Enzymatic hydrolysis of carbohydrates in by-products of processed rice

Hidrólise enzimática de carboidratos em coprodutos do arroz beneficiado

Camila da Silva Turini Roberta Martins Nogueira Evaldo Martins Pires Juliana da Silva Agostini About the authors

ABSTRACT:

Over post-harvest steps of rice, from pre-cleaning to processing, a large amount of by-product is generated. Some of these by-products, due to their high starch and fiber content can be used in ethanol production. The objective was to evaluate the effect of enzymatic hydrolysis conditions on the production of reducing sugars, from pre-cleaning residue and type III paddy rice, as well as the effect of the pre-treatment of its fibers, targeting the use of these residues in ethanol fuel production. The proximate analysis was performed, followed by the pre-treatment of samples. Enzymatic hydrolysis was conducted in two ways: using one enzyme at a time or applying them simultaneously. The starch content was 41.18 and 53.41%; the fibers were 30.44 and 23.39%, of which 6.53 and 4.41% were lignin, for the pre-cleaning residue and paddy rice, respectively. Alkaline pre-treatment reduce lignin content by 47.94 and 18.23% for the pre-cleaning residue and type III paddy rice, respectively. Hydrolysis efficiency was 22.61 and 15.32% for the cellulase enzyme, and 82.18 and 87.07% for the amylolytic enzymes in the pre-cleaning residue and type III paddy rice, respectively. The hydrolysis with the separated enzymes presented higher reducing sugar yields. Therefore, the pre-cleaning residue and type III paddy rice can be used for ethanol production by its enzymatic hydrolysis, aiming to add value and to increase the sustainability of the rice production chain.

Key words:
enzyme; Oryza sativa; reducing sugar; residue; starch

RESUMO:

Durante o processamento do arroz são gerados grandes volumes de resíduos, desde a pré-limpeza até o beneficiamento. Alguns destes resíduos, por apresentarem elevado teor de amido e fibras, podem ser utilizados na produção de etanol. O objetivo foi avaliar a viabilidade técnica do resíduo da pré-limpeza e do arroz em casca tipo III como matéria-prima para a produção de etanol, a partir da hidrólise enzimática. Foi analisada a composição centesimal dos resíduos e, em seguida, o pré-tratamento. A hidrólise enzimática foi realizada por dois procedimentos, o primeiro com as enzimas separadas e o segundo com as enzimas atuando simultaneamente. O teor de amido foi de 41,18 e 53,41%; de fibras 30,44% e 23,39%, dentre as quais 6,53 e 4,41% foi de lignina, para o resíduo da pré-limpeza e o arroz em casca, respectivamente. Com o pré-tratamento alcalino, a lignina reduziu em 47,94 e 18,23% para o resíduo da pré-limpeza e para o arroz em casca tipo III, respectivamente. A eficiência da hidrólise foi de 22,61 e 15,32% para a enzima celulase, e 82,18 e 87,07% para as enzimas amilolíticas no resíduo da pré-limpeza e no arroz em casca tipo III, respectivamente. A hidrólise com as enzimas separadas apresentou maior rendimento em açúcar redutor. Sendo assim, o resíduo da pré-limpeza e o arroz em casca do tipo III podem ser considerados matérias-primas viáveis para produção de etanol, visando agregação de valor e o aumento da sustentabilidade na cadeia produtiva do arroz.

Palavras-chave:
açúcar redutor; amido; enzima; Oryza sativa; resíduo

INTRODUCTION:

Oryza sativa (rice) is one of the most important cereals for the food chain. Brazil is the 9th largest producer in the world and the largest in Latin America, having produced 11.4 million tons during the 2018 harvest (FAO, 2018FAO - Food and Agriculture Organization of the United Nations. Rice market monitor. v.XXI, n.1, 2018. Available from: <Available from: http://www.fao.org/3/I9243EN/i9243en.pdf >. Accessed: Oct. 19, 2019.
http://www.fao.org/3/I9243EN/i9243en.pdf...
; IBGE, 2019IBGE - Instituto Brasileiro de Geografia e Estatística. Levantamento sistemático da produção agrícola - dezembro 2018. 2019. Available from: -<Available from: -https://sidra.ibge.gov.br/home/lspa/brasil >. Accessed: Oct. 19, 2019.
https://sidra.ibge.gov.br/home/lspa/bras...
).

The grains are composed of proteins, lipids, fibers, ashes, minerals, vitamins and starch. The latter represents the largest amount in the grain composition (WALTER, et al., 2008WALTER, M. et al. Rice: composition and nutritional characteristics. Ciência Rural, Santa Maria v.38, p.1184-1192, 2008. Available from: <Available from: https://doi.org/10.1590/S0103-84782008000400049 >. Accessed: Jul. 7, 2019. doi: 10.1590/S0103-84782008000400049.
https://doi.org/10.1590/S0103-8478200800...
; ZIEGLER et al., 2017ZIEGLER, V. et al. Effects of storage temperature of whole rice grains with brown, black and red pericarps, on the physicochemical and pasting properties. Brazilian Journal of Food Technology, v.20, e2016051, 2017. Available from: <Available from: http://dx.doi.org/10.1590/1981-6723.5116 >. Accessed: Jul. 2, 2019. doi: 10.1590/1981-6723.5116.
http://dx.doi.org/10.1590/1981-6723.5116...
). Factors such as cultivar, soil preparation, seed quality, climate, storage conditions and harvest time can influence the composition, yield and quality of the grain (KAMINSKI et al., 2013KAMINSKI, T. A. et al. Chemical composition and structural changes of irrigated rice during storage. Semina: Ciências Agrárias, v.34, p.1167-1184, 2013. Available from: <Available from: http://dx.doi.org/10.5433/1679-0359.2013v34n3p1167 >. Accessed: May, 20, 2019. doi: 10.5433/1679-0359.2013v34n3p1167.
http://dx.doi.org/10.5433/1679-0359.2013...
; ZIEGLER et al., 2017ZIEGLER, V. et al. Effects of storage temperature of whole rice grains with brown, black and red pericarps, on the physicochemical and pasting properties. Brazilian Journal of Food Technology, v.20, e2016051, 2017. Available from: <Available from: http://dx.doi.org/10.1590/1981-6723.5116 >. Accessed: Jul. 2, 2019. doi: 10.1590/1981-6723.5116.
http://dx.doi.org/10.1590/1981-6723.5116...
).

Before it is marketed, polished rice undergoes pre-cleaning, drying, storage, classification and processing operations inside a processing plant. During the pre-cleaning phase, impurities such as straw, husks, broken grains, stones and other materials are removed. Impurities can slow down the drying process, accelerate the development of microorganisms, and facilitate insects’ proliferation (HALBERSTADT et al., 2015HALBERSTADT, K. F. et al. Sustainable practices on waste resulting from the production rice Revista Eletrônica em Gestão, Educação e Tecnologia Ambiental, Santa Maria, v.19, p.298-312, 2015. Available from: <Available from: http://dx.doi.org/10.5902/2236117015816 >. Accessed: Feb. 10, 2019. doi: 10.5902/2236117015816.
http://dx.doi.org/10.5902/2236117015816...
). From field to market, about 2 million tons of by-products are generated annually in Brazil (IPEA, 2012IPEA - Instituto de Pesquisa Econômica Aplicada. Diagnósticos dos resíduos orgânicos do setor agrossilvopastoril e agroindústrias associadas. Relatório de Pesquisa, setembro, 2012. Available from: <Available from: http://www.ipea.gov.br/portal/index.php?option=com_content&view=article&id=15493&catid=222&Itemid=7 >. Accessed: Jan. 12, 2020.
http://www.ipea.gov.br/portal/index.php?...
). Some of them are currently used for animal feed or energy production.

Domestic and International markets have requirements for paddy or processed grain, as well for its fragments. For the Brazilian market, the Normative Instruction number 6 of 2009, from the Ministry of Agriculture, Livestock and Supply (BRASIL, 2009BRASIL Ministério da Agricultura, Pecuária e Abastecimento. Instrução normativa Nº 6, de 16 de fevereiro, 2009.) defines classes based on the grain quality. Rice is classified according to the occurrence of defects, from type I to non-compliant. Lower quality rice has a reduced market value, which may turn its processing unfeasible.

Thus, by-products or low-quality grains can become a co-product, considering the development of technologies that can use it alternatively, adding value to the production chain (NASCIMENTO FILHO & FRANCO, 2015NASCIMENTO FILHO, W. B.; FRANCO, C. R. Potential Assessment of Waste Produced Through the Agro-Industrial Processing in Brazil. Revista Virtual de Química, 7, 1968-1987, 2015. Available from: <Available from: http://dx.doi.org/10.5935/1984-6835.20150116 > Accessed: Mar. 20, 2019. doi: 10.5935/1984-6835.20150116.
http://dx.doi.org/10.5935/1984-6835.2015...
).

The transformation of by-products from agricultural raw materials processing has been widely studied, emphasizing biofuel production, that can enhance profits of the production chains and its sustainability, not to mention that the by-products may not compete with food production (NONES et al., 2017NONES, D. L. et al. Quantification of agricultural and forestry waste biomass to production of compacts for power generation. Revista de Ciências Agroveterinárias, 16, 155-164, 2017. Available from: <Available from: http://dx.doi.org/10.5965/223811711622017155 >. Accessed: Apr. 17, 2019. doi: 10.5965/223811711622017155.
http://dx.doi.org/10.5965/22381171162201...
; TAKANO & HOSHINO, 2018TAKANO, M.; HOSHINO, K. Bioethanol production from rice straw by simultaneous saccharification and fermentation with statistical optimized cellulase cocktail and fermenting fungus. Journal Bioresources and Bioprocessing, 5, 16, 2018. Available from: <Available from: http://dx.doi.org/10.1186/s40643-018-0203-y >. Accessed: Apr. 12, 2019.
http://dx.doi.org/10.1186/s40643-018-020...
). Brazil stands out as one of the largest ethanol producers in the world, having produced approximately 33.14 billion liters in the 2018/2019 season (55.7% of global production). However, sugar cane remains the country’s main raw material (CONAB, 2019CONAB. Cana de açúcar. Acompanhamento da safra brasileira. v.6. n.3. 2019. Available from: <Available from: https://www.conab.gov.br/info-agro/safras/cana >. Accessed: Jan. 25, 2020.
https://www.conab.gov.br/info-agro/safra...
), which can create a critical dependence on this input.

Low-quality paddy rice and the by-product gathered from the pre-cleaning can be considered for ethanol production, after its hydrolysis that allows the formation of reducing sugar and the subsequent alcoholic fermentation (RAUL et al., 2016RAUL, J. et al. Enzymatic saccharification and fermentation of rice processing residue for ethanol production at constant temperature. Biosystems Engineering, 42, 110-116, 2016. Available from: <Available from: http://dx.doi.org/10.1016/j.biosystemseng.2015.12.013 >. Accessed: Mar. 03, 2019. 10.1016/j.biosystemseng.2015.12.013.
http://dx.doi.org/10.1016/j.biosystemsen...
). The high content of starch and fiber of those by-products can render their transformation into fermentable sugars technically and economically feasible.

The enzymatic hydrolysis of starch and fibers results in higher conversion into sugar than the acid hydrolysis process (FERREIRA et al., 2013FERREIRA, S. M. et al. Production of reducing sugars by acid and enzimatic hydrolysis of broken rice flour. Revista Brasileira de Produtos Agroindustriais, 15, 383-390, 2013. Available from: <Available from: https://doi.org/10.15871/1517-8595/rbpa.v15n4p383-390 > Accessed: Jun. 16, 2019. doi: 10.15871/1517-8595/rbpa.v15n4p383-390.
https://doi.org/10.15871/1517-8595/rbpa....
). The selective action of the enzyme on the molecule is the key factor for this result. Cellulase acts selectively on the fiber, while amyloglucosidase and alpha-amylase act only on starch, under specific conditions of pH, temperature, concentration and reaction time; reducing the formation of secondary products (BISSWANGER, 2014BISSWANGER, H. Enzyme assays. Perspectives in Science, 1, s41-55, 2014. Available from: <Available from: http://dx.doi.org/10.1016/j.pisc.2014.02.005 >. Accessed: Jun. 16, 2019. doi: 10.1016/j.pisc.2014.02.005.
http://dx.doi.org/10.1016/j.pisc.2014.02...
).

Starch is composed of two polysaccharides: amylose and amylopectin and its hydrolysis produce oligosaccharides, dextrins and glucose (ECKERT et al., 2018ECKERT, C. T. et al. Maize ethanol production in Brazil: Characteristics and perspectives. Renewable and Sustainable Energy Reviews, 82, 3907-3912, 2018. Available from: <Available from: https://doi.org/10.1016/j.rser.2017.10.082 >. Accessed: Aug. 01, 2019. doi: 10.1016/j.rser.2017.10.082.
https://doi.org/10.1016/j.rser.2017.10.0...
). For fibers, cellulose hydrolysis generates glucose and cellobiose, and hemicellulose degradation produces arabinose, xylose, mannose, rhamnose, galactose and glucose (BALAT, 2011BALAT, M. Production of bioethanol from lignocellulosic materials via the biochemical pathway: A review. Energy Conversion and Management, 5, 858-875, 2011. Available from: <Available from: http://dx.doi.org/10.1016/j.enconman.2010.08.013 >. Accessed: Oct. 25, 2019. doi: 10.1016/j.enconman.2010.08.013.
http://dx.doi.org/10.1016/j.enconman.201...
).

The objective was to evaluate the technical feasibility of the use of pre-cleaning residue and paddy rice type III as raw materials for ethanol production from enzymatic hydrolysis.

MATERIALS AND METHODS:

Sample preparation

The pre-cleaning residue (PCR) and the paddy rice classified as type III (PRT3) were obtained from a rice processing plant located in Lagoa da Confusão, State of Tocantins, Brazil. Pampeira and IRGA 425 cultivars comprised the samples.

Samples were dried in an oven operating by forced circulation of air (Model SL-102, Solab) at 60 ºC for 48 h (INSTITUTO ADOLFO LUTZ, 2018INSTITUTO ADOLFO LUTZ. Métodos físicos-químicos para análise de alimentos. São Paulo: Instituto Adolfo Lutz, 2008.). After drying, samples were crushed and sieved at 1 mm in a knife mill (Model MA 340, Marconi). The prepared material was stored in sealed containers prior to being analyzed and hydrolyzed.

Proximate analysis

The proximate analysis was performed for both by-products (PCR and PRT3) according to the AOAC international standards (AOAC, 2000AOAC - Association of Official Analytical Chemists. 2000 Official methods of analysis of the Association of the Analytical Chemists. 17th ed. Virginia, 2000.), for the following parameters: Moisture (AOAC 934.01), ash (AOAC 924.05), crude fat (AOAC 920.39C) and crude protein (AOAC 920.87), using the conversion factor of 6.25. Total fiber and lignin content were determined by the dietary fiber method (VAN SOEST et al., 1991VAN SOEST, P. J.; et al. Methods for dietary fiber, neutral detergent fiber, and nonstarch polysaccharides in relation to animal nutrition. Journal of Dairy Science, 74, 3583-3597, 1991. Available from: <Available from: http://dx.doi.org/10.3168/jds.S0022-0302(91)78551-2 >. Accessed: Feb. 15, 2019. doi: 10.3168/jds.S0022-0302(91)78551-2.
http://dx.doi.org/10.3168/jds.S0022-0302...
) and the starch content by the Fehling method (AOAC, 2016AOAC - Association of Official Analytical Chemists. Official methods of analysis of the Association of the Analytical Chemists. 20th ed. Maryland/USA, 2016.). Cellulose concentration was estimated using the difference of total fiber, lignin content and the neutral detergent fibers - NDF.

The non-fibrous carbohydrate content was estimated by equation 1 (SNIFFEN et al., 1992SNIFFEN, C. J. et al. A net carbohydrate and protein system for evaluating cattle diets: II. Carbohydrate and protein availability. Journal of Animal Science, 70, 3562-3577, 1992. Available from: <Available from: http://dx.doi.org/10.2527/1992.70113562x >. Accessed: Jan. 15, 2019. 10.2527/1992.70113562x.
http://dx.doi.org/10.2527/1992.70113562x...
).

NFC = 100 - (CP + CF + ASH + TF) (1)

where:

NFC = Non-Fibrous Carbohydrates (%);

CP = Crude protein (%);

CF = Crude Fat (%);

ASH = Ash (%);

TF = Total fibres (%).

Pre-treatment of samples

Once both by-products were characterized, a part of the samples of PCR and PRT3 were submitted to the pre-treatment to break its fibers. It aimed the hydrolysis of the lignocellulosic complex for the exposure of the cellulose molecules to the action of the specific enzymes. To do so, 10 g of sample was added in 100 mL of 0.1 Mol.L-1 NaOH solution and then autoclaved (vertical AV analog model, Phoenix) for 1 h at 121 oC. The samples were taken out from the autoclave and left to stand for 24 h at room temperature. The material was filtered and the solid portion retained in the filter was washed by distilled water until reaching pH 7.6 at 26.1 oC. Washed, the solid material was dried in an oven operating by forced circulation of air (Model SL-102, Solab) at 80 ºC for 30 h (SUKUMARAN et al., 2009SUKUMARAN, R. K. et al. Cellulase production using biomass feed stock and its application in lignocellulose saccharification for bio-ethanol production. Renewable Energy, 34, 421-424, 2009. Available from: <Available from: http://dx.doi.org/10.1016/j.renene.2008.05.008 >. Accessed: Mar. 03, 2019. doi: 10.1016/j.renene.2008.05.008.
http://dx.doi.org/10.1016/j.renene.2008....
).

After pre-treatment, lignin (VAN SOEST et al., 1991VAN SOEST, P. J.; et al. Methods for dietary fiber, neutral detergent fiber, and nonstarch polysaccharides in relation to animal nutrition. Journal of Dairy Science, 74, 3583-3597, 1991. Available from: <Available from: http://dx.doi.org/10.3168/jds.S0022-0302(91)78551-2 >. Accessed: Feb. 15, 2019. doi: 10.3168/jds.S0022-0302(91)78551-2.
http://dx.doi.org/10.3168/jds.S0022-0302...
) and starch contents were determined based on the Fehling method (AOAC, 2016) in order to evaluate the pre-treatment efficiency for lignin removal.

The remaining part of the samples, which were not submitted to pre-treatment, was taken to enzymatic hydrolysis and the consequent production of reducing sugars by the action of enzymes: alpha-amylase (StarMax 300®), amyloglucosidase (StarMax GA 300®) and cellulase (CeluMax C®), produced and marketed by PROZYN®. As the environmental and the medium conditions, such as pH, temperature (T) and concentration (E) strongly influence the efficiency of the enzymatic process, they were set during a pre-test stage.

Enzymatic hydrolisys

Once ideal values for pH, temperature and concentration were determined, the samples were submitted to hydrolysis. The three enzymes were tested, individually, to set the optimal time of reaction. Yields were determined at 24 h, 48 h and 72 h from the beginning of reaction for cellulase and amyloglucosidase and after 90, 120 and 150 minutes for alpha-amylase. The range for reaction times and for the chemical-physical characteristics of the medium, were set based on the guidelines from the enzyme technical sheet.

The part of the samples that were hydrolyzed by the action of each enzyme at a time were submitted to the following procedure: 20 g of untreated sample was added to 0.4% of cellulase diluted in 50 mL of sodium acetate buffer solution. The pH of buffer solution was stabilized in 5.0. The mixture of the solution, cellulase and sample was placed inside a water bath and stirred for 48 h at 50 oC. After cellulase acted over samples, 50 mL of sodium acetate buffer solution (pH 4.5) and 0.1% of amyloglucosidase was added to the mixture. It stirred again this mixture in a water bath for 24 h at 58 oC. Finally, the mixture received 0.3% of the alpha-amylase diluted in 50 mL of phosphate buffer solution (pH 6.0) and it was stirred in the water bath at 90 °C, for 90 minutes.

As each enzyme is highly specific and its optimal performance can occur at conditions close to those for another enzyme, the reducing sugar yield was also tested for cellulase and amyloglucosidase acting simultaneously, in order to reduce the total reaction time, optimizing the process.

For the analysis of the simultaneous activity of the cellulase and amyloglucosidase followed by the alpha-amylase action, 0.4% of cellulase and 0.1% of amyloglucosidase were diluted in 50 mL of sodium acetate buffer solution (pH 4.5) which received 20 g of no-pretreated sample. This mixture was stirred in a water bath for 48 h at 60 oC followed by the addition of 0.3% of alpha-amylase enzyme diluted in 50 mL of phosphate buffer solution (pH 6.0), which was stirred in a water bath at 90 °C for 90 minutes.

After each reaction time for the individual or simultaneous action of enzymes, 2 mL of hydrolysate was taken out. 2 mL of NaOH 0.05 Mol.L-1 was added to hydrolysate for enzyme inactivation. This inactivated hydrolysate was centrifuged (Model 206, Fanem) at 3600 rpm for 10 min and the supernatant was used to determine the reducing sugars content by 3.5-dinitrosalicylic acid - DNS method (MILLER, 1959MILLER, G. L. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Analytical Chemistry, 31, 426-428, 1959. Available from: <Available from: http://dx.doi.org/10.1021/ac60147a030 >. Accessed: Sept. 15, 2019. doi: 10.1021/ac60147a030.
http://dx.doi.org/10.1021/ac60147a030...
).

The efficiency of the hydrolysis process for amyloglucosidase and alpha-amylase was calculated by equation 2 (KOWALSKI et al., 2017KOWALSKI, R. L. et al. Production of second generation ethanol from avocado seed (Persea americana Mill.) Revista Brasileira de Energias Renováveis, 6, 665-677, 2017. Available from: <Available from: http://dx.doi.org/10.1017/CBO9781107415324.004 >. Accessed: Jul. 16, 2019. doi: 10.1017/CBO9781107415324.004.
http://dx.doi.org/10.1017/CBO97811074153...
) and for cellulase by equation 3 (ANDRADE et al., 2019ANDRADE, T. C. C. et al. Enzymatic Cellulose Hydrolysis for Glucose Obtainment Using Ionic Liquid as a Solvent Medium Revista Virtual Química, 11, 310-325, 2019. Available from: <Available from: https://doi.org/10.21577/1984-6835.20190022 >. Accessed: Apr. 19, 2019. doi: 10.21577/1984-6835.20190022.
https://doi.org/10.21577/1984-6835.20190...
):

η = [ Glu ] [ Stc ] fs. 100

(2)

where:

ƞ: hydrolysis yield (%);

[Glu]: glucose concentration (%);

[Stc]: starch concentration (%);

fs: conversion factor for starch (1.11).

η = [ Glu ] [ Cel ] fc. 100

(3)

where:

ƞ: hydrolysis yield (%);

[Gli]: glucose concentration (%);

[Cel]: cellulose concentration (%);

fc: conversion factor for celulose (0.9).

The total reducing sugar concentration obtained after the hydrolysis of fiber and starch was analyzed for each fraction: arabinose, cellobiose, galactose, glucose, mannose, xylose and sucrose. The individual sugar concentrations were determined by high-performance liquid chromatography (HPLC) using a refractive index (RI) detector, according to AOAC 977.20 (AOAC, 2016AOAC - Association of Official Analytical Chemists. Official methods of analysis of the Association of the Analytical Chemists. 20th ed. Maryland/USA, 2016.).

Statistical analysis

The data from proximate analysis were submitted to descriptive statistics. Other data were submitted to the normality test followed by ANOVA. The means were compared by the Tukey’s test. All statistical procedures were performed by Action Stat Pro software (ESTATCAMP, 2017ESTATCAMP. Action Stat Pro. Version 3.1. São Carlos-SP. Brazil: Estatcamp - Consultoria Estatística e Qualidade; 2017.).

RESULTS:

The proximate analysis of PCR revealed that 48.60% of the material are non-fibrous carbohydrates; 30.44 ± 0.90% represented total fibers, being 11.50% cellulose; 9.63 ± 0.23% of crude protein; 5.92 ± 0.36% of crude fat; 5.91 ± 0.25% of moisture and 5.41 ± 0.39% of ash.

The results for PRT3 shows 59.73% of non-fibrous carbohydrates; 23.39 ± 0.86% of total fibers, of which 8.34% are cellulose; 7.40 ± 0.13% of moisture; 7.18 ± 0.49% of crude protein; 6.56 ± 0.27% of crude fat and 3.14 ± 0.11% of ash.

The alkaline pre-treatment reduced 47.94% of the lignin content of PCR samples and 18.60% of PRT3. But 77.76 and 75.55% of starch were also removed from PCR e PRT3, respectively, by the alkaline pre-treatment (Table 1).

Table 1
Lignin and starch contents (Mean ± SD g/100g) for pre-cleaning residue (PCR) and for type III paddy rice (PRT3) before and after pre-treatment.

The optimal values of pH, temperature (T) and concentration (E) for enzymatic hydrolysis using cellulase, amyloglucosidase and alpha-amylase did not differ among PCR and PRT3 (Table 2).

Table 2
Optimum pH, temperature (T) and enzyme concentration (E) for enzymes.

The time of exposure to some enzymes influenced the conversion rate of carbohydrates into reducing sugar for both by-products. The influence of time was observed for cellulase and for alpha-amylase, not for amyloglucosidase. For cellulase, the best results for reducing sugar yields were achieved at a time of reaction of 48h. For alpha-amylase, the optimal time was 90 min, and 24h for amyloglucosidase (Table 3).

Table 3
Reducing sugar concentration (mg.mL-1) (Mean ± SD) after enzymatic hydrolysis by cellulase, amyloglucosity and alpha-amylase at different reaction times.

Amyloglucosidase was responsible for most of the reducing sugar produced during the hydrolysis using one enzyme at a time, followed by alpha-amylase and cellulase, likewise for the hydrolysis efficiency. The yields calculated for the simultaneous action of amyloglucosidase and cellulase were lower than the results of its isolated action (Table 4).

Table 4
Reducing sugar (SC) and efficiency (η) for hydrolysis by cellulase, amyloglucosidase, alpha-amylase, and simultaneously by cellulose plus amyloglucosidase.

The concentration of total reducing sugar was higher for the hydrolysis by the separate enzymes (Figure 1) and glucose was the only sugar retrieved from the enzymatic hydrolysis of starch and fiber from PCR and PRT3 (Table 5).

Figure 1
Reducing sugar concentration (Mean ± SD) after enzymatic hydrolysis of PRT3 (A) and PCR (B) by the action of enzymes separately or simultaneously. Means followed by the same letter do not differ statistically by the F test at p < 0.05.

Table 5
Sugars concentration of hydrolyzed samples, determined by HPLC-RI.

DISCUSSION:

The starch content determines the feasibility of a raw material to get good yields from its hydrolysis and subsequent fermentation for ethanol production. Concentrations of 41.18% in PCR and 53.41% in PRT3 qualifies these by-products for ethanol production, since other traditionally used raw materials such as maize, sorghum and cassava have 67, 70 and 85% starch, respectively (HILL, et al., 2012HILL, H. et al. Variation in sorghum starch synthesis genes associated with differences in starch phenotype. Food Chemistry, 131, 175-183, 2012. Available from: <Available from: http://dx.doi.org/10.1016/j.foodchem.2011.08.057 >. Accessed: Feb. 10, 2019. doi: 10.1016/j.foodchem.2011.08.057.
http://dx.doi.org/10.1016/j.foodchem.201...
; SOARES et al., 2017SOARES, J. S.; ATUI, M. B.; MARCIANO, M. A. M.; LORINI, I. Microscopic analysis of the starch extracted from conventional and transgenic corn (Zea mays). Revista Instituto Adolfo Lutz, 76, 1718, 2017. Available from: <Available from: http://www.ial.sp.gov.br/resources/insituto-adolfo-lutz/publicacoes/rial/10/rial76_completa/artigos-separados/1718.pdf >. Accessed: Feb. 08, 2019.
http://www.ial.sp.gov.br/resources/insit...
; URBANO et al., 2017URBANO, L. H. et. al. Kinetic and fermentative study of cassava starch. Revista Raízes e Amidos Tropicais, 13, 46-55, 2017. Available from: <Available from: http://dx.doi.org/10.17766/1808-981X.2017v13n1p46-55 >. Accessed: Mar. 18, 2019. doi: 10.17766/1808-981X.2017v13n1p46-55.
http://dx.doi.org/10.17766/1808-981X.201...
). Further, ethanol production may be greater if fibers can be converted into reducing sugars too, after hydrolysis of these polysaccharides (RAELE et al., 2014RAELE, R. et al. Scenarios for the second generation ethanol in Brazil. Technological Forecasting and Social Change, 87, 205-223, 2014. Available from: <Available from: http://dx.doi.org/10.1016/j.techfore.2013.12.010 >. Accessed: Dec. 02, 2019. doi: 10.1016/j.techfore.2013.12.010.
http://dx.doi.org/10.1016/j.techfore.201...
).

The proximate analysis of PCR shows similar results found for other rice strains, grown in different Brazilian regions (KAMINSKI et al., 2013KAMINSKI, T. A. et al. Chemical composition and structural changes of irrigated rice during storage. Semina: Ciências Agrárias, v.34, p.1167-1184, 2013. Available from: <Available from: http://dx.doi.org/10.5433/1679-0359.2013v34n3p1167 >. Accessed: May, 20, 2019. doi: 10.5433/1679-0359.2013v34n3p1167.
http://dx.doi.org/10.5433/1679-0359.2013...
; AMAGLIANI et al., 2017AMAGLIANI, L. et al. The composition, extraction, functionality and applications of rice proteins: A review. Trends in Food Science & Technology, 64, 1-12, 2017. Available from: <Available from: http://dx.doi.org/10.1016/j.tifs.2017.01.008 >. Accessed: Apr. 17, 2019. doi: 10.1016/j.tifs.2017.01.008.
http://dx.doi.org/10.1016/j.tifs.2017.01...
). Therefore, these results can be replicated for other producing regions in Brazil or perhaps in the world. However, it must be considered that there are many materials that are part of PCR and it can vary among different processing plants, such as leaves, bark, soil, other parts of the plant and so on. Therefore, its chemical composition may be different depending on the cultivar, harvest time, weather conditions or processing procedures.

The reduction in lignin content via alkaline pre-treatment indicates the rupture of the lignocellulosic complex (KIM, et al., 2016KIM, J. S.; et al. A review on alkaline pretreatment technology for bioconversion of lignocellulosic biomass. Bioresource Technology, 199, 42-48, 2016. Available from: <Available from: http://dx.doi.org/10.1016/j.biortech.2015.08.085 >. Accessed: Apr. 16, 2019. doi: 10.1016/j.biortech.2015.08.085.
http://dx.doi.org/10.1016/j.biortech.201...
). Similar results for delignification of rice husk and straw were presented by other authors (AZEVEDO et al., 2016AZEVEDO, V. Q. et al. Characterization of biomass aiming second generation ethanol production. Revista Brasileira de Engenharia e Sustentabilidade, 2, 61-65, 2016. Available from: <Available from: https://doi.org/10.18540/2446941603032017561 >. Accessed: May, 15, 2019. doi: 10.18540/2446941603032017561.
https://doi.org/10.18540/244694160303201...
, TAKANO & HOSHINO, 2018TAKANO, M.; HOSHINO, K. Bioethanol production from rice straw by simultaneous saccharification and fermentation with statistical optimized cellulase cocktail and fermenting fungus. Journal Bioresources and Bioprocessing, 5, 16, 2018. Available from: <Available from: http://dx.doi.org/10.1186/s40643-018-0203-y >. Accessed: Apr. 12, 2019.
http://dx.doi.org/10.1186/s40643-018-020...
). The highest reduction for lignin content in PCR samples is due to the presence of layers of silica, constituting a kind of shield that prevents the action of alkali (MARIN et al., 2015MARIN, D. C. et al. Revalorization of rice husk waste as a source of cellulose and silica. Fibers and Polymers, 16, 285-293, 2015. Available from: <Available from: http://dx.doi.org/10.1007/s12221-015-0285-5 >. Accessed: Nov. 17, 2019. doi: 10.1007/s12221-015-0285-5.
http://dx.doi.org/10.1007/s12221-015-028...
). Conversely, the pre-treatment caused an undesired effect, by reducing the starch content once hydrolyzed with alkali. It minimizes the yield of reducing sugar, so the use of pre-treatment was not recommended for the tested by-products.

Cellulase is an enzymatic complex that acts in synergism, so the hydrolysis process required a longer reaction time. However, if glucose and cellobiose formed by hydrolysis remained in the same medium for a long time, they inhibited the action of cellulase, decreasing the reducing sugar concentrations (SINGH, et al., 2014SINGH, A. et al. Enzymatic hydrolysis of microwave alkali pretreated rice husk for ethanol production by Saccharomyces cerevisiae, Scheffersomyces stipitis and their co-culture. Journal Fuel, 116, 699-702, 2014. Available from: <Available from: http://dx.doi.org/10.1016/j.fuel.2013.08.072 >. Accessed: Oct. 01, 2019. doi: 10.1016/j.fuel.2013.08.072.
http://dx.doi.org/10.1016/j.fuel.2013.08...
).

Hydrolysis by amyloglucosidase can occur for long periods, since it acts on the non-reducing end, breaking both connections: α-1,4 and α-1,6 (TORRES, et al., 2012TORRES, L. M., et al. Amylolitic enzymes concentration in the starch hydrolisis of ginger. Ciência Rural, Santa Maria 42, 327-1332, 2012. Available from: <Available from: http://dx.doi.org/10.1590/s0103-84782012005000043 >. Accessed: Feb. 03, 2019. doi: 10.1590/s0103-84782012005000043.
http://dx.doi.org/10.1590/s0103-84782012...
). Conversely, alpha-amylases hydrolyze starch faster, as these enzymes only break the α-1.4 bonds along the amylose and amylopectin chains (TORRES et al., 2012TORRES, L. M., et al. Amylolitic enzymes concentration in the starch hydrolisis of ginger. Ciência Rural, Santa Maria 42, 327-1332, 2012. Available from: <Available from: http://dx.doi.org/10.1590/s0103-84782012005000043 >. Accessed: Feb. 03, 2019. doi: 10.1590/s0103-84782012005000043.
http://dx.doi.org/10.1590/s0103-84782012...
). Most enzymes produced by bacteria are stable at 90oC, but they lose stability over time, which may decrease their activity (KHAWLLA et al., 2014KHAWLLA, B. J. et al. Potato peel as feedstock for bioethanol production: A comparison of acidic and enzymatic hydrolysis. Industrial Crops and Products, 52, 144-149, 2014. Available from: <Available from: http://dx.doi.org/10.1016/j.indcrop.2013.10.025 >. Accessed: Apr. 17, 2019. doi: 10.1016/j.indcrop.2013.10.025.
http://dx.doi.org/10.1016/j.indcrop.2013...
).

A higher relation between starch and fibers concentrations, and the occurrence of lignin that hinders enzymatic access to cellulose (CASTRO et al., 2017CASTRO, B.G.F. et al. Alkaline deacetylation as a strategy to improve sugars recovery and ethanol production from rice straw hemicellulose and cellulose. Industrial Crops and Products, 106, 65-73, 2017. Available from: <Available from: http://dx.doi.org/10.1016/j.indcrop.2016.08.053 >. Accessed: Apr. 17, 2019. doi: 10.1016/j.indcrop.2016.08.053.
http://dx.doi.org/10.1016/j.indcrop.2016...
), explains the higher conversion rate into reducing sugar by the action of amylolitic enzymes (TORRES et al., 2012TORRES, L. M., et al. Amylolitic enzymes concentration in the starch hydrolisis of ginger. Ciência Rural, Santa Maria 42, 327-1332, 2012. Available from: <Available from: http://dx.doi.org/10.1590/s0103-84782012005000043 >. Accessed: Feb. 03, 2019. doi: 10.1590/s0103-84782012005000043.
http://dx.doi.org/10.1590/s0103-84782012...
).

The higher efficiency of amyloglucosidase can be justified by the high concentration of amylopectin compared to amylose in rice (DENARDIN et al., 2012DENARDIN, C. C. et al. Amylose content in rice (Oryza sativa) affects performance, glycemic and lipidic metabolism in rats. Ciência Rural, Santa Maria 42, 2381-387, 2012. Available from: <Available from: http://dx.doi.org/10.1590/S0103-84782012005000002 >. Accessed: May, 30, 2019. doi: 10.1590/S0103-84782012005000002.
http://dx.doi.org/10.1590/S0103-84782012...
). However, the lower reducing sugar yield for simultaneous hydrolysis is due to the pH and the temperature of the medium. They were adjusted to an intermediary condition for both enzymes, as close as possible to the optimal values, but it may reduce enzymatic activity (BISSWANGER, 2014BISSWANGER, H. Enzyme assays. Perspectives in Science, 1, s41-55, 2014. Available from: <Available from: http://dx.doi.org/10.1016/j.pisc.2014.02.005 >. Accessed: Jun. 16, 2019. doi: 10.1016/j.pisc.2014.02.005.
http://dx.doi.org/10.1016/j.pisc.2014.02...
). For cellulase, the reducing sugar yield was lower due to the low concentration of cellulose in the tested by-products, that results in the dispensable use of this enzyme for ethanol production using the rice by-products.

The absence of arabinose, galactose, xylose and mannose in sugar analysis demonstrated that hemicellulose was not degraded (Balat, 2011BALAT, M. Production of bioethanol from lignocellulosic materials via the biochemical pathway: A review. Energy Conversion and Management, 5, 858-875, 2011. Available from: <Available from: http://dx.doi.org/10.1016/j.enconman.2010.08.013 >. Accessed: Oct. 25, 2019. doi: 10.1016/j.enconman.2010.08.013.
http://dx.doi.org/10.1016/j.enconman.201...
). Since the action of enzymes is specific, the environmental and the medium conditions were not sufficient for the hydrolysis of the hemicellulose.

The cellobiose formed by cellulose hydrolysis was degraded into glucose by the action of β-glucosidase that compose the enzymatic complex (ODEGA & PETRI, 2010ODEGA, T. L.; PETRI, D.F.S. Biomass Enzymatic Hydrolysis. Química Nova, 33, 1549-1558, 2010. Available from: <Available from: https://doi.org/10.1590/S0100-40422010000700023 >. Accessed: May, 10, 2019. doi: 10.1590/S0100-40422010000700023.
https://doi.org/10.1590/S0100-4042201000...
). The action of amylolitic and cellulolytic enzymes on the material was confirmed by the exclusive presence of glucose in the hydrolysate, which may increase the yield of alcoholic fermentation by Saccharomyces sp. yeasts.

CONCLUSION:

Alkaline pre-treatment was effective for lignin reduction but caused the loss of a part of the starch in by-products. Therefore, the use of non-pretreated by-products ensures higher sugar concentrations for ethanol production.

Enzymatic hydrolysis using one enzyme at a time presented a higher yield in reducing sugars. Acting simultaneously, the enzymes had its action influenced by the pH and the temperature. Optimum temperature, pH, concentration and time of reaction can increase the performance of enzymatic hydrolysis of rice by-products.

The use of amyloglucosidase and alpha-amylase to hydrolyze pre-cleaning residue and low-quality paddy rice can make feasible the use of these by-products for ethanol production. As a result, increasing the diversity of sustainable raw materials can supply this fuel chain.

ACKNOWLEDGEMENTS

We thank the technical and financial support of Evidência Agrícola Ltda and of Universidade Federal do Mato Grosso - Campus of Sinop.

REFERENCES

  • CR-2020-0522.R2

Publication Dates

  • Publication in this collection
    21 June 2021
  • Date of issue
    2021

History

  • Received
    03 June 2020
  • Accepted
    11 Feb 2021
  • Reviewed
    14 Apr 2021
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