Ethanol production from enzymatic hydrolysis of sugarcane bagasse pretreated with lime and alkaline hydrogen peroxide

In this work we evaluated ethanol production from enzymatic hydrolysis of sugarcane bagasse. Two pretreatments agents, lime and alkaline hydrogen peroxide, were compared in their performance to improve the susceptibility of bagasse to enzymatic action. Mild conditions of temperature, pressure and absence of acids were chosen to diminish costs and to avoid sugars degradation and consequent inhibitors formation. The bagasse was used as it comes from the sugar/ethanol industries, without grinding or sieving, and hydrolysis was performed with low enzymes loading (3.50 FPU g⁻¹ dry pretreated biomass of cellulase and 1.00 CBU g⁻¹ dry pretreated biomass of ??-glucosidase). The pretreatment with alkaline hydrogen peroxide led to the higher glucose yield: 691 mg g⁻¹ of glucose for pretreated bagasse after hydrolysis of bagasse pretreated for 1 h at 25 °C with 7.35% (v/v) of peroxide. Fermentation of the hydrolyzates from the two pretreatments were carried out and compared with fermentation of a glucose solution. Ethanol yields from the hydrolyzates were similar to that obtained by fermentation of the glucose solution. Although the preliminary results obtained in this work are promising for both pretreatments considered, reflecting their potential for application, further studies, considering higher biomass concentrations and economic aspects should be performed before extending the conclusions to an industrial process.     

Cellulase production using biomass feed stock and its application in lignocellulose saccharification for bio-ethanol production

A major constraint in the enzymatic saccharification of biomass for ethanol production is the cost of cellulase enzymes. Production cost of cellulases may be brought down by multifaceted approaches which include the use of cheap lignocellulosic substrates for fermentation production of the enzyme, and the use of cost efficient fermentation strategies like solid state fermentation (SSF). In the present study, cellulolytic enzymes for biomass hydrolysis were produced using solid state fermentation on wheat bran as substrate. Crude cellulase and a relatively glucose tolerant BGL were produced using fungi Trichoderma reesei RUT C30 and Aspergillus niger MTCC 7956, respectively. Saccharification of three different feed stock, i.e. sugar cane bagasse, rice straw and water hyacinth biomass was studied using the enzymes. Saccharification was performed with 50 FPU of cellulase and 10 U of β-glucosidase per gram of pretreated biomass. Highest yield of reducing sugars (26.3 g/L) was obtained from rice straw followed by sugar cane bagasse (17.79 g/L). The enzymatic hydrolysate of rice straw was used as substrate for ethanol production by Saccharomyces cerevisiae. The yield of ethanol was 0.093 g per gram of pretreated rice straw.     

The addition of hydrolyzed rice straw in xylose fermentation by Pichia stipitis to increase bioethanol production at the pilot-scale

The pretreatment of agricultural biomass by diluted acid is often employed to facilitate the release of monosaccharide for the subsequent enzyme hydrolysis for lignocellulosic ethanol production. However, furfural and hydroxymethylfurfural are usually generated and markedly decrease the yield of pentose fermentation during this pretreatment. In the present study, the enhancement of lignocellulosic ethanol production was successfully demonstrated at pilot scale with extra addition of hydrolyzed rice straw into pentose fermentation by Pichia stiptis. This way has resulted into the increase of P. stiptis cell mass was shown to play a positive role. The ethanol yield, 0.45 g_p/g_s, with the addition of hydrolyzed rice straw in hemicellulosic hydrolysate from plywood, bagasse and bamboo were increase 20–51% to demonstrate the applicability of this technology in a variety of lignocellulosic ethanol processes due to the efficient conversion of xylose.     

Effect of nutrient supplementation on ethanol production in different strategies of saccharification and fermentation from acid pretreated rice straw

The effect of nutrient supplementation on ethanol production by recently selected thermotolerant yeast (Kluyveromyces marxianus NRRL Y-6860) was investigated in different strategies of saccharification and fermentation employing rice straw pretreated by dilute acid. Among the evaluated strategies, similar ethanol yields (Y_P/S ∼ 0.23 g g⁻¹) were obtained with or without nutrient addition. However, considering the whole process time, the strategy based on simultaneous saccharification and fermentation (SSF), without pre-hydrolysis, was assigned as the most suitable configuration due to the highest ethanol volumetric productivity (1.4 g L⁻¹ h⁻¹), about 2-fold higher in relation to the others. The impact of enzymatic preparation employed in this study was also evaluated on glucose fermentation in semi-synthetic medium. The enzymatic preparation affected both glucose consumption and ethanol production by K. marxianus NRRL Y-6860, but just in the absence of nutrients. Therefore, the enzyme type and loading should be carefully defined, not only by the capital costs involved, but also by the possibility of increasing the fermentation inhibitors.     

Ethanol production from agroindustrial biomass using a crude enzyme complex produced by Aspergillus niger

This study investigates ethanol production from simultaneous fermentation and saccharification (SFS) and separated hydrolysis and fermentation (SHS) using enzyme complexes produced by Aspergillus niger strains (ATCC 16404, ATCC 1057, ATCC 9029). The enzyme complexes were produced by solid-state fermentation (SSF) on inexpensive and readily available agroindustrial products: rice byproduct (composed of AFEX-treated rice rust and rice bran), whey and sugarcane bagasse. The ethanol was produced by Saccharomyces cerevisiae Y904 using whey and rice byproduct as the substrate and the enzyme complex produced by A. niger. The best result for solid-state fermentation (40 U/g of dry substrate, A. niger ATCC 16404) was obtained in a 0.5 L rotating drum bioreactor at 40 °C filled half filled with solid biomass composed of rice byproduct (86% wt/wt), whey (12% wt/wt) and CaCl₂ (2.0% wt/wt). The best result for ethanol fermentation (11.7 g/L of ethanol) was obtained after 12 h of SFS at pH 4.5 and 35 °C. A comparative study of ethanol production by Trichoderma reesei CCT 2768 and A. niger ATCC 16404 complexes under the same optimised SFS and SSF conditions was also performed, revealing that ethanol production by the A. niger enzyme complex was 2.25 times higher than that by T. reesei. These findings suggest that the ethanol production using crude enzymatic complexes produced by A. niger and agroindustrial biomass described in this paper is very promising in terms of disposal of the whey produced by cheese-making and other dairy food processing.     

Enhanced bio-hydrogen production from sugarcane juice by immobilized Clostridium butyricum on sugarcane bagasse

Immobilized Clostridium butyricum TISTR 1032 on sugarcane bagasse improved hydrogen production rate (HPR) approximately 1.2 times in comparison to free cells. The optimum conditions for hydrogen production by immobilized C. butyricum were initial pH 6.5 and initial sucrose concentration of 25 g COD/L. The maximum HPR and hydrogen yield (HY) of 3.11 L H₂/L substrate·d and 1.34 mol H₂/mol hexose consumed, respectively, were obtained. Results from repeated batch fermentation indicated that the highest HPR of 3.5 L H₂/L substrate·d and the highest HY of 1.52 mol H₂/mol hexose consumed were obtained at the medium replacement ratio of 75% and 50% respectively. The major soluble metabolites in both batch and repeated batch fermentation were butyric and acetic acids.     

Lipid production from sweet sorghum bagasse through yeast fermentation

Cryptococcus curvatus has great potential in fermenting unconditioned hydrolysates of sweet sorghum bagasse. With hydrolysates obtained by enzymatic hydrolysis of the solid pretreated by microwave with lime, the maximal yeast cell dry weight and lipid content were 10.83 g/l and 73.26%, respectively. For hydrolysates obtained in the same way but without lime, these two parameters were 15.50 g/l and 63.98%, respectively. During yeast fermentation, glucose and xylose were consumed simultaneously while cellobiose was released from the residual bagasse. The presence of lime, on one hand, made cellulose more accessible to enzymes as evidenced by higher total reducing sugar release compared to that without during enzymatic hydrolysis step; on the other hand, it caused the degradation of sugars to non-sugar chemicals during pretreatment step. As a result, higher lipid yield of 0.11 g/g bagasse or 0.65 ton/hectare of land was achieved from the pathway of microwave pretreatment and enzymatic hydrolysis while 0.09 g/g bagasse or 0.51 ton/hectare of land was attained from the process of lime-assisted microwave pretreatment followed by the same enzymatic saccharification.     

Bioethanol production from sweet potato by co-immobilization of saccharolytic molds and Saccharomyces cerevisiae

To investigate the bioethanol production from sweet potato, the saccharification and fermentation conditions of co-immobilization of saccharolytic molds (Aspergillus oryzae and Monascus purpureus) with Saccharomyces cerevisiae were analyzed. The immobilized yeast cells showed that at 10% glucose YPD (yeast extract peptone dextrose) the maximum fermentation rate was 80.23%. Viability of yeasts cells were 95.70% at a final ethanol concentration of 6%. Immobilization enhanced the ethanol tolerance of yeast cells. In co-immobilization of S. cerevisiae with A. oryzae or M. purpureus, the optimal hardening time of gel beads was between 15 and 60 min. Bioethanol production was 3.05-3.17% (v v⁻¹) and the Y_E/s (yield of ethanol production/starch consumption) was 0.31-0.37 at pH 4, 30 °C and 150 rpm during 13 days fermentation period. Co-immobilization of S. cerevisiae with a mixed cultures of A. oryzae and M. purpureus at a ratio of 2:1, the bioethanol production was 3.84% (v v⁻¹), and the Y_E/s was 0.39 for a 11 days incubation. However a ratio of A. oryzae and M. purpureus at 1:2 resulted a bioethanol production rate of 4.08% (v v⁻¹), and a Y_E/s of 0.41 after 9 days of fermentation.     

Biological saccharification by Clostridium thermocellum and two-stage hydrogen and methane production from hydrogen peroxide-acetic acid pretreated sugarcane bagasse

The present work aimed at establishing an efficient degradation and energy recovery system form sugarcane bagasse (SCB) through hydrogen peroxide-acetic acid (HPAC) pretreatment, thermophilic hydrogen production and mesophilic methane production. The degradation ratio of HPAC pretreated SCB (HPAC-SCB, 2%, w/v) exceeded 90% under the biological hydrolysis of C. thermocellum without enzyme addition. The hydrogen yield in the co-culture fermentation of T. thermosaccharolyticum and C. thermocellum from HPAC-SCB (2%, w/v) reached 226 mL/g substrate. The long-term hydrogen fermentation was successfully established with 1.59 L/(L·d), 0.159 L/g substrate for average hydrogen productivity and yield, respectively. Methane production of 0.341 L/g COD (chemical oxygen demand)_added was recovered by semi-continuous methane fermentation from hydrogen-producing effluent at 12 days of hydraulic retention time (HRT). Average energy recovery of 8.79 MJ/kg SCB was obtained under the optimal conditions. The present work indicated the promising application of the established process in valorization of lignocellulosic bio-waste.     

Hydrogen production from water hyacinth through dark- and photo- fermentation

This article discusses the method of producing hydrogen from water hyacinth. Water hyacinth was pretreated with microwave heating and alkali to enhance the enzymatic hydrolysis and hydrogen production in a two-step process of dark- and photo- fermentation. Water hyacinth with various concentrations of 10–40 g/l was pretreated with four methods: (1) steam heating; (2) steam heating and microwave heating/alkali pretreatment; (3) steam heating and enzymatic hydrolysis; (4) steam heating, microwave heating/alkali pretreatment and enzymatic hydrolysis. Water hyacinth (20 g/l) pretreated with method 4 gave the maximum reducing sugar yield of 30.57 g/100 g TVS, which was 45.6% of the theoretical reducing sugar yield (67.0 g/100 g TVS). The pretreated water hyacinth was used to produce hydrogen by mixed H₂-producing bacteria in dark fermentation. The maximum hydrogen yield of 76.7 ml H₂/g TVS was obtained at 20 g/l of water hyacinth. The residual solutions from dark fermentation (mainly acetate and butyrate) were used to further produce hydrogen by immobilized Rhodopseudomonas palustris in photo fermentation. The maximum hydrogen yield of 522.6 ml H₂/g TVS was obtained at 10 g/l of water hyacinth. Through a combined process of dark- and photo- fermentation, the maximum hydrogen yield from water hyacinth was dramatically enhanced from 76.7 to 596.1 ml H₂/g TVS, which was 59.6% of the theoretical hydrogen yield.     

Evaluation of an adapted inhibitor-tolerant yeast strain for ethanol production from combined hydrolysate of softwood

In order to evaluate the potential of an adapted inhibitor-tolerant yeast strain developed in our lab to produce ethanol from softwood, the effect of furfural and HMF presented in defined medium and pretreatment hydrolysate on cell growth was investigated. And the efficiency of ethanol production from enzymatic hydrolysate mixed with pretreatment hydrolysate of softwood by bisulfite and sulfuric acid pretreatment process was reported. The results showed that in the combined treatments of the two inhibitors, cell growth was not affected at 1 g/L each of furfural and HMF. When 3 g/L each of furfural and HMF was applied, the adapted strain responded with an extended lag phase of 24 h. Both in batch and fed-batch runs of combined hydrolysate fermentation, the final ethanol concentrations were above 20.0 g/L and the ethanol yields (Y_p/s) on the total amount of fermentable sugar presented in the pretreated materials were above 0.40 g/g. It implies the great promise of the yeast strain for improving ethanol production from softwood due to its high ability of metabolizing inhibitor compounds of furfural and HMF.     

Ethanol and co-product generation from pressurized batch hot water pretreated T85 bermudagrass and Merkeron napiergrass using recombinant Escherichia coli as biocatalyst

Pretreatment of grasses is required to maximize ethanol yield during fermentation. T85 bermudagrass and Merkeron napiergrass (Pennisetum purpureum Schumach.) were either left untreated or were pressurized batch hot water (PBHW) pretreated for 2 min at 230 °C at 5% w/v whole grass solids loading. Following a 24 h enzymatic digestion, untreated and PBHW pretreated grasses were evaluated for ethanol production and co-product generation including potential fermentation inhibitors. Fermentations of PBHW pretreated grasses with E. coli LY01 produced twice the ethanol of their untreated counterparts. PBHW pretreated Merkeron napiergrass produced 224.5 mg/g grass ethanol (73% maximum theoretical yield) and PBHW pretreated T85 bermudagrass reached 213.0 mg/g grass (70% maximum theoretical ethanol yield). Pretreatment by PBHW resulted in increased solubilization of hemicelluloses. PBHW pretreatment also produced potential fermentation inhibitors such as acetic, formic, cinnamic acids, and aldehydes. Despite some of these inhibitors remaining with the solids after PBHW pretreatment, there was more efficient hydrolysis of the cellulose and remaining hemicellulose during the enzymatic digestion of the grasses prior to fermentation when compared to the untreated grasses. This increase in digestibility observed with enzymes prior to fermentation resulted in increased ethanol yields during bioconversion using E. coli LY01 as the biocatalyst.     

Parametric gasification process of sugarcane bagasse for syngas production

This research focuses on parametric influence on product distribution and syngas production from conventional gasification. Three experimental parameters at three different levels of temperature (700, 800 and 900 °C), sugarcane bagasse loading (2, 3 and 4 g) and residence time (10, 20 and 30 min) were studied using horizontal axis tubular furnace. Response Surface Methodology supported by central composite design was adopted in order to investigate parameters impact on product distribution (i.e., gas, tar and char) and gaseous products (i.e., H₂, CO, CO₂ and CH₄). The highest H₂ fraction obtained was 42.88 mol% (36.91 g-H₂ kg-biomass⁻¹) at 3 g of sugarcane bagasse loading, 900 °C and 30 min reaction time. The temperature was identified as the most influential parameter followed by reaction time for H₂ production and diminishing the bio-tar and char yields. An increase in sugarcane bagasse loading, on other hand, favored the production of bio-tar, CO₂ and CH₄ production. The statistical analysis verified temperature as most significant (p-value 0.0008) amongst the parameters investigated for sugarcane bagasse biomass gasification.     

Cell degeneration and performance decline of immobilized Clostridium acetobutylicum on bagasse during hydrogen and butanol production by repeated cycle fermentation

The cell degeneration and the fermentation performance decline during repeated cycle fermentation with immobilized Clostridium acetobutylicum on bagasse for hydrogen and butanol production was studied. The cell degeneration has been characterized in abnormality of a long-chain morphology through 7 cycles of repeated fermentation. The fermentation performance decline has been indicated by decrease of glucose consumption rate from 0.82 g/L/h to 0.22 g/L/h, reduction of hydrogen production and productivity from 6 L/L to 2.5 L/L and 170 mL/L/h to 40 mL/L/h, as well as the decrease of butanol production and butanol productivity from 6.5 g/L to 1.0 g/L and 0.18 g/L/h to 0.02 g/L/h, respectively. The ratio of hydrogen production and butanol production was in the range of 6–10 and 17–20 during the earlier three and later four cycles, respectively. The carbon flow directed to ethanol was higher during the later period of fermentation.     

Cane molasses fermentation for continuous ethanol production in an immobilized cells reactor by Saccharomyces cerevisiae

Sodium-alginate immobilized yeast was employed to produce ethanol continuously using cane molasses as a carbon source in an immobilized cell reactor (ICR). The immobilization of Saccharomyces cerevisiae was performed by entrapment of the cell cultured media harvested at exponential growth phase (16 h) with 3% sodium alginate. During the initial stage of operation, the ICR was loaded with fresh beads of mean diameter of 5.01 mm. The ethanol production was affected by the concentration of the cane molasses (50, 100 and 150 g/l), dilution rates (0.064, 0.096, 0.144 and 0.192 h^?1) and hydraulic retention time (5.21, 6.94, 10.42 and 15.63 h) of the media. The pH of the feed medium was set at 4.5 and the fermentation was carried out at an ambient temperature. The maximum ethanol production, theoretical yield (Y_E/S), volumetric ethanol productivity (Q_P) and total sugar consumption was 19.15 g/l, 46.23%, 2.39 g l^?1 h^?1 and 96%, respectively.     

Coproduction of hydrogen and butanol by Clostridium acetobutylicum with the biofilm immobilized on porous particulate carriers

The porous particulate carriers of activated carbon, bagasse and brick were used for Clostridium acetobutylicum immobilization for coproduction of hydrogen and butanol. The dense microbial population was growing on the carrier surface with the biofilms formed during fermentation. The homogeneous array of the microbial cells on the surface looks some interesting behaviors. The cells have the ability to shuttle between holes in bagasse. Higher efficiency of cell immobilization could be achieved accordingly. The cell concentration during immobilized fermentation was about one order magnitude higher than that during free cell fermentation. Enhanced fermentation for hydrogen and butanol has been achieved during the immobilized fermentation. The highest yield of hydrogen was 1.81 mol/mol when brick was used as immobilization carrier, while the highest butanol yield of 0.22 g/g was achieved during fermentation with bagasse as immobilization carrier. Hydrogen productivity and butanol productivity were up to 403.2 ml/L/h and 0.44 g/L/h, respectively. Hydrogen and butanol production behaved differently in organic and inorganic carrier materials.     

Biohydrogen production from pure and natural lignocellulosic feedstock with chemical pretreatment and bacterial hydrolysis

Pretreatment and saccharification of lignocellulosic materials is the key technology affecting the efficiency of cellulosic biohydrogen production. In this work, two pure cellulosic materials (i.e., carboxymethyl-cellulose (CMC) and xylan) were directly hydrolyzed (without pretreatment) by a cellulolytic isolate Cellulomonas uda E3-01 able to release extracellular cellulolytic enzymes. Natural cellulosic feedstock (i.e., sugarcane bagasse) was chemically pretreated prior to the bacterial hydrolysis.A temperature-shift strategy (35 °C for cellulolytic enzymes production and 45 °C for hydrolysis reaction) was used to increase the production of reducing sugars during the bacterial hydrolysis. The hydrolysates of CMC, xylan, and bagasse were efficiently converted to H₂ via dark fermentation with Clostridium butyricum CGS5. The maximum hydrogen yield was 8.80 mmol H₂/g reducing sugar (i.e., 1.58 mol H₂/mol hexose) for CMC, 6.03 mmol H₂/g reducing sugar (i.e., 0.91 mol H₂/mol pentose) for xylan, and 6.01 mmol H₂/g reducing sugar for bagasse.     

Simultaneous saccharification and fermentation (SSF) of very high gravity (VHG) potato mash for the production of ethanol

Simultaneous saccharification and fermentation (SSF) of very high gravity (VHG) potato mash, containing 304 g L^?1 of dissolved carbohydrates, was carried out for ethanol production. Potato tubers were ground into a mash, which was highly viscous. Mash viscosity was reduced by the pretreatment with mixed enzyme preparations of pectinase, cellulase and hemicellulase. The enzymatic pretreatment established the use of VHG mash with a suitable viscosity. Starch in the pretreated mash was liquefied to maltodextrins by the action of thermo-stable α-amylase at 85 °C. SSF of liquefied mash was performed at 30 °C with the simultaneous addition of glucoamylase, yeast (Saccharomyces cerevisiae) and ammonium sulfate as a nitrogen source for the yeast. The optimal glucoamylase loading, ammonium sulfate concentration and fermentation time were 1.65 AGU g^?1, 30.2 mM and 61.5 h, respectively, obtained using the response surface methodology (RSM). Ammonium sulfate supplementation was necessary to avoid stuck fermentation under VHG condition. Using the optimized condition, ethanol yield of 16.61% (v/v) was achieved, which was equivalent to 89.7% of the theoretical yield.     

Preliminary exploration on pretreatment with metal chlorides and enzymatic hydrolysis of bagasse

Converting biomass to fermentable sugar is the critical step in the biomass refinery. Moreover, pretreatment of biomass plays an important role in improving the conversion of biomass to sugar. In this study, sugarcane bagasse was pretreated by metal chloride Lewis acids (0.1 mol L⁻³ CrCl₃, FeCl₃, FeCl₂, ZnCl₂ and AlCl₃ solution) for cellulase hydrolysis. The effects of pretreatments on the yield, chemical components, and sequential cellulase hydrolysis of pretreated bagasse were investigated. The results indicated that metal chlorides with different pKa values could efficiently remove the hemicellulose in bagasse during pretreatment. Furthermore, an inhibition factor (IF) quantitatively reflecting difficulty of cellulase hydrolysis was proposed. The low IF means the facile cellulase hydrolysis. The IF of Fe (III)-pretreated bagasse could decrease to 1.35. In this case, the enzymatic digestibility of bagasse approached to 100%.     

Influence of impregnation with lactic acid on sugar yields from steam pretreatment of sugarcane bagasse and spruce, for bioethanol production

Lignocellulosic biomass can be utilized to produce ethanol, a promising alternative energy source produced through fermentation of sugars. However, in order to achieve high sugar and ethanol yields, the lignocellulosic material must be pretreated before the enzymatic hydrolysis and fermentation. Dilute acid pretreatment, using SO₂, is one of the most promising methods of pretreatment for softwood and agricultural residues. However, handling the high acidity of the slurry obtained from pretreatment and difficulty in recycling/degradation of the impregnating agent are some of the drawbacks of the dilute acid processes. In the present study the influence of utilization of a weak organic acid (lactic acid), as impregnating agent, on the sugar yield from pretreatment, with and without addition of SO₂, was investigated. The efficiency of pretreatment was assessed by enzymatic hydrolysis of the slurry obtained by pretreatment, using sugarcane bagasse and spruce, stored for one and two months in the presence of lactic acid separately, as feedstocks. Pretreatment of bagasse after storage with 0.5% lactic acid resulted in an overall glucose yield, i.e. after enzymatic hydrolysis, of 79% of theoretical based on the amount available in the raw material. This was as good as pretreatment using SO₂ as impregnating agent. However, storage of spruce with lactic acid before pretreatment, with and without addition of SO₂, was not efficient and resulted in lower sugar yields than pretreatment using SO₂ only.