. Ouyang, 2018. Co-production of ethanol, xylooligosaccharides and magnesium lignosulfonate from wheat straw by a controlled magnesium bisulfite pretreatment (MBSP). Industrial Crops and Products. 113: 128-134. 16. Sluiter, A., Hames, B., Ruiz, R., Scarlata, C., Sluiter, J., Templeton, D., Crocker, D., 2008. Determination of Structure Carbohydrates and Lignin in Biomass; National Renewable Energy Laboratory(NREL): Golden, CO, NREL/TP-510-42618. 17. Shurson, G. C., 2018. Yeast and yeast derivatives in feed additives and ingredients: Sources, characteristics, animal responses, and quantification methods. Animal Feed Science and Technology. 235, 60-76. 18. Wang, Y. H., J. Zhang., Y. S. Qu and H. Q. Li., 2018. Removal of chromophore in enzymatic hydrolysis by acid precipitation to improve the quality of xylooligosaccharides from corn stalk. Bioresour Technol. 249, 751-757. 19. Xiao, X., J. Bian., X. P. Peng., H. Xu., B. Xiao and R. C. Sun., 2013. Autohydrolysis of bamboo (Dendrocalamus giganteus Munro) culm for the production of xylooligosaccharides. Bioresour Technol. 138: 63-70. 20. Zhao, C., Wu, Y., Liu, X., Liu, B., Cao, H., Yu, H., Sarker, S., Nahar, L., Xiao, J., 2017. Functional properties, structural studies and chemo-enzymatic synthesis of oligosaccharides. Trends in Food Science and Technology. 66, 135-145.
21. Zhang, Y., Mu, X., Wang, H., Li, B., Peng, H., 2014. Combined Deacetylation and PFI Refining Pretreatment of Corn Cob for the Improvement of a Two-Stage Enzymatic Hydrolysis. J. Agric. Food Chem. 62(20), 4661-4667. Figures: b)
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Fig. 1 Effect of SHF and SSF on the composition of fermented broth. Fig. 1a is the process of SHF without pre-hydrolysate, Fig. 1b is the process of SHF with pre-hydrolysate; Fig. 1c is the process of SSF without pre-hydrolysate, and Fig. 1d is the process of SSF with pre-hydrolysate. Highlights: 1. Reed sawdust from pulp mill was used for the production of yeast protein feed 2. Yeast protein feed production from SHF and SSF with or without pre-hydrolysate were compared. 3. The purification process of xylo-oligosaccharide can be omitted.
4. Final glucan and xylan utilization efficiencies were 85.45%, 91.03%, respectively.
S0960-8524(18)31380-4 https://doi.org/10.1016/j.biortech.2018.09.127 BITE 20543
To appear in:
Bioresource Technology
Received Date: Revised Date: Accepted Date:
27 July 2018 22 September 2018 25 September 2018
Please cite this article as: Chen, M., Li, Q., Zhang, Y., Li, H., Lu, J., Cheng, Y., Wang, H., Xylo-oligosaccharides enriched yeast protein feed production from reed sawdust, Bioresource Technology (2018), doi: https://doi.org/ 10.1016/j.biortech.2018.09.127
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Xylo-oligosaccharides enriched yeast protein feed production from reed sawdust Meixia Chena #, Qiang Lib #, Ya Zhanga, Haiming Lia, Jie Lua, Yi Chenga and Haisong Wanga* aLiaoning
Key Laboratory of Pulp and Papermaking Engineering, Dalian Polytechnic
University, Dalian 116034, Liaoning, China. bDepartment
of Plant Pathology and Microbiology, Texas A&M University, College
Station, TX 77840, USA. #These
authors contributed equally in this work.
*Corresponding
authors: Haisong Wang. Tel: +86 532 80662725. E-mail address:
[email protected] Abstract The aim of this study was to convert the cellulose and hemicellulose, in reed sawdust from the pulp mills, into yeast protein and Xylo-oligosaccharide, then functionalize xylooligosaccharide as yeast feed. Both synchronous saccharification and fermentation and separate hydrolysis and fermentation of cellulase and Candida utilis were investigated to produce protein feed. By optimizing the fermentation conditions, 6.1 g/L of protein with 76.1 % (7.1 g/L) Xylo-oligosaccharide as the sugar was obtained. The final glucan and xylan utilization efficiencies in reed sawdust were 85.45% and 91.03%, respectively. Xylo-oligosaccharide enriched yeast protein feed from reed sawdust was thus realized by pretreatment, enzymatic hydrolysis and synchronous saccharification and fermentation. Keywords: Reed sawdust; synchronous saccharification and fermentation; xylooligosaccharide; protein feed.
1. Introduction Reed, as a rhizomatous perennial graminoid plant, can absorb toxic pollutants in water and maintain wetland ecology, which brings in high adaptability to extremely harsh and cruel conditions such as saline land and heavy metal soil (Chu, et al., 2017). In the past decades, reed was mainly used for construction and making daily products like thatching and mats. Besides these applications, reed has recently become an sought after energy crop for papermaking due to its high content of celluloses (40.52%) and hemicelluloses (25.86%) (Ge, et al., 2016; Lu, et al., 2013). In China, pulp and paper industries could annually consume 2.5×106~3×106 tons of reeds, generating about 2×105 tons reed sawdust (RS) as a byproduct. RS thereby represented a waste stream to be upgraded. Nutritional yeast could be used as feed protein, since it has high contents of protein and vitamin B. In addition, many researchers have reported that nutritional yeast could prevent important diseases from animals, regulate metabolic balance and improve the feed efficiency to animals (Shurson, et al., 2018; Dias, et al., 2018). All these functions make nutrition yeast a promising nutritional ingredient to be utilized in feed (Dias, et al., 2018). All current yeast proteins were produced from starch and sucrose, which competed with food supplies (Shurson, et al., 2018). Lignocellulosic biomass has high polysaccharides content, which could be an alternative of traditional starch and sucrose for yeast protein production. Moreover, antibiotics in animal feed has been world-widely restricted, which provides biomass-based alternatives opportunity to replace the traditional yeast protein (Carvalho, et al., 2013). Xylo-oligosaccharides (XOS), a functional sugar, has great potential in various applications such as food and health care
products. XOS has the functions of immune, antimicrobial and other health benefits (Moure et al., 2006). XOS could not be digested or absorbed in the gastrointestinal tract (Zhao, et al., 2017; Boonchuay, et al., 2018), which means it can reach colon and promote the growth of beneficial bacteria, and further to enhance short chain fatty acid production and improve the immunity of animals (Carvalho, et al., 2013). Nevertheless, the commercialization of XOS is still impractical due to the single feedstock and the high cost of the purification process (Wang, et al., 2017). This paper thus exploited the production of yeast protein from XOS. Previous study has shown that RS is a promising feedstock for XOS production. 0.136 g XOS, 0.274 g glucose and other pentose yielded were 0.105 g /g of RS, respectively. The impurities in the mixture liquor were mainly monosaccharides at 73.59%. However, the produced XOS accounted for only 26.41% of the total sugars, which was significant below the usual purity of commercial XOS＞ 50% as required by the yeast feed. The reason of this low purity could be attributed to the similar structure characteristics between xylose and glucose, and makes XOS difficult to be separated from the glucose in the subsequent purification process (Li, et al., 2015; Chang, et al., 2017). To address the problem, the commercial Candida utilis was used to convert the fermentable sugars into animal nutrient feed proteins and improve the purity of XOS in this study. Utilization of XOS as the animal protein feed could thus add into the traditional animal feed and boost the application of functional XOS in feed field. 2. Materials and methods 2.1 Materials and pretreatment
RS in this study was provided by a reed pulp mill (Jinzhou in Liaoning Kim Paper Co, Ltd, Liaoning province, China). The moisture content of the RS after air-dried at room temperature was 9.80%. Liquid hot water (LHW) pretreatment was conducted in a 15 L digester with four small tanks. About 100 g oven-dried RS and 800 mL working volume were loaded in each (RSto-liquid ratio was 1:8). The cooking mixture was heated to 170ºC and hold for 30 min, then separated by 300 mesh nylon gauze. The processed solids were air-dried for chemical composition analysis and enzymatic hydrolysis. The pre-hydrolysate was detoxificated with sodium hydroxide at 30°C after neutralized by adjusting the pH to above 7.0, then stored at 4ºC before use. 2.3 Microorganism and growth conditions The yeast strain (Candida utilis), a commercial strain cocktails, procured from Cangzhou Xingye Biotechnology Co, Ltd. Hebei province, China. The activated culture medium used was YPD (contained 20 g of glucose, 20 g of peptone, 10 g of yeast extract in 1 L of deionized water) culture medium. Seeds were cultured at 32 ± 2ºC and in a shaking incubator at 200 rpm / min (ZWY-240, Shanghai, China) for 20 h. 2.4 Procedures of separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) for consuming glucose. For SSF process, enzymatic hydrolysis experiments were carried out in 250 mL flask loaded with 4g processed solid, 66 mL sodium citrate buffer (PH 4.8) and 0.25 mL xylanase (1000 IU/ mL). The experiments were performed at 50ºC and 120 rpm / min for 8h, then followed by boiling water bath to inactivate enzyme, after that, adding the pre-
hydrolysate at a ratio of 5:6 (v/v) to xylanase solution, then the mixture was concentrated to 50 mL before the pH was adjusted to 5.0 with solid citric acid. 2.5 mL activated yeast seed culture, 0.3 mL cellulase (80 FPU/ mL) and the formulated nutrient (8 g/L of yeast extract powder, 1.5 g/L of MgSO4, and 1 g/L KH2PO4) were inoculated to start the fermentation. Another parallel experiment was without pre-hydrolysate. The process of SHF was like SSF except for the cellulase hydrolysis and fermentation was operated respectively. The xylanase enzymatic residue was washed and further hydrolyzed by cellulase for 30h. The two stages hydrolysate were followed by inactivate enzyme, then adding pre-hydrolysate as SSF. The pH of fermentation was adjusted to 5.5. The fermentation temperature was maintained at 32°C, agitation at 200 rpm. The samples were withdrawn every 12h to determine residual sugars by harvesting entire flasks. 2.5 Analytical methods 2.5.1 Compositional analysis of processed solid The chemical components in processed solid was determined by the NREL/TP-51042618 standard analytical procedure (Sluiter et al, 2008). 2.5.2 Compositional analysis of the residual sugars and by-products in fermented broth Fermented broth samples were filtered by 0.2-μm syringe filters. The residual glucose and xylose were identified using the SBA-40D Biological Sensing Analyzer (Biology Institute of the Shandong Academy of Sciences, Jinan, China). XOS content was measured by ion chromatography with the CarboPacTMPA1 column (Dionex-300, Dionex Corporation, USA). The eluent was a mixture of NaOH and CH3COONa, and the
flow rate was 1 mL / min. 0.5 mL fermented broth was collected and washed with 0.9% normal saline for 3 times, and then the precipitate was diluted 5 times for measuring the activity of the yeast (colony forming units, cfu) and dry cell weight (DCW, g/L). The viable cell density of different periods was calculated accordingly by the values of optical density (OD600), and the XOS ratio was determined by the data of produced XOS accounted for total sugars. All the experiments were done in triplicate and the data shown were the average ± standard deviation. 3. Results and Discussion 3.1 Compositional analysis of processed solid and pre-hydrolysate In a previous work, the optimal condition for the LHW pretreatment was at the temperature of 170ºC have been found. About 0.24 g acetic acid was obtained, meanwhile, the hydrolysis of hemicellulose mainly produced 10.56 g XOS and about 0.95 g xylose / 100g of RS, the furfural was only 0.07 g/100g of RS. All these degraded productions corresponded to 64.4% of initial xylan (based on the xylan in RS). Moreover, by degradation of xylan into XOS, xylose and furfural, the glucan content in processed solid was enriched (49.88%). The components in processed solid will be further hydrolyzed and utilized with the enzymatic hydrolysis and the followed fermentation. 3.2 Compositional analysis of fermented broth 3.2.1 Comparison of the SHF process with and without pre-hydrolysate SHF was carried out in a 250 mL flask. As shown in Fig. 1a and 1b, glucose was rapidly
consumed and the data of OD600 was increased during 24h, due to that the glucose was assimilated prior to xylose (Boonchuay, et al., 2018). By applying the pre-hydrolysate, the logarithmic growth phase was delayed 12~24h, and the data of the OD600 was about 0.43 at 36h (Fig. 1b), which was lower than that of the process without pre-hydrolysate (0.71). The furfural content we detected in this medium was 0.10 g / L. Moreover, extractions such as pigments, lipids, formic acid, etc (data not shown), which were dissolved into pre-hydrolysate during pretreatment could be the inhibitors of fermentation (Li, et al., 2018). The existence of these solubilized inhibitors could explain the reasons that Candida utilis cannot grow well in a medium of containing pre-hydrolysate. However, Candida utilis could assimilate pentose and XOS (Koyama, et al., 2014), leading to rapid decrement of the XOS after 36h incubation. Therefore, the optimized terminal fermentation period was defined at 36h. Interestingly, XOS have an increased point at 24h (Fig. 1a and 1b), which was probably due to that some xylose chain with higher degree of polymerization can be hydrolyzed by Candida utilis metabolites. As a result, for the process of SHF with 36h incubation, 94% of glucose can be assimilated and 5.60 g/L of yeast protein obtained, and XOS ratio with pre-hydrolysate (76.1%) was much higher than that without pre-hydrolysate (67.7%). 3.2.2 Comparison of the SSF process with and without pre-hydrolysate Besides SHF, SSF was further used to produce the yeast feed. As shown in Fig. 1c, the glucose content was increased to 17 g/L at 12h, due to that the glucose release rate during SSF was faster than the assimilate of Candida utilis, while the increament of glucose was
not obvious in the medium with pre-hydrolysate (Fig. 1d), although the data of OD600 was even lower. For example, the residual of glucose and xylose was only about 1.0 g/L at 36h, and the data of OD600 was only 0.35, which was 2-fold lower than that of without pre-hydrolysate (0.75). However, significant difference was not found in final DCW between with and without hydrolysate, which could be explained as the sugar metabolic was mainly used to provide enough Adenosine-triphosphate (ATP) for survival with the cumulative of metabolites led to the yeast cells were dying more rapidly (Yin, et al., 2018). Similar to SHF, the logarithmic growth phase of SSF with pre-hydrolysate was delayed 12~24h, which could also be rendered by the soluble XOS as an inhibitor to cellulase (Limayem, et al., 2012). The cellulase enzymatic hydrolysis could be lowered by the XOS inhibition. Furthermore, XOS could also be the substrate of the cellulase (Zhang, et al., 2014). For example, in SSF, the XOS was decreased from 10.4 to 7.1 g/L and the xylose was increased from 5.5 to 6.4 g/L at 24h (Fig. 1d). At last, the consumption of glucose urged the assimilation of xylose and XOS, leading to a rapid decrement in XOS from 7.0 to 2.6 g/L after 36h to 60h incubation (Fig. 1d). Meanwhile, the data of OD600 (from 0.37 to 0.45) was slightly increased, however, the assimilation and reduction of XOS is not expected. Based on these results, 36h of incubation was defined as the terminal time. Overall, in the process of SSF, XOS ratio with pre-hydrolysate (76.30%) was relatively higher than that without pre-hydrolysate (37.61%). 3.3 Comparison of the composition in ultimate fermented broth of both SHF and SSF. The effect of feedback inhibition on cellulase by glucose and cellobiose could be
elucidated from the DCW data of SSF and SHF. For both with and without the prehydrolysate, the DCW data of SSF was higher than that of SHF process. The higher DCW of SSF could be attributed to that the assimilation of glucose decreased the inhibition of glucose on cellulase (Qi, et al., 2018) and improved the utilization of the components in RS. The improved fermentation could also be observed from the relatively higher DCW. As a result, 76.3% (7.1 g/L) XOS ratio was also the highest in this condition. More importantly, SSF with pre-hydrolysate can omit purification process of XOS and could thereby significantly reduce the processing cost. Overall, the data of DCW and XOS ratio in the process of SSF with pre-hydrolysate were 6.1 g/L and 76.3% (7.1 g/L), respectively, the recovery of XOS based on the initial XOS before fermentation was 67.3%, the Candida utilis viability was about 9.38 log cfu / mL, and the final glucan and xylan utilization efficiencies were 85.45%, 91.03%, respectively. In another word, when 100g RS (35.90g glucan, 21.21g xylan and 20.76g lignin) was pretreated with LHW for 30min and followed by xylanase enzymatic hydrolysis and 36h SSF of cellulase and fermentation, 6.21g XOS, 5.50g protein and 1.05g xylose can be obtained. Overall, the research paved a new way that using lignocellulosic biomass as material for the yeast protein and XOS enriched feed production. Such utilization could overcome the main challenging in using XOS as the feed additive, which have a high-cost in the purification process (Wang, et al., 2017), and finally render it luxurious to use in feed field. XOSderived yeast protein and XOS enriched feed thus could be an alternative to nutritional yeast and has great potential to transform both RS pulping industry and nutritional yeast
industry. 4. Conclusion RS as the waste from reed pulping mills was pretreated with LHW and then fermented for yeast feed production. Processes of SSF and SHF with or without pre-hydrolysate were investigated. SSF process with pre-hydrolysate for 36h incubation was optimized, and 6.1 g/L of protein with 76.3% XOS as the sugar was obtained. In addition, the recovery of XOS based on the initial XOS before fermentation was 67.3%, and the final glucan and xylan utilization efficiencies were 85.45% and 91.03%, respectively. By producing RS into XOS enriched yeast feed, both XOS and yeast proteins were enriched in the same product. Acknowledgments The authors are grateful for the financial support from the Natural Science Foundation of China (No. 31370582 and No. 31770624), the Natural Science Foundation of Liaoning (No. 20170540069) and Liaoning Provincial Department of Education science and technology research projects (No. LZ2015005 and 2017J002). Appendix A. Supplementary data E-supplementary data for this work can be found in e-version of this paper online References 1. Boonchuay, P., Techapun, C., Leksawasdi, N., Seesuriyachan, P., Hanmoungjai P., Watanabe, M., Takenaka, S., Chaiyaso, T., 2018. An integrated process for xylooligosaccharide and bioethanol production from corncob. Bioresour Technol. 256, 399-407.
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purification and applications of xylo-oligosaccharides as food additives and nutraceuticals. Process Biochemistry. 41(9), 1913–1923 15. Ren, J., L. Liu, J. Zhou, X. Li and J. Ouyang, 2018. Co-production of ethanol, xylooligosaccharides and magnesium lignosulfonate from wheat straw by a controlled magnesium bisulfite pretreatment (MBSP). Industrial Crops and Products. 113: 128-134. 16. Sluiter, A., Hames, B., Ruiz, R., Scarlata, C., Sluiter, J., Templeton, D., Crocker, D., 2008. Determination of Structure Carbohydrates and Lignin in Biomass; National Renewable Energy Laboratory(NREL): Golden, CO, NREL/TP-510-42618. 17. Shurson, G. C., 2018. Yeast and yeast derivatives in feed additives and ingredients: Sources, characteristics, animal responses, and quantification methods. Animal Feed Science and Technology. 235, 60-76. 18. Wang, Y. H., J. Zhang., Y. S. Qu and H. Q. Li., 2018. Removal of chromophore in enzymatic hydrolysis by acid precipitation to improve the quality of xylooligosaccharides from corn stalk. Bioresour Technol. 249, 751-757. 19. Xiao, X., J. Bian., X. P. Peng., H. Xu., B. Xiao and R. C. Sun., 2013. Autohydrolysis of bamboo (Dendrocalamus giganteus Munro) culm for the production of xylooligosaccharides. Bioresour Technol. 138: 63-70. 20. Zhao, C., Wu, Y., Liu, X., Liu, B., Cao, H., Yu, H., Sarker, S., Nahar, L., Xiao, J., 2017. Functional properties, structural studies and chemo-enzymatic synthesis of oligosaccharides. Trends in Food Science and Technology. 66, 135-145.
21. Zhang, Y., Mu, X., Wang, H., Li, B., Peng, H., 2014. Combined Deacetylation and PFI Refining Pretreatment of Corn Cob for the Improvement of a Two-Stage Enzymatic Hydrolysis. J. Agric. Food Chem. 62(20), 4661-4667. Figures: b)
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Fig. 1 Effect of SHF and SSF on the composition of fermented broth. Fig. 1a is the process of SHF without pre-hydrolysate, Fig. 1b is the process of SHF with pre-hydrolysate; Fig. 1c is the process of SSF without pre-hydrolysate, and Fig. 1d is the process of SSF with pre-hydrolysate. Highlights: 1. Reed sawdust from pulp mill was used for the production of yeast protein feed 2. Yeast protein feed production from SHF and SSF with or without pre-hydrolysate were compared. 3. The purification process of xylo-oligosaccharide can be omitted.
4. Final glucan and xylan utilization efficiencies were 85.45%, 91.03%, respectively.