Ethanol Production from the Organic Fraction Obtained After Thermal Pretreatment of Municipal Solid Waste

In this work, the use of organic fraction from municipal solid waste (MSW) as substrate for ethanol production based on enzymatic hydrolysis was evaluated. MSW was subjected to a thermal pretreatment (active hygienization) at 160?°C from 5 to 50 min. The organic fiber obtained after 30 min was used as substrate in a simultaneous saccharification and fermentation (SSF) and fed-batch SSF process using cellulases and amylases. In a fed-batch mode with 25% (w/w) substrate loading, final ethanol concentration of 30 g/L was achieved (60% of theoretical). In these conditions, more than 160 L of ethanol per ton of dry matter could be produced from the organic fraction of MSW.     

Ethanol production from lignocellulosic byproducts of olive oil extraction

The recent implementation of a new two-step centrifugation process for extracting olive oil in Spain has substantially reduced water consumption, thereby eliminating oil mill wastewater. However, a new high sugar content residue is still generated. In this work the two fractions present in the residue (olive pulp and fragm ented stones) were assayed as substrate for ethanol production by the simultaneous saccharification and fermentation (SSF) process. Pretreatment of fragmented olive stones by sulfuric acid-catalyzed steam explosion was the most effective treatment for increasing enzymatic digestibility; however, a pretreatment step was not necessary to bioconvert the olive pulp into ethanol. Theolive pulp and fragmented olive stones were tested by the SSF process using a fed-batch procedure. By adding the pulp three times at 24-h intervals, 76% of the theoretical SSF yield was obtained. Experiments with fed-batch pretreated olive stones provided SSF yields significantly lower than those obtained at standard SSF procedure. The preferred SSF conditions to obtain ethanol from olives stones (61% of theoretical yield) were 10% substrate and addition of cellulases at 15 filter paper units/g of substrate.     

Effects of temperature and moisture on dilute-acid steam explosion pretreatment of corn stover and cellulase enzyme digestibility

Corn stover is emerging as a viable feedstock for producing bioethanol from renewable resources. Dilute-acid pretreatment of corn stover can solubilize a significant portion of the hemicellulosic component and enhance the enzymatic digestibility of the remaining cellulose for fermentation into ethanol. In this study, dilute H₂SO₄ pretreatment of corn stover was performed in a steam explosion reactor at 160°C, 180°C, and 190°C, approx 1 wt% H₂SO₄, and 70-s to 840-s residence times. The combined severity (Log₁₀ [R _o ] - pH), an expression relating pH, temperature, and residence time of pretreatment, ranged from 1.8 to 2.4. Soluble xylose yields varied from 63 to 77% of theoretical from pretreatments of corn stover at 160 and 180°C. However, yields >90% of theoretical were found with dilute-acid pretreatments at 190°C. A narrower range of higher combined severities was required for pretreatment to obtain high soluble xylose yields when the moisture content of the acid-impregnated feedstock was increased from 55 to 63 wt%. Simultaneous saccharification and fermentation (SSF) of washed solids from corn stover pretreated at 190°C, using an enzyme loading of 15 filter paper units (FPU)/g of cellulose, gave ethanol yields in excess of 85%. Similar SSF ethanol yields were found using washed solid residues from 160 and 180°C pretreatments at similar combined severities but required a higher enzyme loading of approx 25 FPU/g of cellulose.     

Thermophilic Bacillus coagulans Requires Less Cellulases for Simultaneous Saccharification and Fermentation of Cellulose to Products than Mesophilic Microbial Biocatalysts

Ethanol production from lignocellulosic biomass depends on simultaneous saccharification of cellulose to glucose by fungal cellulases and fermentation of glucose to ethanol by microbial biocatalysts (SSF). The cost of cellulase enzymes represents a significant challenge for the commercial conversion of lignocellulosic biomass into renewable chemicals such as ethanol and monomers for plastics. The cellulase concentration for optimum SSF of crystalline cellulose with fungal enzymes and a moderate thermophile, Bacillus coagulans, was determined to be about 7.5 FPU g^?1 cellulose. This is about three times lower than the amount of cellulase required for SSF with Saccharomyces cerevisiae, Zymomonas mobilis, or Lactococcus lactis subsp. lactis whose growth and fermentation temperature optimum is significantly lower than that of the fungal cellulase activity. In addition, B. coagulans also converted about 80% of the theoretical yield of products from 40 g/L of crystalline cellulose in about 48 h of SSF with 10 FPU g^?1 cellulose while yeast, during the same period, only produced about 50% of the highest yield produced at end of 7 days of SSF. These results show that a match in the temperature optima for cellulase activity and fermentation is essential for decreasing the cost of cellulase in cellulosic ethanol production.     

Bioconversion of Kraft Paper Mill Sludges to Ethanol by SSF and SSCF

Paper mill sludge is a solid waste material composed of pulp residues and ash generated from pulping and paper making processes. The carbohydrate portion of the sludge has chemical and physical characteristics similar to pulp. Because of its high carbohydrate content and well-dispersed structure, the sludges can be biologically converted to value-added products without pretreatment. In this study, two different types of paper mill sludges, primary sludge and recycle sludge, were evaluated as a feedstock for bioconversion to ethanol. The sludges were first subjected to enzymatic conversion to sugars by commercial cellulase enzymes. The enzymatic conversion was inefficient because of interference by ash in the sludges with the enzymatic reaction. The main cause was that the pH level is dictated by CaCO₃ in ash, which is two units higher than the pH optimum of cellulase. To alleviate this problem, simultaneous saccharification and cofermentation (SSCF) using cellulase (Spezyme CP) and recombinant Escherichia coli (ATCC-55124), and simultaneous saccharification and fermentation (SSF) using cellulase and Saccharomyces cerevisiae (ATCC-200062) were applied to the sludges without any pretreatment. Ethanol yields of 75–81% of the theoretical maximum were obtained from the SSCF on the basis of total carbohydrates. The yield from the SSF was also found to be in the range of 74–80% on the basis of glucan. The SSCF and SSF proceeded under stable condition with the pH staying near 5.0, close to the optimum for cellulase. Decrease of pH occurred due to carbonic acid and other organic acids formed during fermentation. The ash was partially neutralized by the acids produced from the SSCF and SSF and acted as a buffer to stabilize the pH during fermentation. When the SSF and SSCF were operated in fed-batch mode, the ethanol concentration in the broth increased from 25.5 and 32.6 g/L (single feed) to 45 and 42 g/L, respectively. The ethanol concentration was limited by the tolerance of the microorganism in the case of SSCF. The ethanol yield in fed-batch operation decreased to 68% for SSCF and 70% for SSF. The high-solids condition in the bioreactor appears to create adverse effects on the cellulase reaction.     

Solid-State Fermentation of Palm Kernel Cake with <Emphasis Type="Italic">Aspergillus flavus</Emphasis> in Laterally Aerated Moving Bed Bioreactor

Solid-state fermentation (SSF) was employed to enhance the nutritive values of palm kernel cake (PKC) for poultry feeding. Aspergillus flavus was isolated from local PKC and utilized to increase the mannose content of PKC via the degradation of β-mannan in PKC; evaluation was done for batch SSF in Erlenmeyer flasks and in a novel laterally aerated moving bed (LAMB) bioreactor. The optimum condition for batch SSF in flasks was 110% initial moisture content, initial pH 6.0, 30 °C, 855 μm particle size, and 120 h of fermentation, yielding 90.91 mg mannose g⁻¹ dry PKC (5.9-fold increase). Batch SSF in the LAMB at the optimum condition yielded 79.61 mg mannose g⁻¹ dry PKC (5.5-fold increase) within just 96 h due to better heat and mass transfer when humidified air flowed radially across the PKC bed. In spite of a compromise of 12% reduction in mannose content when compared with the flasks, the LAMB facilitated good heat and mass transfer, and improved the mannose content of PKC in a shorter fermentation period. These attributes are useful for batch production of fermented PKC feed in an industrial scale.     

Production of Cellulase from Kraft Paper Mill Sludge by Trichoderma Reesei Rut C-30

Paper mill sludge is a solid waste material generated from pulping and papermaking operations. Because of high glucan content and its well-dispersed structure, paper mill sludges are well suited for bioconversion into value-added products. It also has high ash content originated from inorganic additives used in papermaking, which causes hindrance to bioconversion. In this study, paper mill sludges from Kraft process were de-ashed by a centrifugal cleaner and successive treatment by sulfuric acid and sodium hydroxide, and used as a substrate for cellulase production. The treated sludge was the only carbon source for cellulase production, and predominantly inorganic nutrients were used as the nitrogen source for this bioprocess. The cellulase enzyme produced from the de-ashed sludge exhibited cellulase activity of 8 filter paper unit (FPU)/mL, close to that obtainable from pure cellulosic substrates. The yield of cellulase enzyme was 307 FPU/g glucan of de-ashed sludge. Specific activity was 8.0 FPU/mg protein. In activity tests conducted against the corn stover and α-cellulose, the xylanse activity was found to be higher than that of a commercial cellulase. Relatively high xylan content in the sludge appears to have induced high xylanase production. Simultaneous saccharification and fermentation (SSF) was performed using partially de-ashed sludge as the feedstock for ethanol production using Sacharomyces cerevisiae and the cellulase produced in-house from the sludge. With 6% (w/v) glucan feed, ethanol yield of 72% of theoretical maximum and 24.4 g/L ethanol concentration were achieved. These results were identical to those of the SSF using commercial cellulases.     

Effect of Initial Particle Size and Densification on AFEX-Pretreated Biomass for Ethanol Production

Switchgrass (SG), corn stover (CS), and prairie cordgrass (PCG) pretreated with ammonia fiber expansion (AFEX) were densified using a novel low-temperature, low-pressure densification method. Simultaneous saccharification and fermentation (SSF) and separate hydrolysis and fermentation (SHF) were performed with loose and densified AFEX-treated biomass to determine the effect of post-AFEX densification. Biomass particle size reduction before pretreatment increased 144-h SSF ethanol yields from densified material by 8–9 % although no significant differences were seen in the first 72 h. Grinding material after densification had no impact on final ethanol yields but increased production rates in the first 24–48 h. Low-pressure, post-AFEX densification had no adverse effects on SSF ethanol yields from SG or CS but reduced yields from densified PCG by 16 %. Glucose concentrations after hydrolysis (SHF) showed similar trends. Ethanol yields after SHF, however, showed that densification had no significant impact on CS or PCG but reduced final ethanol yields from SG.     

Effect of Resistant Starch on Hydrolysis and Fermentation of Corn Starch for Ethanol

Starch samples with 0% or 30% amylose were subjected to four different liquefaction enzyme treatments (at various temperature and pH conditions) followed by simultaneous saccharification and fermentation (SSF). Resistant starch (RS) measurements were conducted for the initial starch sample, after liquefaction and after SSF. Initial RS was higher for 30% amylose starch samples (16.53 g/100 g sample) compared with 0% amylose (0.76 g/100 g sample). Higher initial RS resulted in lower conversion of starch into sugars and lower final ethanol yields. The four enzymes hydrolyzed RS, but in varying amounts. Higher temperature liquefaction hydrolyzed a larger portion of RS, resulting in higher ethanol concentrations and lower final residual solids (non-fermentables), whereas lower temperature liquefaction hydrolyzed a smaller portion of RS and resulted in lower ethanol concentrations and higher final residual solids. Decreases in resistant starch after high temperature liquefaction were 55% to 74%, whereas low temperature liquefaction decreases were 11% to 43%. For all enzyme treatments, RS content of starch samples decreased further after SSF.     

Enzymatic Fractionation of SAA-Pretreated Barley Straw for Production of Fuel Ethanol and Astaxanthin as a Value-Added Co-Product

Barley straw was used to demonstrate an integrated process for production of fuel ethanol and astaxanthin as a value-added co-product. Barley straw was pretreated by soaking in aqueous ammonia using the previously determined optimum conditions, which included 77.6 °C treatment temperature, 12.1 h treatment time, 15 wt% ammonia concentration, and 1:8 solid-to-liquid ratio. In the newly developed process, the pretreated barley straw was first hydrolyzed with ACCELLERASE® XY (a commercial hemicellulase product) to generate a xylose-rich solution, which contained 3.8 g/l glucose, 22.9 g/l xylose, and 2.4 g/l arabinose, with 96 % of the original glucan being left intact. The xylose-rich solution was used for production of astaxanthin by the yeast Phaffia rhodozyma without further treatment. The resulting cellulose-enriched solid residue was used for ethanol production in a fed-batch simultaneous saccharification and fermentation using ACCELLERASE® 1500 (a commercial cellulase product) and the industrial yeast Saccharomyces cerevisiae. At the end of the fermentation, 70 g/l ethanol was obtained, which was equivalent to 63 % theoretical yield based on the glucan content of the solid substrate.     

Ethanol Production from Sugarcane Bagasse by Zymomonas mobilis Using Simultaneous Saccharification and Fermentation (SSF) Process

Considerable efforts have been made to utilize agricultural and forest residues as biomass feedstock for the production of second-generation bioethanol as an alternative fuel. Fermentation utilizing strains of Zymomonas mobilis and the use of simultaneous saccharification and fermentation (SSF) process has been proposed. Statistical experimental design was used to optimize the conditions of SSF, evaluating solid content, enzymatic load, and cell concentration. The optimum conditions were found to be solid content (30%), enzymatic load (25 filter paper units/g), and cell concentration (4 g/L), resulting in a maximum ethanol concentration of 60 g/L and a volumetric productivity of 1.5 g L^?1?h^?1.     

Phytosterols Elevation in Bamboo Shoot Residue through Laboratorial Scale Solid-State Fermentation Using Isolated Aspergillus niger CTBU

Aspergillus niger CTBU isolated from local decayed bamboo shoot residue was employed to solid-state fermentation (SSF) of bamboo shoot residue to elevate the content of phytosterols. Strain acclimatization was carried out under the fermentation condition using bamboo shoot as substrate for fermentation performance improvement. The optimal fermentation temperature and nitrogen level were investigated using acclimatized strain, and SSF was carried out in a 500-ml Erlenmeyer flask feeding 300-mg bamboo shoot residue chips under the optimal condition (33 °C and feeding 4 % urea), and 1,186 mg (100 g)^?1 of total phytosterol was attained after 5-day fermentation, in comparison, only 523 mg (100 g)^?1 of phytosterol was assayed in fresh shoots residue. HPLC analysis of the main composition of total phytosterols displays that the types of phytosterols and composition ratio of main sterols keep steady. This laboratorial scale SSF unit could be scaled up for raw phytosterols production from discarded bamboo shoot residue and could reduce its cost.     

Valorization of By-Products from Palm Oil Mills for the Production of Generic Fermentation Media for Microbial Oil Synthesis

This study demonstrates the production of a generic nutrient-rich feedstock using by-product streams from palm oil production that could be used as a substitute for commercial fermentation supplements. Solid-state fermentations of palm kernel cake (PKC) and palm-pressed fiber (PPF) were conducted in tray bioreactors and a rotating drum bioreactor by the fungal strain Aspergillus oryzae for the production of crude enzymes. The production of protease was optimized (319.3 U/g) at an initial moisture content of 55 %, when PKC was used as the sole substrate. The highest free amino nitrogen (FAN) production (5.6 mg/g) obtained via PKC hydrolysis using the crude enzymes produced via solid-state fermentation was achieved at 50 °C. Three initial PKC concentrations (48.7, 73.7, and 98.7 g/L) were tested in hydrolysis experiments, leading to total Kjeldahl nitrogen to FAN conversion yields up to 27.9 %. Sequential solid-state fermentation followed by hydrolysis was carried out in the same rotating drum bioreactor, leading to the production of 136.7 U/g of protease activity during fermentation and 196.5 mg/L of FAN during hydrolysis. Microbial oil production was successfully achieved with the oleaginous yeast strain Lipomyces starkeyi DSM 70296 cultivated on the produced PKC hydrolysate mixed with commercial carbon sources, including glucose, xylose, mannose, galactose, and arabinose.     

Pilot-Scale Fermentation of Aqueous-Ammonia-Soaked Switchgrass

Aqueous-ammonia-steeped switchgrass was subject to simultaneous saccharification and fermentation (SSF) in two pilot-scale bioreactors (50- and 350-L working volume). Switchgrass was pretreated by soaking in ammonium hydroxide (30%) with solid to liquid ratio of 5 L ammonium hydroxide per kilogram dry switchgrass for 5 days in 75-L steeping vessels without agitation at ambient temperatures (15 to 33 °C). SSF of the pretreated biomass was carried out using Saccharomyces cerevisiae (D₅A) at approximately 2% glucan and 77 filter paper units per gram cellulose enzyme loading (Spezyme CP). The 50-L fermentation was carried out aseptically, whereas the 350-L fermentation was semiaseptic. The percentage of maximum theoretical ethanol yields achieved was 73% in the 50-L reactor and 52–74% in the 350-L reactor due to the difference in asepsis. The 350-L fermentation was contaminated by acid-producing bacteria (lactic and acetic acid concentrations approaching 10 g/L), and this resulted in lower ethanol production. Despite this problem, the pilot-scale SSF of aqueous-ammonia-pretreated switchgrass has shown promising results similar to laboratory-scale experiments. This work demonstrates challenges in pilot-scale fermentations with material handling, aseptic conditions, and bacterial contamination for cellulosic fermentations to biofuels.     

Thermophilic Dry Methane Fermentation of Distillation Residue Eluted from Ethanol Fermentation of Kitchen Waste and Dynamics of Microbial Communities

Thermophilic dry methane fermentation is advantageous for feedstock with high solid content. Distillation residue with 65.1 % moisture content was eluted from ethanol fermentation of kitchen waste and subjected to thermophilic dry methane fermentation, after adjusting the moisture content to 75 %. The effect of carbon to nitrogen (C/N) ratio on thermophilic dry methane fermentation was investigated. Results showed that thermophilic dry methane fermentation could not be stably performed for >10 weeks at a C/N ratio of 12.6 and a volatile total solid (VTS) loading rate of 1 g/kg sludge/d; however, it was stably performed at a C/N ratio of 19.8 and a VTS loading rate of 3 g/kg sludge/d with 83.4 % energy recovery efficiency. Quantitative PCR analysis revealed that the number of bacteria and archaea decreased by two orders of magnitude at a C/N ratio of 12.6, whereas they were not influenced at a C/N ratio of 19.8. Microbial community analysis revealed that the relative abundance of protein-degrading bacteria increased and that of organic acid-oxidizing bacteria and acetic acid-oxidizing bacteria decreased at a C/N ratio of 12.6. Therefore, there was accumulation of NH₄ ⁺ and acetic acid, which inhibited thermophilic dry methane fermentation.     

Aqueous Ammonia Soaking of Switchgrass Followed by Simultaneous Saccharification and Fermentation

Simultaneous saccharification and fermentation (SSF) of switchgrass was performed following aqueous ammonia pretreatment. Switchgrass was soaked in aqueous ammonium hydroxide (30%) with different liquid–solid ratios (5 and 10 ml/g) for either 5 or 10 days. The pretreatment was carried out at atmospheric conditions without agitation. A 40–50% delignification (Klason lignin basis) was achieved, whereas cellulose content remained unchanged and hemicellulose content decreased by approximately 50%. The Sacccharomyces cerevisiae (D₅A)-mediated SSF of ammonia-treated switchgrass was investigated at two glucan loadings (3 and 6%) and three enzyme loadings (26, 38.5, and 77 FPU/g cellulose), using Spezyme CP. The percentage of maximum theoretical ethanol yield achieved was 72. Liquid–solid ratio and steeping time affected lignin removal slightly, but did not cause a significant change in overall ethanol conversion yields at sufficiently high enzyme loadings. These results suggest that ammonia steeping may be an effective method of pretreatment for lignocellulosic feedstocks.     

Bioethanol Production from Soybean Residue via Separate Hydrolysis and Fermentation

Bioethanol was produced using polysaccharide from soybean residue as biomass by separate hydrolysis and fermentation (SHF). This study focused on pretreatment, enzyme saccharification, and fermentation. Pretreatment to obtain monosaccharide was carried out with 20% (w/v) soybean residue slurry and 270 mmol/L H₂SO₄ at 121 °C for 60 min. More monosaccharide was obtained from enzymatic hydrolysis with a 16 U/mL mixture of commercial enzymes C-Tec 2 and Viscozyme L at 45 °C for 48 h. Ethanol fermentation with 20% (w/v) soybean residue hydrolysate was performed using wild-type and Saccharomyces cerevisiae KCCM 1129 adapted to high concentrations of galactose, using a flask and 5-L fermenter. When the wild type of S. cerevisiae was used, an ethanol production of 20.8 g/L with an ethanol yield of 0.31 g/g consumed glucose was obtained. Ethanol productions of 33.9 and 31.6 g/L with ethanol yield of 0.49 g/g consumed glucose and 0.47 g/g consumed glucose were obtained in a flask and a 5-L fermenter, respectively, using S. cerevisiae adapted to a high concentration of galactose. Therefore, adapted S. cerevisiae to galactose could enhance the overall ethanol fermentation yields compared to the wild-type one.     

Effect of Phosphate on Glucosamine Production by Ethanolic Fungus Mucor indicus

In this study, the effect of phosphorous compound concentration on the production of glucosamine by Mucor indicus was investigated. Changes in the yield of ethanol, the major metabolite of the fungus, were also followed besides. The alkali insoluble material of the biomass of the fungus mainly contained phosphates and polymers of glucosamine and N-acetyl glucosamine, i.e., chitin and chitosan. Yields of glucosamine (78–113 g/kg dry fungal biomass) and ethanol (200–370 g/kg glucose) were significantly affected by the phosphorous concentration. The results showed that lower concentrations of phosphorous favored the production of glucosamine while higher ethanol as well as biomass yields was obtained at higher concentrations. The best concentration was 0.5 g/l where glucosamine yield was 0.37 g/l (11 % of the biomass). At this phosphate concentration, ethanol and biomass yields were 360 and 76 g/kg glucose, respectively. On average, proteins comprised 51.5 % of the biomass. Glycerol was the second important metabolite during the fermentation by the fungus which appeared at lower yields (20–34 g/kg glucose).     

Ethanol production from jerusalem artichoke by inulinase and zymomonas mobilis

Ethanol production from Jerusalem artichoke was studied using inulinase and Z.mobilis by simultaneous saccharification and fermentation (SSF) process. The SSF process showed higher ethanol yield and productivity than the acid or enzymatic prehydrolyzed two-step process. The optimum temperature and inulinase concentration for SSF were 35°C and 0.25% (v/w, 4.4 units/g of sugar), respectively. In order to operate the SSF process in a continuous mode, inulinase and Z.mobilis cells were coimmobilized in alginate beads, using chitin as a matrix for enzyme immobilization. The maximum ethanol productivity of the continuous SSF process was 55.1 g/L/h, with 55% conversion yield. At the conversion yield of 90%, the productivity was 32.7 g/L/h. The continuous SSF system could be operated stably over 2 wk with an ethanol concentration of 48.6 g/L (95% of theoretical yield).     

Ethanol production from corn starch in a fluidized-bed bioreactor

The production of ethanol from industrial dry-milled corn starch was studied in a laboratory-scale fluidized-bed bioreactor using immobilized biocatalysts. Saccharification and fermentation were carried out either simultaneously or separately. Simultaneous saccharification and fermentation (SSF) experiments were performed using small, uniform κ-carrageenan beads (1.5–2.5 mm in diameter) of co-immobilized glucoamylase and Zymomonas mobilis. Dextrin feeds obtained by the hydrolysis of 15% drymilled corn starch were pumped through the bioreactor at residence times of 1.5–4h. Single-pass conversion of dextrins ranged from 54–89%, and ethanol concentrations of 23–36 g/L were obtained at volumetric productivities of 9–15 g/L-h. Very low levels of glucose were observed in the reactor, indicating that saccharification was the rate-limiting step. In separate hydrolysis and fermentation (SHF) experiments, dextrin feed solutions of 150–160 g/L were first pumped through an immobilized-glucoamylase packed column. At 55°C and a residence time of 1 h, greater than 95% conversion was obtained, giving product streams of 162–172 g glucose/L. These streams were then pumped through the fluidized-bed bioreactor containing immobilized Z. mobilis. At a residence time of 2 h, 94% conversion and ethanol concentration of 70 g/L were achieved, resulting in an overall process productivity of 23 g/L-h. Atresidence times of 1.5 and 1 h, conversions of 75 and 76%, ethanol concentrations of 49 and 47 g/L, and overall process productivities of 19 and 25 g/L-h, respectively, were achieved.