scholarly journals Rational engineering of Saccharomyces cerevisiae towards improved tolerance to multiple inhibitors in lignocellulose fermentations

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Bianca A. Brandt ◽  
Maria D. P. García-Aparicio ◽  
Johann F. Görgens ◽  
Willem H. van Zyl
Keyword(s):  
Xylose Fermentation ◽  
Ethanol Yield ◽  
Specific Gene ◽  
Biomass Growth ◽  
Inhibitor Tolerance ◽  
Rational Engineering ◽  
Ethanol Yields ◽  
Membrane Changes ◽  
A Minor ◽  
Inhibitor Resistance

Abstract Background The fermentation of lignocellulose hydrolysates to ethanol requires robust xylose-capable Saccharomycescerevisiae strains able to operate in the presence of microbial inhibitory stresses. This study aimed at developing industrial S.cerevisiae strains with enhanced tolerance towards pretreatment-derived microbial inhibitors, by identifying novel gene combinations that confer resistance to multiple inhibitors (thus cumulative inhibitor resistance phenotype) with minimum impact on the xylose fermentation ability. The strategy consisted of multiple sequential delta-integrations of double-gene cassettes containing one gene conferring broad inhibitor tolerance (ARI1, PAD1 or TAL1) coupled with an inhibitor-specific gene (ADH6, FDH1 or ICT1). The performances of the transformants were compared with the parental strain in terms of biomass growth, ethanol yields and productivity, as well as detoxification capacities in a synthetic inhibitor cocktail, sugarcane bagasse hydrolysate as well as hardwood spent sulphite liquor. Results The first and second round of delta-integrated transformants exhibited a trade-off between biomass and ethanol yield. Transformants showed increased inhibitor resistance phenotypes relative to parental controls specifically in fermentations with concentrated spent sulphite liquors at 40% and 80% v/v concentrations in 2% SC media. Unexpectedly, the xylose fermentation capacity of the transformants was reduced compared to the parental control, but certain combinations of genes had a minor impact (e.g. TAL1 + FDH1). The TAL1 + ICT1 combination negatively impacted on both biomass growth and ethanol yield, which could be linked to the ICT1 protein increasing transformant susceptibility to weak acids and temperature due to cell membrane changes. Conclusions The integration of the selected genes was proven to increase tolerance to pretreatment inhibitors in synthetic or industrial hydrolysates, but they were limited to the fermentation of glucose. However, some gene combination sequences had a reduced impact on xylose conversion.

scholarly journals Rational Engineering of Saccharomyces Cerevisiae Towards Improved Tolerance to Multiple Inhibitors in Lignocellulose Fermentations

2020 ◽  
Author(s):  
Bianca A. Brandt ◽  
Maria D.P. García-Aparicio ◽  
Johann F. Görgens ◽  
Willem Heber Van Zyl
Keyword(s):  
Xylose Fermentation ◽  
Ethanol Yield ◽  
Specific Gene ◽  
Biomass Growth ◽  
Inhibitor Tolerance ◽  
Rational Engineering ◽  
Ethanol Yields ◽  
Membrane Changes ◽  
A Minor ◽  
Inhibitor Resistance

Abstract Background: The fermentation of lignocellulose hydrolysates to ethanol require robust xylose capable Saccharomyces cerevisiae strains able to operate in the presence of microbial inhibitory stresses. This study aimed at developing industrial S. cerevisiae strains with enhanced tolerance towards pretreatment-derived microbial inhibitors, by identifying novel gene combinations that confer resistance to multiple inhibitors (thus cumulative inhibitor resistance phenotype) with minimum impact on the xylose fermentation ability. The strategy consisted of multiple sequential delta-integrations of double gene cassettes containing one gene conferring broad inhibitor tolerance (ARI1, PAD1 or TAL1) coupled with an inhibitor specific gene (ADH6, FDH1 or ICT1). The performances of the transformants were compared with the parental strain in terms of biomass growth, ethanol yields and productivity, as well as detoxification capacities in a synthetic inhibitor cocktail, sugarcane bagasse hydrolysate as well as hardwood spent sulphite liquor.Results: The first and second round of delta-integrated transformants exhibited a trade-off between biomass and ethanol yield. Transformants showed increased inhibitor resistance phenotypes relative to parental controls specifically in fermentations with concentrated spent sulphite liquors at 40% and 80% v/v concentrations in 2% SC media. Unexpectedly, the xylose fermentation capacity of the transformants was reduced compared to the parental control, but certain combinations of genes had a minor impact (e.g. TAL1 + FDH1). The TAL1+ ICT1 combination negatively impacted on both biomass growth and ethanol yield, which could be linked to the ICT1 protein increasing transformant susceptibility to weak acids and temperature due to cell membrane changes. Conclusions: The integration of the selected genes was proven to increase tolerance to pretreatment inhibitors in synthetic or industrial hydrolysates, but they were limited to the fermentation of glucose. However, some genes combination sequences had a reduced impact on xylose conversion.

scholarly journals Metabolic Engineering of a Phosphoketolase Pathway for Pentose Catabolism in Saccharomyces cerevisiae

2004 ◽  
Vol 70 (5) ◽  
pp. 2892-2897 ◽  
Author(s):  
Marco Sonderegger ◽  
Michael Schümperli ◽  
Uwe Sauer
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plant Material ◽  
Xylose Fermentation ◽  
Ethanol Yield ◽  
Limited Capacity ◽  
Fermentation Rate ◽  
Phosphoketolase Pathway ◽  
Ethanol Yields ◽  
Acetate Accumulation ◽  
Acetate Formation

ABSTRACT Low ethanol yields on xylose hamper economically viable ethanol production from hemicellulose-rich plant material with Saccharomyces cerevisiae. A major obstacle is the limited capacity of yeast for anaerobic reoxidation of NADH. Net reoxidation of NADH could potentially be achieved by channeling carbon fluxes through a recombinant phosphoketolase pathway. By heterologous expression of phosphotransacetylase and acetaldehyde dehydrogenase in combination with the native phosphoketolase, we installed a functional phosphoketolase pathway in the xylose-fermenting Saccharomyces cerevisiae strain TMB3001c. Consequently the ethanol yield was increased by 25% because less of the by-product xylitol was formed. The flux through the recombinant phosphoketolase pathway was about 30% of the optimum flux that would be required to completely eliminate xylitol and glycerol accumulation. Further overexpression of phosphoketolase, however, increased acetate accumulation and reduced the fermentation rate. By combining the phosphoketolase pathway with the ald6 mutation, which reduced acetate formation, a strain with an ethanol yield 20% higher and a xylose fermentation rate 40% higher than those of its parent was engineered.

scholarly journals Deletion ofFPS1, Encoding Aquaglyceroporin Fps1p, Improves Xylose Fermentation by Engineered Saccharomyces cerevisiae

2013 ◽  
Vol 79 (10) ◽  
pp. 3193-3201 ◽  
Author(s):  
Na Wei ◽  
Haiqing Xu ◽  
Soo Rin Kim ◽  
Yong-Su Jin
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Xylose Fermentation ◽  
Plant Biomass ◽  
Ethanol Yield ◽  
Xylose Reductase ◽  
Enzymatic Reactions ◽  
Xylose Metabolism ◽  
Xylose Consumption ◽  
Content Type ◽  
Ethanol Yields

ABSTRACTAccumulation of xylitol in xylose fermentation with engineeredSaccharomyces cerevisiaepresents a major problem that hampers economically feasible production of biofuels from cellulosic plant biomass. In particular, substantial production of xylitol due to unbalanced redox cofactor usage by xylose reductase (XR) and xylitol dehydrogenase (XDH) leads to low yields of ethanol. While previous research focused on manipulating intracellular enzymatic reactions to improve xylose metabolism, this study demonstrated a new strategy to reduce xylitol formation and increase carbon flux toward target products by controlling the process of xylitol secretion. Using xylitol-producingS. cerevisiaestrains expressing XR only, we determined the role of aquaglyceroporin Fps1p in xylitol export by characterizing extracellular and intracellular xylitol. In addition, whenFPS1was deleted in a poorly xylose-fermenting strain with unbalanced XR and XDH activities, the xylitol yield was decreased by 71% and the ethanol yield was substantially increased by nearly four times. Experiments with our optimized xylose-fermenting strain also showed thatFPS1deletion reduced xylitol production by 21% to 30% and increased ethanol yields by 3% to 10% under various fermentation conditions. Deletion ofFPS1decreased the xylose consumption rate under anaerobic conditions, but the effect was not significant in fermentation at high cell density. Deletion ofFPS1resulted in higher intracellular xylitol concentrations but did not significantly change the intracellular NAD+/NADH ratio in xylose-fermenting strains. The results demonstrate that Fps1p is involved in xylitol export inS. cerevisiaeand present a new gene deletion target,FPS1, and a mechanism different from those previously reported to engineer yeast for improved xylose fermentation.

scholarly journals POTENCIAL DE CLONES EXPERIMENTAIS DE BATATA-DOCE PARA PRODUÇÃO DE ETANOL

Nativa ◽  
2018 ◽  
Vol 6 (4) ◽  
pp. 352
Author(s):  
Adriano Mendes Lourenço ◽  
Aline Torquato Tavares ◽  
Tiago Alves Ferreira ◽  
Danilo Alves da Silva Porto Lopes ◽  
João Victor Gonçalves Carline ◽  
...  
Keyword(s):  
Ethanol Production ◽  
Sweet Potato ◽  
Ipomoea Batatas ◽  
Ethanol Yield ◽  
Starch Content ◽  
Great Ability ◽  
Ethanol Yields ◽  
Root Starch ◽  
Breeding Selection ◽  
Starch Yield

A batata-doce (Ipomoea batatas (L.) Lam.) tem sido reportada como uma das espécies de planta com grande capacidade de converter biomassa em matéria prima para produção de etanol. O objetivo do trabalho foi avaliar o potencial de clones de batata-doce para produção de etanol. Foram avaliados 60 clones de batata-doce para produtividade de raízes, teor de amido nas raízes, produtividade de amido, coloração da casca e da polpa e o rendimento de etanol. O clone BDTO#122,32 e as cultivares Ana Clara e Carolina Vitória com média de 46,77; 42,75 e 41,25 t ha-¹, respectivamente, foram os que mais conseguiram acumular biomassa na forma de raiz. Os clones que apresentam as maiores médias de produtividade de amido por hectare foram BDTO#144.22 e BDTO#100.23, com valores de 15,46 e 14,16% t ha-1, com rendimentos de etanol de 8,33 e 7,63 m³ ha-¹. Os clones BDTO#144.22 e BDTO#100.23 apresentaram as maiores médias de produtividade de amido por hectare e rendimento de etanol, sendo, portanto, os mais promissores para a produção de etanol.Palavras-chave: Ipomoea batatas (L.) Lam, melhoramento genético, seleção, biocombustível. POTENTIAL OF EXPERIMENTAL CLONES OF SWEET POTATO FOR ETHANOL PRODUCTION ABSTRACT:Sweet potato (Ipomoea batatas (L.) Lam.) Has been reported as one of the plant species with great ability to convert biomass into feedstock for ethanol production. The objective of this work was to evaluate the potential of sweet potato clones for ethanol production. Twenty-six sweet potato clones were evaluated for root productivity, root starch content, starch yield, bark and pulp color, and ethanol yield. Clone BDTO # 122.32 and cultivars Ana Clara and Carolina Vitória averaging 46.77; 42.75 and 41.25 t ha-1, respectively, were the ones that were able to accumulate biomass in the root form. The clones presenting the highest starch productivity per hectare were BDTO # 144.22 and BDTO # 100.23, with values of 15.46 and 14.16% t ha-1, with ethanol yields of 8.33 and 7.63 m³ ha-¹. The clones BDTO # 144.22 and BDTO # 100.23 showed the highest averages of starch productivity per hectare and yield of ethanol, thus being the most promising for the production of ethanol.Keywords: Ipomoea potatoes (L.) Lam, breeding, selection, biofuel.

scholarly journals High Productivity Ethanol Fermentation of Glucose & Xylose Using Membrane Assisted Continuous Cell Recycle

2021 ◽  
pp. 8-19
Author(s):  
Gautam Degweker ◽  
Arvind Lali
Keyword(s):  
Ethanol Fermentation ◽  
Ethanol Yield ◽  
Continuous Fermentation ◽  
Cost Effective ◽  
High Yield ◽  
High Cell ◽  
Cell Recycle ◽  
High Productivity ◽  
Ethanol Yields ◽  
Cell Densities

Rapid and high yield conversion of xylose to ethanol remains a signi cant bottleneck in the cost-effective production of ethanol using mixed sugars derived from lignocellulosic biomass (LBM). The present study attempts to circumvent this by separate continuous fermentation of glucose and xylose using high cell densities of a Saccharomyces cerevisiae mutant (ICT-1) and a Scheffersomyces stipitis mutant (M1CD), respectively with the help of external micro ltration membrane assisted cell recycle. Different cell densities and aeration rates for xylose fermentation were studied for optimizing continuous fermentation. Consistent high ethanol yields and productivities of 0.46 g/g and 5.19 g/L/h with glucose; and 0.38 g/g and 1.62 g/L/h with xylose; were achieved in simple media. This provided an average ethanol yield of 0.44 g/g on combined sugars, and average productivity of 3.4 g/L/h which is higher than typical molasses-based batch ethanol fermentation. The study thus highlights the potential of high cell density recycle strategy as an effective approach for separate ethanol fermentation of LBM derived sugars.

QUALIDADE INDUSTRIAL E RENDIMENTO DE ETANOL DE GENÓTIPOS DE SORGO SACARINO EM PLANTA E REBROTA

2018 ◽  
Vol 17 (2) ◽  
pp. 216
Author(s):  
TASSIANO MAXWELL MARINHO CÂMARA ◽  
RAFAEL AUGUSTO DA COSTA PARRELLA ◽  
HERÁCLITON DE ÉFESO DA SILVA
Keyword(s):  
Sorghum Bicolor ◽  
Sweet Sorghum ◽  
Ethanol Yield ◽  
Block Design ◽  
Production Costs ◽  
Randomized Block Design ◽  
Ethanol Yields ◽  
Sorghum Genotypes ◽  
Industrial Yield ◽  
Lower Production

 RESUMO - O rendimento de cultivares de sorgo sacarino em planta de primeiro corte e na rebrota tem sido estudado para viabilizar o cultivo com menores custos de produção. Entretanto, dados qualitativos sobre a qualidade de caldo e rendimento de etanol entre safra principal e rebrota são escassos. Objetivou-se com este trabalho comparar genótipos de sorgo sacarino quanto à qualidade industrial e avaliar o efeito do tipo de colheita e biomassa processada no rendimento industrial para produção de etanol. Foi conduzido um experimento em blocos ao acaso com três repetições em esquema de parcela subdividida, sendo as parcelas constituídas por 20 genótipos de sorgo e as subparcelas pelos tipos de amostras processadas ou colheitas realizadas. Os maiores rendimentos de etanol, em termos absolutos, foram obtidos para CMSXS646 e BRS 511 na colheita de primeiro corte com colmos limpos. A utilização de colmos limpos promoveu, em média, um incremento de 13,5, 14,2, 13,4, 3,3, 7,9, 7,0, 8,0 e 16,4% para etanol, ART, ATR, umidade, ARC, Pol, °Brix, quantidade de caldo, respectivamente, e redução de 17,3% para fibra, em relação às amostras de colmos com folhas. O rendimento médio de etanol por tonelada de caldo extraído foi 28% superior no primeiro corte comparativamente à rebrota.Palavras-chave: Bioenergia, biomassa, °Brix, Sorghum bicolor.  INDUSTRIAL QUALITY AND ETHANOL YIELD OF SWEET SORGHUM GENOTYPES IN MAIN AND RATOON CROPS  ABSTRACT - The yield of sweet sorghum cultivars in first cut and ratoon crops have been studied to enable the cultivation with lower production costs. However, qualitative data on broth quality and yield of ethanol on main crop and ratoon are scarce. The aim of this work was to compare sweet sorghum genotypes regarding the industrial quality and to evaluate the effects of harvests and processed biomass types in the industrial yield for ethanol production. A randomized block design experiment was carried out with three replicates in a split plot design, being the plots composed by 20 sorghum genotypes and the subplots by the types of processed samples or harvests. The highest ethanol yields, in absolute terms, were obtained for CMSXS646 and BRS 511 in first cut with clean stalks. The use of clean stalks promoted, on average, an increase of 13.5, 14.2, 13.4, 3.3, 7.9, 7.0, 8.0 and 16.4% for ethanol, ART, ATR, moisture, ARC, Pol, ° Brix, juice amount, respectively, and reduction of 17.3% for fiber, in relation to samples with stalks and leafs. The average yield of ethanol per extracted juice ton was 28% higher in first cut compared to ratoon.Keywords: bioenergy, biomass, °Brix, Sorghum bicolor.

Photo-additions of aromatic hydrocarbons—indene and naphthalene with acrylonitrile

1969 ◽  
Vol 47 (5) ◽  
pp. 757-766 ◽  
Author(s):  
John J. McCullough ◽  
C. W. Huang
Keyword(s):  
Butyl Alcohol ◽  
Major Product ◽  
Minor Amount ◽  
Strong Base ◽  
Magnetic Resonance Data ◽  
Cis And Trans Isomers ◽  
Resonance Data ◽  
Ethanol Yields ◽  
A Minor ◽  
Cis And Trans

The photolyses of indene and naphthalene with acrylonitrile have been studied, and the products characterized. Indene reacts with acrylonitrile when the hydrocarbon absorbs light, or when a triplet sensitizer (acetophenone) is absorbing. Photolysis under the former conditions in ethanol yields 2-(1-indenylpropionitrile) (70%), 2-(3-indenylpropionitrile) (10%), and 6-cyano-2,3-benzobicyclo[3,2,0]hept-2-ene (20%). Base-catalyzed isomerizations of the substituted indenes are reported. On sensitization, cis- and trans-isomers of 7-cyano-2,3-benzobicyclo[3,2,0]hept-2-ene are formed, with a minor amount of indene dimer. The former isomers can be equilibrated with strong base. Adducts of 1,1-dimethyl- and 1,1-diphenylindene with acrylonitrile are also described, and nuclear magnetic resonance data reported. Naphthalene adds acrylonitrile on photolysis in t-butyl alcohol (pyrex filter) to afford trans-8-cyano-2,3-benzobicyclo-[4,2,0]-octa-2,4-diene (90%) and 1- and 2-naphthyl-2-propionitrile (10%). Sensitization with acetophenone gave no detectable products. Irradiation of the major product at 253.7 mμ through Vycor gave naphthalene. The preparative value of these reactions is considered and possible mechanisms are proposed.

scholarly journals Decreased Xylitol Formation during Xylose Fermentation in Saccharomyces cerevisiae Due to Overexpression of Water-Forming NADH Oxidase

2011 ◽  
Vol 78 (4) ◽  
pp. 1081-1086 ◽  
Author(s):  
Guo-Chang Zhang ◽  
Jing-Jing Liu ◽  
Wen-Tao Ding
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Xylose Fermentation ◽  
Nadh Oxidase ◽  
Xylose Reductase ◽  
Control Strain ◽  
Content Type ◽  
Ethanol Yields ◽  
Glycerol Yield ◽  
Set Up ◽  
And Control

ABSTRACTThe recombinant xylose-fermentingSaccharomyces cerevisiaestrain harboring xylose reductase (XR) and xylitol dehydrogenase (XDH) fromScheffersomyces stipitisrequires NADPH and NAD+, creates cofactor imbalance, and causes xylitol accumulation during growth ond-xylose. To solve this problem,noxE, encoding a water-forming NADH oxidase fromLactococcus lactisdriven by thePGK1promoter, was introduced into the xylose-utilizing yeast strain KAM-3X. A cofactor microcycle was set up between the utilization of NAD+by XDH and the formation of NAD+by water-forming NADH oxidase. Overexpression ofnoxEsignificantly decreased xylitol formation and increased final ethanol production during xylose fermentation. Under xylose fermentation conditions with an initiald-xylose concentration of 50 g/liter, the xylitol yields for of KAM-3X(pPGK1-noxE) and control strain KAM-3X were 0.058 g/g xylose and 0.191 g/g, respectively, which showed a 69.63% decrease owing tonoxEoverexpression; the ethanol yields were 0.294 g/g for KAM-3X(pPGK1-noxE) and 0.211 g/g for the control strain KAM-3X, which indicated a 39.33% increase due tonoxEoverexpression. At the same time, the glycerol yield also was reduced by 53.85% on account of the decrease in the NADH pool caused by overexpression ofnoxE.

scholarly journals Bioconversion process of source-separated organic waste for ethanol production

2021 ◽  
Author(s):  
Valeriy Bekmuradov
Keyword(s):  
Kinetic Model ◽  
Ethanol Production ◽  
Lignocellulosic Biomass ◽  
Substrate Concentration ◽  
Ethanol Yield ◽  
Ethanol Conversion ◽  
Ethanol Yields ◽  
Environmental Goals ◽  
Ethanol Washing ◽  
Experimental Values

Production of biofuel such as ethanol from lignocellulosic biomass is a beneficial way to meet sustainability, energy security, and environmental goals. Lignocellulosic biomass such as source-separated organic (SSO) waste is particularly attractive since it is widely available, often at a negative cost, reduce the land depletion from using food-based biomass for ethanol production and reduce the amount of generated waste. Therefore, in order to meet the future fuel demands and cope with increasing volume of municipal waste this study was a first attempt to use SSO as a feedstock for ethanol production. The main objectives of the study were: a) to compare standard and modified celluloseorganic- solvent-based lignocellulosic fractionation (COSLIF) pretreatment of SSO waste for ethanol production in terms of enzyme savings, sugar formation and ethanol yields; b) to produce ethanol from SSO by using modified COSLIF pretreatment and fermentation with two different recombinant strains: Z. mobilis 8b and S. cerevisiae DA2416; and c) to develop experimental kinetic model capable of predicting behavior of batch SSCF on SSO waste with different SSO substrate concentrations using Berkeley Madonna program. Based on the obtained results, it was found that SSO is an excellent feedstock material for ethanol conversion. The efficiency of modified COSLIF pretreatment was improved by 20% compared to standard method using ethanol washing of pretreated SSO samples during the experimental procedures instead of acetone. On average, glucose yield from SSO samples pretreated by modified COSLIF was about 90% compared to 10% for untreated samples. S. cerevisiae DA2416 outperformed Z. mobilis 8b on ethanol yields during the fermentation process, with 0.50 g ethanol/g potential sugar fed on SSO in less than 5 days, with a 96% cellulose conversion, totalling in 150 g/L ethanol produced. A kinetic model with newly integrated values of experimentally defined SSO feedstock constants was proven to predict the ethanol yield accurately with substrate concentration ranges of 20 g/L - 50 g/L. Model prediction at higher substrate concentration (e.g. 100 g/L) deviated from the experimental values, suggesting that ethanol inhibition is a major factor in bioethanol conversion.

scholarly journals Assessing Ethanol Yield from Fermented Juice of Local and Exotic Sweet Sorghum Varieties Grown in Sudan

2021 ◽  
Vol 12 ◽  
pp. 11-17
Author(s):  
Salma I. Abdalbagi ◽  
Maarouf I. Mohammed
Keyword(s):  
Regression Analysis ◽  
Sweet Sorghum ◽  
Cultural Practices ◽  
Ethanol Yield ◽  
Correlation And Regression Analysis ◽  
Juice Yield ◽  
Ethanol Yields ◽  
Significant Regression ◽  
Theoretical Ethanol Yield ◽  
Fermented Juice

An experiment was conducted in 2017 at Alwaha Project, Khartoum State to study the potential of ethanol yield from fermented juice of local and exotic sweet sorghums. 40 genotypes were arranged in RCB design. Juice yield, ethanol yield as percentage of fermented juice, actual and theoretical ethanol yields were studied. The Baker’s yeast (Saccharomyces cerevisiae) was used in the fermentation process. ANOVA tests, correlation and regression analysis of actual vs theoretical ethanol yield were performed. Significant differences were detected among genotypes for ethanol yields. High ethanol yields as percentage from fermented juice were obtained, but ethanol yield per hectare was relatively low due to low juice yields. The genotypes BlueRibbon and KensasCollies from exotic materials; 5AbjSG51 and S.154 Ab70 from local materials were identified as promising for ethanol production. Correlation between actual ethanol yield and Juice yield was positive and highly significant. Regression analysis indicated that theoretical ethanol yield was little bit overestimated. It was concluded that juice yield is more crucial than ethanol concentration in maximizing ethanol yield of sweet sorghum. More efforts are needed to develop high juice-yielding genotypes. Juice maximization can also be achieved by using efficient milling devices and optimizing cultural practices.

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