Research article
Ethanol from rice byproduct using amylases secreted by Rhizopus microsporus var. oligosporus. Enzyme partial purification and characterization

https://doi.org/10.1016/j.jenvman.2020.110591Get rights and content

Highlights

  • Definition of a three-steps bioprocess to produce ethanol from broken rice.

  • First step involved amylases production from Rhizopus microsporus var. oligosporus.

  • In second step amylases were used to yield glucose from hydrolysis of broken rice.

  • Third stage aimed to ferment glucose by Saccharomyces cerevisiae to make ethanol.

  • The enzyme purification and its biochemical characterization were also addressed.

Abstract

A three-stage bioethanol bioprocess was developed. Firstly, amylases were obtained from Rhizopus microsporus var. oligosporus using wheat bran in solid-state fermentation. Secondly, amylases hydrolyzed a rice byproduct to make a glucose-rich solution, and this sugar was finally metabolized by Saccharomyces cerevisiae to produce bioethanol. Besides, the secreted enzymes were also partially purified and characterized. The amylase activity (AA) in the crude extract was 358 U/g substrate, and the partially purified enzyme showed the best activity in the 4.0–5.5 pH range. A wide pH stability range (3.5–8.5) was confirmed. The amylase was thermostable up to 60 °C. The ion Mn+2 (10 mM) improved by 60% the AA. There was a 54.9% yield in the conversion of rice residues into reducing sugars in 10 h. The glucose-rich solution was undergone fermentation by S. cerevisiae and showed high ethanol efficiency, 95.8% of the theoretical value. These results suggested a promising technology for bioethanol production.

Introduction

Food and agro-industrial wastes are suitable raw materials to produce biofuels because these residues reduce the need for new agricultural cultivation areas with this end. The improvement of technologies that use biological catalysts and agro-industrial byproducts for biofuels production has received significant attention in science and industry. Bioethanol has proven to be a reasonable alternative source of energy and less aggressive to the environment since this biofuel is mainly obtained by fermentation of a renewable feedstock and has a relatively low level of greenhouse gas emission. Thus, the number of manufacturers producing in a feasible way this commodity is increasing worldwide (Nayak and Bhushan, 2019).

Lignocellulosic materials together with starch residues are an alternative source for the production of ethanol (Nosratpour et al., 2018). Agribusiness generates a large amount of these products that can be reused, among them are: wheat bran, type II wheat flour, broken rice, cassava bagasse (Barchi et al., 2016; Escaramboni et al., 2018; Freitas et al., 2014; Nunes et al., 2017).

The world rice production in 2017 was estimated at 759.6 million tons. Rice is cultivated on every continent, highlighting Asia with 90.4% of the world production. Following in descending order Americas with 4.8%, Africa with 4.2%, Europe with 0.5% and Oceania with 0.1% of world rice production (FAO, 2018). Broken rice is a byproduct generated during rice milling when once of the grains are broken, which is usually used for animal feed. Therefore, this residue is less expensive than rice and can be applied in fermentation processes (Abd Razak et al., 2019; Li et al., 2013; Moin et al., 2016; Myburgh et al., 2019). About 10–15% of rice grain originally harvested becomes broken rice during rice milling processing (Li et al., 2019). Rice bran is another byproduct generated from the rice milling process, containing about 13.2–17.3% protein, 17.0–22.9% lipid, 9.5–13.2% crude fiber, 16.1% starch, vitamin B complexes, and minerals including iron, potassium, calcium, chlorine, magnesium, and manganese, and most of all, dietary fiber (27.6–33.3%) (Dang and Vasanthan, 2019; Kim et al., 2013). This material represents 10% of the rice stock and it is usually used as livestock feed, although additional amounts of rice bran are frequently available (Tsuchiya and Yoshida, 2017). Thus, alternative technologies for cost-effective use of these materials to contribute to the growing demand of the global bioenergy sector are needed.

The global revenue for industrial enzymes is estimated at US$ 7.2 billion a year in 2020 (Silveira et al., 2019a). Amylases stand out in the industrial sector and they find application in the manufacturing of detergent, food, textile, chemistry, paper, bakery, pharmaceutical, ethanol as well as clinical analysis (Di Donato et al., 2019; Kumari et al., 2019; Silveira et al., 2019b; Zhang et al., 2017). Amylases are part of the class of hydrolases acting on starch structure with the hydrolysis of the glycosidic bonds present in the chains of amylose and amylopectin. The most commonly used amylases in the industry are α-amylases, β-amylases, and glucoamylases (de Souza et al., 2019; Pandey et al., 2006). Several bacteria and fungi have been mainly identified as optimal amylase producers (Gopinath et al., 2017). Specifically, filamentous fungi have been extensively used to produce amylases. They are prolific makers of extracellular proteins, and their associated manufacturing process is cost-effective when Solid State Fermentation (SSF) is used. Besides, the fungal amylases are preferred over other microbial sources due to their GRAS (Generally Recognized as Safe) status (John, 2017).

SSF processes stand out and are valued as an important alternative process to the Submerged Fermentation (SmF). SSF is growing popularity as waste management technology, among its applications are bioremediation, detoxification, bioleaching and biopulping (Arora et al., 2018). In general, SSF uses low-cost substrates such as agricultural and industrial residues and contributing also to saving water, as well as demands lower capital and operating costs, reduced downstream process and higher enzymatic productivity for many enzymes have been reported (Soccol and Costa, 2017a).

The fungus Rhizopus microspores var oligosporus is amylolytic, used in Asian cooking for the production of fermented foods such as tempeh (Hartanti et al., 2015), and recognized as safe for food activity (Freitas et al., 2014). When this strain is used in the SSF processes with wheat bran as substrate, amylases for industrial purposes are yielded (Escaramboni et al., 2018; Fernández Nunes et al., 2017). These enzymes are responsible for significant hydrolysis of starch (liquefaction and saccharification), and glucose with a high potential for ethanol production is obtained (Escaramboni et al., 2018; Freitas et al., 2014).

This work aimed to define a three-steps bioprocess to produce ethanol from a rice byproduct. The first stage aimed at producing of amylases from Rhizopus microsporus var. oligosporus using wheat bran in SSF. Secondly, fungal amylases were used to yield glucose-rich solution from hydrolysis of rice residues, and, finally, this sugar was metabolized by Saccharomyces cerevisiae to produce bioethanol. The enzyme purification from SSF and its biochemical characterization was also addressed.

Section snippets

Microorganisms and maintenance of strains

Rhizopus microsporus var. oligosporus (CCT 3762) was obtained by André Tosello Culture Collection (Campinas, Brazil) and maintained on Potato Dextrose Agar medium (PDA) at 4 °C. The yeast strain Saccharomyces cerevisiae M-26 was previously isolated from bioethanol distillery (de Oliva Neto et al., 2004) and a commercial strain of S. cerevisiae (Fleishmann Royal Co., Sao Paulo, Brazil) were used in the ethanol fermentation. Yeasts were stored at −75 °C in Eppendorf on YM medium containing 2%

Amylase production and purification

The production of crude enzyme was made by 27 cultures with Rhizopus microspores var. oligosporus in SSF using wheat bran as a substrate and the amylase activity was 39.8 U mL−1 or 251.6 U/g substrate. This enzyme was previously pre-purified by filtration steps, centrifugation, and concentration, for subsequent downstream steps. After tangential ultrafiltration using hollow fiber cartridge 10 kDa, amylase activity increased 1.66 times resulting in 236 mL of concentrate enzyme with 66.2 U mL−1

Discussion

In the present study, SSF with the fungus R. oligosporus was successfully applied using wheat bran as a substrate to produce amylases. Tests conducted by Freitas et al. (2014) compared the production in SmF of amylases from R. oryzae and R. oligosporus at the same time and conditions. They demonstrated a higher amylase production (28.6%) for the second microorganism. This justifies the use of this strain for amylase production by our team in later works including the current one. However, SSF

Conclusions

This study showed the broken rice, a residue of the rice industry can be considered for ethanol production through a fast and direct enzymatic hydrolysis, since do not need pre-treatment, using glucoamylases from R. oligosporus. High production of glucose was obtained per ton of this product if compared to sugar cane. The ethanol efficiency and yeast viability were high demonstrating this substrate is suitable for ethanol fermentation by S. cerevisiae.

Author contributions

Pedro de Oliva Neto and Hamilton Cabral conceived and designed the study. Fabiane Fernanda de Barros Ranke, Thais Yumi Shinya and Franciane Cristina de Figueiredo performed the experimental work. Fabiane Fernanda de Barros Ranke and Eutimio Gustavo Fernández Núñez wrote the paper. Eutimio Gustavo Fernández Núñez reviewed and edited the manuscript. All authors read and approved the manuscript.

Funding

This study was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq grant number 134523/2013-0) and Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, grant number 2014/24188-1).

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this manuscript.

Acknowledgments

The authors would like to thank the Culture Collection of Tropical Research Foundation and Technology "André Tosello” – Campinas/SP - by giving strain of Rhizopus microsporus var oligosporus (CCT 3762), Cerealista Rampazzo Ltda-ME, Cândido Mota, Sao Paulo State, Brazil and Moinho Nacional (Assis, SP, Brazil) for the donation of the starch substrates.

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