Low-cost Biofuel Research Articles

Hydrogen-free deoxygenation of oleic acid on acidic and basic ZSM-5 and Y-zeolites: Products for biofuel and reaction pathways

A large volume of highly acidic vegetable oils generated as waste could be utilized to produce low-cost biofuels without competing with food production. This study investigated the conversion of oleic acid (OA), used as a model fatty compound, in a micropyrolysis apparatus under conditions similar to those employed in fluidized catalytic cracking (FCC) of petroleum feedstocks. The influence of small-micropore size zeolites (ZSM-5) and large-micropore size zeolites (Y), in both their acidic H- and basic Na-forms, on the product distribution was evaluated. Oleic acid was pre-adsorbed onto the zeolites at a mass ratio Catalyst:OA = 10:1. Thermal cracking of pure oleic acid was limited, predominantly producing linear 1-alkenes and carboxylic acids. Conversion in the presence of catalysts was enhanced, resulting in the formation of a greater variety of hydrocarbons. Branched and cyclic alkanes, as well as polyaromatics hydrocarbons, were produced in greater quantities on Y zeolites compared to ZSM-5, due to the larger micropore diameter of the Y zeolite. Among the catalysts, Na-Y produced the highest number of hydrocarbons, predominantly within the gasoline range. These results are promising for the co-processing of fatty residues in the FCC, promoting the production of second-generation drop-in biofuels and bio-based chemicals, and contributing to industrial decarbonization.

Enhancement of biomass productivity and biochemical composition of alkaliphilic microalgae by mixotrophic cultivation using cheese whey for biofuel production

The growth of microalgae under alkaline conditions ensures an ample supply of CO2 from the atmosphere, with a low risk of crashing due to contamination and predators. The present study investigated the mixotrophic cultivation of two alkaliphilic microalgae (Tetradesmus obliquus and Cyanothece sp.) using cheese whey as an organic carbon source. The variation in cheese whey concentration (0.5–4.5% (v/v)), culture pH (7–11), and NaNO3 concentrations (0–2 gL−1) was evaluated using central composite design in response to biomass productivity and the contents of lipids, total proteins, and soluble carbohydrates. Both investigated microalgae effectively utilized cheese whey as an organic carbon source. The optimum conditions for simultaneously maximizing biomass and lipid productivity in T. obliquus were 3.5% (v/v) whey, pH 10.0, and 0.5 g L−1 NaNO3. Under these conditions, the biomass, lipid, soluble carbohydrate, and protein productivities were 48.69, 20.64, 7.02, and 10.97 mg L−1 day−1, respectively. Meanwhile, Cyanothece produced 52.78, 11.42, 4.31, and 7.89 mg L−1 day−1 of biomass, lipid, carbohydrate, and protein, respectively, at 4.5% (v/v) whey, pH 9.0, and 1.0 g L−1 NaNO3. The lipids produced under these conditions were rich in saturated fatty acids (FAs) and monounsaturated FAs, with no polyunsaturated FAs in both microalgae. Moreover, several biodiesel characteristics were estimated, and results fell within the ranges specified by international standards. These findings indicate that the mixotrophic cultivation of alkaliphilic microalgae could open new avenues for promoting microalgae productivity through low-cost biofuel production.

Transforming Fecal Sludge into an Affordable Biofuel Alternative: A Sustainable Solution for Developing Countries

Developing countries are facing challenges due to rapid urbanization and insufficient sanitation facilities. However, valorizing treated fecal sludge as a fuel source presents an opportunity to recover energy and mitigate environmental impacts. This experimental study aimed to produce low-cost biofuel from dried fecal sludge and enhance its energy efficiency by incorporating locally available organic matters. Various organic materials like rice husk, cow dung, sawdust, and coal were carbonized and mixed with the sludge to enhance calorific value. Eight sludge and organic matter mixtures were formed into briquettes. The blend of 50% sludge and 50% coal yielded the highest calorific value of 14618 KJ/kg and a boiling time of 14 minutes. The second-highest result was for 50% sludge and 50% cow dung, with a calorific value of 14427 KJ/kg and a boiling time of 23 minutes. The study found that blending sludge with organic materials enhances energy output. Briquettes with 50% sludge and 50% coal cost 19.87 BDT/kg, while those with 50% sludge and 50% cow dung cost 14.37 BDT/kg, proving more economical. The latter blend emerged as the most efficient and cost-effective biofuel, offering a sustainable eco-friendly solution for Bangladesh’s rural energy market.

Co-Fermentation of Chlorella vulgaris with Oleaginous Yeast in Starch Processing Effluent as a Carbon-Reducing Strategy for Wastewater Treatment and Biofuel Feedstock Production

Low biomass yield and nutrient removal efficiency are problems challenging the employment of microorganisms for wastewater remediation. Starch processing effluent (SPE) was used as a fermentation substrate to co-culture Chlorella vulgaris and Rhodotorula glutinis for biofuel feedstock production. Co-culture options were compared, and the optimal conditions were identified. The result shows that microalgae and yeast should be inoculated simultaneously at the beginning of SPE-based fermentation to achieve high biomass yield and the optimal inoculation ratio, light intensity, and temperature should be 2:1, 150 μmol/m2/s, and 25 °C, respectively. Under the optimal conditions, the lipid yield of microorganisms was 1.81 g/L and the carbon–conversion ratio reached 82.53% while lipid yield and the carbon–conversion ratio in a monoculture fell in the range of 0.79–0.81 g/L and 55.93–62.61%, respectively. Therefore, compared to the monoculture model, the co-fermentation of Chlorella vulgaris and Rhodotorula glutinis in starch processing effluent could convert nutrients to single-cell oil in a more efficient way. It should be noted that with the reduced concentration of residual organic carbon in effluent and the increased carbon–conversion ratio, co-fermentation of microalgae and yeast can be regarded as a promising and applicable strategy for starch processing effluent remediation and low-cost biofuel feedstock production.

Effective hydrodeoxygenation bio-oil via natural zeolite supported transition metal oxide catalyst

Bio-oil from biomass pyrolysis is promising to be used as a sustainable biofuel and high-value-added chemical. However, the presence of high acid, water, and oxygenate causes corrosive properties, low higher heating value (HHV), and instability of the bio-oil component. Therefore, refining the bio-oil is essential to improve its quality. In this study, we introduced natural zeolite (HZ) impregnated with transition metal oxide (TMO) to refine the bio-oil using the hydrodeoxygenation method (HDO) at various catalyst ratios and temperatures. We find that ZnO/HZ 5% wt. shows the best catalytic performance, with the conversion of organic phase reaching ∼ 50%. The refined bio-oil from Fe2O3, ZnO, and CuO has high-quality physicochemical properties with carbon, oxygen, water level, and HHV values are 37–52%, 40–53%, 8–27%, and 17–21 MJ/kg, respectively. This result represents a high catalytic performance for the hydrodeoxygenation process of bio-oil using natural zeolite-based transition metal oxide for better and low-cost biofuel production.

Full-Chain FeCl3 Catalyzation Is Sufficient to Boost Cellulase Secretion and Cellulosic Ethanol along with Valorized Supercapacitor and Biosorbent Using Desirable Corn Stalk

Cellulosic ethanol is regarded as a perfect additive for petrol fuels for global carbon neutralization. As bioethanol conversion requires strong biomass pretreatment and overpriced enzymatic hydrolysis, it is increasingly considered in the exploration of biomass processes with fewer chemicals for cost-effective biofuels and value-added bioproducts. In this study, we performed optimal liquid-hot-water pretreatment (190 °C for 10 min) co-supplied with 4% FeCl3 to achieve the near-complete biomass enzymatic saccharification of desirable corn stalk for high bioethanol production, and all the enzyme-undigestible lignocellulose residues were then examined as active biosorbents for high Cd adsorption. Furthermore, by incubating Trichoderma reesei with the desired corn stalk co-supplied with 0.05% FeCl3 for the secretion of lignocellulose-degradation enzymes in vivo, we examined five secreted enzyme activities elevated by 1.3-3.0-fold in vitro, compared to the control without FeCl3 supplementation. After further supplying 1:2 (w/w) FeCl3 into the T. reesei-undigested lignocellulose residue for the thermal-carbonization process, we generated highly porous carbon with specific electroconductivity raised by 3-12-fold for the supercapacitor. Therefore, this work demonstrates that FeCl3 can act as a universal catalyst for the full-chain enhancement of biological, biochemical, and chemical conversions of lignocellulose substrates, providing a green-like strategy for low-cost biofuels and high-value bioproducts.

Growth-uncoupled propanediol production in a Thermoanaerobacterium thermosaccharolyticum strain engineered for high ethanol yield

Cocultures of engineered thermophilic bacteria can ferment lignocellulose without costly pretreatment or added enzymes, an ability that can be exploited for low cost biofuel production from renewable feedstocks. The hemicellulose-fermenting species Thermoanaerobacterium thermosaccharolyticum was engineered for high ethanol yield, but we found that the strains switched from growth-coupled production of ethanol to growth uncoupled production of acetate and 1,2-propanediol upon growth cessation, producing up to 6.7 g/L 1,2-propanediol from 60 g/L cellobiose. The unique capability of this species to make 1,2-propanediol from sugars was described decades ago, but the genes responsible were not identified. Here we deleted genes encoding methylglyoxal reductase, methylglyoxal synthase and glycerol dehydrogenase. Deletion of the latter two genes eliminated propanediol production. To understand how carbon flux is redirected in this species, we hypothesized that high ATP levels during growth cessation downregulate the activity of alcohol and aldehyde dehydrogenase activities. Measurements with cell free extracts show approximately twofold and tenfold inhibition of these activities by 10 mM ATP, supporting the hypothesized mechanism of metabolic redirection. This result may have implications for efforts to direct and maximize flux through alcohol dehydrogenase in other species.

Co-Processing Agricultural Residues and Wet Organic Waste Can Produce Lower-Cost Carbon-Negative Fuels and Bioplastics.

Scalable, low-cost biofuel and biochemical production can accelerate progress on the path to a more circular carbon economy and reduced dependence on crude oil. Rather than producing a single fuel product, lignocellulosic biorefineries have the potential to serve as hubs for the production of fuels, production of petrochemical replacements, and treatment of high-moisture organic waste. A detailed techno-economic analysis and life-cycle greenhouse gas assessment are developed to explore the cost and emission impacts of integrated corn stover-to-ethanol biorefineries that incorporate both codigestion of organic wastes and different strategies for utilizing biogas, including onsite energy generation, upgrading to bio-compressed natural gas (bioCNG), conversion to poly(3-hydroxybutyrate) (PHB) bioplastic, and conversion to single-cell protein (SCP). We find that codigesting manure or a combination of manure and food waste alongside process wastewater can reduce the biorefinery's total costs per metric ton of CO2 equivalent mitigated by half or more. Upgrading biogas to bioCNG is the most cost-effective climate mitigation strategy, while upgrading biogas to PHB or SCP is competitive with combusting biogas onsite.

Biofuel production, study & characterisation from macro-algae (Azolla pinnata)

The demands for energy and the scarcity in fossil fuel are constantly increasing. This has resulted in the search for sustainable, renewable, and low cost biofuel that has triggered the search for potential bioenergy crops. Aquatic plants that can grow rapidly with minimum resources and can produce biomass in bulk amounts are driving the attention of scientists and researchers throughout the world. The production of biofuels from such organic materials and waste components can result in developing of sustainable alternative that will not only be beneficial to the environment but also to public health. In this study, one such aquatic macro algae Azolla pinnata proved to be potential source for biofuel production. The evaluation of its growth was done and trans-esterification of Azolla pinnata lipid was carried out to produce biofuel. The species have a unique combination of physical, chemical and nutrients composition that makes it a boon to mankind. This macro algae was subjected to series of laboratory testing and evaluation for its characterization such as acid value test, trans-esterification, fatty acid methyl esters (FAMEs) test, gas chromatography which showed the feasibility of algal based biofuel. The comparison of properties of extracted biofuel (physicochemical) from Azollla pinnta was done with standardized ASTM D6751 values. The outcome of produced biofuel was very close to conventional fuel.

Experimental and optimization studies on phycoremediation of dairy wastewater and biomass production efficiency of Chlorella vulgaris isolated from Ganga River, Haridwar, India.

Dairy wastewaters (DWW) are rich in several pollutants, including high biochemical oxygen demand (BOD) and chemical oxygen demand (COD), and their unsafe disposal may cause damage to the environment. In this study, Chlorella vulgaris (identified as NIES:227 strain based on 28s rRNA sequencing) was isolated from the freshwater habitat of the Ganga River at Haridwar, India, and further tested for its efficacy in treating DWW. The phycoremediation experiments were conducted using three different DWW concentrations (0, 50, and 100%), operating temperatures (20, 25, and 30°C), and light intensities (2000, 3000, and 4000lx) using response surface methodology. Results showed that after 16days of experiments, a significant (P < 0.05) reduction in BOD (96.65%) and COD (87.50%) along with a maximum biomass production of 1.757g/L was achieved using 57.72% of dairy industry wastewater, 24.16°C of reactor temperature, and 3874.51lx of light intensity. The RSM models had coefficient of determination (R2) values above 0.9459 with a minimum difference between measured and predicted responses. Therefore, the findings of this study suggest that the isolated C. vulgaris can be effectively used to treat dairy wastewater along with significant production of algal biomass which can be further used for the generation of low-cost biofuel and other materials.

Production of biodiesel from heat-treated edible oil

Among the urgent needs of developing countries are renewable sources of energy and a pollution-free climate. Acid-catalyzed transesterification for production of biodiesel from heat-treated peanut oil was investigated, and the produced biodiesel was characterized. The change in oil properties during the heating process was studied. The results showed an increase in the saponification number with heating time (from 115.47 mg KOH/g for crude oil, to 156.19, 172.7 and 184.68 mg KOH/g after 30, 60 and 120 minutes of heating respectively), a slight decrease in the acid value was observed after heating (from 5.91 to 5.87, 5.87 and 5.88 mg KOH/g after 30, 60, and 120 minutes of heating respectively) There is no obvious change in the refractive index during heating. The peroxide value increased from 2.87 to 3.95 meq O2/kg as the heating time was increased up to 60 minutes. The characteristics of the produced biodiesel were compared to ASTM D 6751 biodiesel standards and ASTM D 975 fossil diesel standards to validate their acceptability as a fuel in diesel engines, and a good agreement was observed. In contrast, to international standards, we notice that ethanol-based fuel has properties that are similar to those of global fuels. Peanut oil can be used to make renewable, low-cost biofuels that can meet a significant portion of the world’s energy needs.

Assessment of the energy potential as a solid biofuel of Sargassum spp. considering sustainability indicator

The present study evaluates the potential use of pelagic Sargassum spp. as a solid biofuel. Massive landings of these brown algae across the Atlantic have produced ecologic and economic problems since 2011. Sargassum biomass valorization could compensate for economic losses and reduce environmental impacts. The production of biofuels could be one of its applications. This research consists of two stages: (a) the physical-energy characterization: morphology, humidity, ash, volatiles, and calorific value, and (b) an estimate of the energy potential of these algae, considering their removal from 600 kilometers of coastline along the Mexican Caribbean coast. An analysis of sustainability indicators considering socioeconomic aspects shows the benefits of using this resource in comparison with other types of low-cost biofuels that produce low environmental impact. The results show the pertinence of using Sargassum spp. as an alternative energy resource with low cost, low environmental impact, high accessibility, and added value for localities along the Mexican Caribbean.

Study of direct synthesis of bio-hydrocarbons from macauba oils using zeolites as catalysts

In line with the aim to minimize climate change and dependence on fossil fuels, there is an increasing demand from different transport sectors for non-oxygenated and low-cost biofuels. In this study, bio-hydrocarbons were synthesized from macauba oils, which is a new and promising biofuel feedstock. These fatty materials were subjected to deoxygenation reactions over beta zeolite and ZSM-5 catalysts with different acidities (Si/Al ratio). Other parameters such as the reaction atmosphere (N2 or H2), catalyst calcination time, feedstock composition, and reaction time were also investigated. A preliminary study of the deoxygenation reaction kinetics indicated a higher conversion rate of hydrocarbons using beta zeolite, achieving a conversion of 100% in 3 h. The products were categorized into different compounds classes, such as linear, cyclic, branched, and aromatic hydrocarbons. The longer calcination time of the non-noble metal catalyst (15 h) and the use of hydrogen resulted in a high degree of deoxygenation. A higher percentage of hydrocarbons was obtained using an already hydrolyzed macauba pulp oil, with high initial acidity (38% m/m), and 10 bar H2. This feedstock and the innovative process provide low-cost alternatives to produce drop-in biofuels on a large scale.

Comparative Study on Pretreatment Processes for Different Utilization Purposes of Switchgrass.

Switchgrass (Panicum virgatum, L., Poaceae) with the advantages of high cellulose yield, and high growth even under low input and poor soil quality, has been identified as a promising candidate for production of low-cost biofuels, papermaking, and nanocellulose. In this study, 12 chemical pretreatments on a laboratory scale were compared for different utilization purposes of switchgrass. It was found that the pretreated switchgrass with sodium hydroxide showed considerable potential for providing mixed sugars for fermentation with 11.10% of residual lignin, 53.85% of residual cellulose, and 22.06% of residual hemicellulose. The pretreatment with 2.00% (v/v) nitric acid was the best method to remove 78.37% of hemicellulose and 39.82% of lignin under a low temperature (125 °C, 30 min), which can be used in the production of nanocellulose. Besides, a completely randomized design analysis of switchgrass pretreatments provided the alternative ethanol organosolv delignification of switchgrass for the papermaking industry with a high residual cellulose of 58.56%. Finally, scanning electron microscopy (SEM), X-ray diffraction (XRD), and Fourier-transform infrared spectroscopy (FT-IR) were carried out to confirm the changes in functional groups, crystallinity, and thermal behavior of the three materials, respectively, from the optimal pretreatments.

Techno-Economic Evaluation of Biorefineries Based on Low-Value Feedstocks Using the BioSTEAM Software: A Case Study for Animal Bedding

Biofuels are still too costly to compete in the energy market and it has been suggested that low-value feedstocks could provide an opportunity for the production of low-cost biofuels; however, the lower quality of these feedstocks requires the introduction of a conditioning step in the biorefinery process. The aim of this study was to evaluate whether feedstock savings cover the cost of conditioning in the case of animal bedding. The BioSTEAM software was used to simulate a wheat straw biorefinery and an animal bedding biorefinery, whose economic performance was compared. The wheat straw biorefinery could deliver ethanol at a minimum selling price of USD 0.61 per liter, which is similar to prices in the literature. The cost of producing ethanol in the animal bedding biorefinery without water recycling was almost 40% higher, increasing the minimum selling price to USD 1.1 per liter of ethanol. After introducing water recycling in the conditioning step, the animal bedding biorefinery could deliver ethanol at a minimum selling price of USD 0.38 per liter, which is 40% lower than in the case of the wheat straw biorefinery. This demonstrates that low-value feedstocks can be used to reduce the biofuel price, as feedstock savings easily cover the additional conditioning cost.

Thermobifida fusca Cellulases Exhibit Increased Endo–Exo Synergistic Activity, but Lower Exocellulase Activity, on Cellulose-III

Cellulose recalcitrance toward saccharification is a barrier for low-cost biofuels production. Ammonia-based pretreatments can alter the native cellulose-I allomorphic state to form an unnatural ce...

The effect of hemicellulose on the binding and activity of cellobiohydrolase I, Cel7A, from Trichoderma reesei to cellulose

Hydrothermal pre-treatments decrease lignocellulose recalcitrance against enzymatic hydrolysis by removing the majority of the hemicellulose, thus increasing cellulase accessibility. However, a small amount of the hemicellulose may remain and become adsorbed to the cellulose, leading to cellulase inhibition. Here, we produced hemicellulose bound cellulose, using glucuronoxylan and galactomannan, to simulate hydrothermally pre-treated hardwoods and softwoods, respectively, and evaluated how this can affect cellulose hydrolysis by Trichoderma reesei derived cellobiohydrolase I (Cel7A). Based on X-ray powder diffraction (XRD), histochemistry, scanning electron microscopy and Simon’s staining, hemicellulose binding onto cellulose affected the physical properties of the biomass, which subsequently affected its hydrolysis rate. As a result of hemicellulose binding onto cellulose, the adsorption of Cel7A was significantly impacted (up to 45%), leading to lowered activities (a 40% reduction), especially for glucuronoxylan. The bound hemicellulose may be released from the cellulose during agitation and hydrolysis. We therefore evaluated the effect of free hemicellulose on Cel7A. Free xylan was more inhibitory to Cel7A than free mannan, demonstrating non-competitive inhibition, while mannan exhibited uncompetitive inhibition. The recalcitrant effect of both bound and free hemicellulose could be relieved by the addition of hemicellulolytic enzymes (i.e. XT6 and Man26A) during cellulose hydrolysis. During the degradation of cellulose in “realistic” woody biomasses by Cel7A, the addition of hemicellulases led to a significant improvement in cellulose hydrolysis. This study showed that hemicellulose remains a critical factor regarding biomass recalcitrance and that the addition of hemicellulolytic activities in commercial enzyme cocktails is required (especially the mannanolytic activities lacking from most commercial enzyme cocktails), in order to realise high sugar yields at low enzyme protein loadings for low-cost biofuel production.

Novel catalytically active Pd/Ru bimetallic nanoparticles synthesized by Bacillus benzeovorans

Bacillus benzeovorans assisted and supported growth of ruthenium (bio-Ru) and palladium/ruthenium (bio-Pd@Ru) core@shell nanoparticles (NPs) as bio-derived catalysts. Characterization of the bio-NPs using various electron microscopy techniques and high-angle annular dark field (HAADF) analysis confirmed two NP populations (1–2 nm and 5–8 nm), with core@shells in the latter. The Pd/Ru NP lattice fringes, 0.231 nm, corresponded to the (110) plane of RuO2. While surface characterization using X-ray photoelectron spectroscopy (XPS) showed the presence of Pd(0), Pd(II), Ru(III) and Ru(VI), X-ray absorption (XAS) studies of the bulk material confirmed the Pd speciation (Pd(0) and Pd(II)- corresponding to PdO), and identified Ru as Ru(III) and Ru(IV). The absence of Ru–Ru or Ru–Pd peaks indicated Ru only exists in oxide forms (RuO2 and RuOH), which are surface-localized. X ray diffraction (XRD) patterns did not identify Pd-Ru alloying. Preliminary catalytic studies explored the conversion of 5-hydroxymethyl furfural (5-HMF) to the fuel precursor 2,5-dimethyl furan (2,5-DMF). Both high-loading (9.7 wt.% Pd, 6 wt.% Ru) and low-loading (2.4 wt.% Pd, 2 wt.% Ru) bio-derived catalysts demonstrated high conversion efficiencies (~95%) and selectivity of ~63% (~20% better than bio-Ru NPs) and 58%, respectively. These materials show promising future scope as efficient low-cost biofuel catalysts.

Influence of diethyl ether on engine performance and emissions characteristics of blends of butanol, pentanol or biodiesel (neem oil methyl ester) in a single cylinder diesel engine

ABSTRACT Biodiesel has become a promising alternative biofuel that exhibits problems like high cost of feedstock, poor atomisation due to high viscosity, stability issues, poor flow properties and high NOx emissions as compared to conventional diesel. Alcohols have recently drawn much attention as a low cost biofuel that, exhibit problems like low cetane number and miscibility issues. Addition of fuel additives, for example, diethyl ether (DEE), may be effective to overcome the problems with biodiesel or alcohols. In this work, the influence of DEE on engine performance and emissions were investigated for diesel/biodiesel blends, diesel/butanol blends or diesel/pentanol blends. Fuel blends of biodiesel (B20), butanol (BU20), pentanol (PN20), biodiesel with DEE (B20D10), butanol with DEE (BU20D10) and pentanol with DEE (PN20D10) were prepared. Experiments were conducted on a single cylinder diesel engine for varying load conditions at constant speed. Engine performance and emission characteristics were determined and compared. Abbreviations: B20: biodiesel blend of 20%; BTE: brake thermal efficiency; BU20: butanol blend of 20%; CO: carbon monoxide; CO2: carbon dioxide; D10: diethyl ether of 10%; DSL: diesel; EGT: exhaust gas temperature; HC: hydrocarbons; NOx: oxides of nitrogen; PN20: pentanol blend of 20%; SFC: specific fuel consumption.

Dynamic bacterial and fungal microbiomes during sweet sorghum ensiling impact bioethanol production

Significant low-cost biofuel production volumes could be achieved from commercial-scale silage by redirecting lactic acid fermentation to ethanol production. A temporal metagenomic analysis on ensiled sweet sorghum inoculated with an ethanologenic yeast has been conducted to understand the underlying microbial processes during bioethanol production. Individual silage buckets approximating silage piles were prepared with freshly harvested material and supplemented with ethanologenic yeast, sulfuric acid or both. The ensiling progress was assessed using high performance liquid chromatography, microbial taxonomic identification and abundance. The combined treatment with Saccharomyces and acid led to a steady reduction of bacterial abundance and microbial diversity with Lactobacillus becoming the dominant genus during the late timepoints. Furthermore, the addition of acid to inhibit bacterial growth hindered Saccharomyces ability to compete with native yeasts like Candida. Knowledge of the response of the in-situ microbial community to the various treatments during ensiling will help improve current methodologies for bioethanol production.