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.     

Conversion of lignocellulosics pretreated with liquid hot water to ethanol

Lignocellulosic materials pretreated using liquid hot water (LHW) (220°C, 5 MPa, 120 s) were fermented to ethanol by batch simultaneous saccharification and fermentation (SSF) usingSaccharomyces cerevisiae in the presence ofTrichoderma reesei cellulase. SSF of sugarcane bagasse (as received), aspen chips (smallest dimension 3 mm), and mixed hardwood flour (−60 +70 mesh) resulted in 90% conversion to ethanol in 2–5 d at enzyme loadings of 15–30 FPU/g. In most cases, 90% of the final conversion was achieved within 75 h of inoculation. Comminution of the pretreated substrates did not affect the conversion to ethanol. The hydrolysate produced from the LHW pretreatment showed slight inhibition of batch growth ofS. cerevisiae. Solids pretreated at a concentration of 100 g/L were as reactive as those pretreated at a lower concentration, provided that the temperature was maintained at 220°C.     

Improvement on Citric Acid Production in Solid-state Fermentation by <Emphasis Type="Italic">Aspergillus niger</Emphasis> LPB BC Mutant Using Citric Pulp

Citric acid (CA) production has been conducted through a careful strain selection, physical–chemical optimization and mutation. The aim of this work was to optimize the physical–chemical conditions of CA production by solid-state fermentation (SSF) using the Aspergillus niger LPB BC strain, which was isolated in our laboratory. The parental and mutant strain showed a good production of CA using citric pulp (CP) as a substrate. The physical–chemical parameters were optimized and the best production was reached at 65% moisture, 30 °C and pH 5.5. The influence of the addition of commercial and alternative sugars, nitrogen sources, salts, and alcohols was also studied. The best results (445.4 g of CA/kg of CP) were obtained with sugarcane molasses and 4% methanol (v/w). The mutagenesis induction of LPB BC was performed with UV irradiation. Eleven mutant strains were tested in SSF where two mutants showed a higher CA production when compared to the parental strain. A. niger LPB B3 produced 537.6 g of CA/kg of CP on the sixth day of fermentation, while A. niger LPB B6 produced 616.5 g of CA/kg of CP on the fourth day of fermentation, representing a 19.5% and 37% gain, respectively.     

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.     

Continuous fermentation of cellulosic biomass to ethanol

Experimental results are presented for continuous conversion of pretreated hardwood flour to ethanol. A simultaneous saccharification and fermentation (SSF) system comprised ofTrichoderma reesei cellulase supplemented with additional β-glucosidase and fermentation bySaccharomyces cerevisiae was used for most experiments, with data also presented for a direct microbial conversion (DMC) system comprised ofClostridium thermocellum. Using a batch SSF system, dilute acid pretreatment of mixed hardwood at short residence time(10 s, 220°C, 1% H₂SO₄) was compared to poplar wood pretreated at longer residence time (20 min, 160°C, 0.45% H₂SO₄). The short residence time pretreatment resulted in a somewhat (10–20%) more reactive substrate, with the reactivity difference particularly notable at low enzyme loadings and/or low agitation. Based on a preliminary screening, inhibition of SSF by byproducts of short residence time pretreatment was measurable, but minor. Both SSF and DMC were carried out successfully in well-mixed continuous systems, with steady-state data obtained at residence times of 0.58–3 d for SSF as well as 0.5 and 0.75 d for DMC. The SSF system achieved substrate conversions varying from 31% at a 0.58-d residence time to 86% at a 2-d residence time. At comparable substrate concentrations (4–5 g/l) and residence times (0.5–0.58 d), substrate conversion in the DMC system (77%) was significantly higher than that in the SSF system (31%). Our results suggest that the substrate conversion in SSF carried out in CSTR is relatively insensitive to enzyme loading in the range 7–25 U/g cellulose and to substrate concentration in the range of 5–60 g/L cellulose in the feed.     

Influence of High Solid Concentration on Enzymatic Hydrolysis and Fermentation of Steam-Exploded Corn Stover Biomass

Steam-exploded corn stover biomass was used as the substrate for fed-batch separate enzymatic hydrolysis and fermentation (SHF) to investigate the solid concentration ranging from 10% to 30% (w/w) on the lignocellulose enzymatic hydrolysis and fermentation. The treatment of washing the steam-exploded material was also evaluated by experiments. The results showed that cellulose conversion changed little with increasing solid concentration, and fermentation by Saccharomyces cerevisiae revealed a nearly same ethanol yield with the water-washed steam-exploded corn stover. For the washed material at 30% substrate concentration, i.e., 30% water insoluble solids (WIS), enzymatic hydrolysis yielded 103.3 g/l glucose solution and a cellulose conversion of 72.5%, thus a high ethanol level up to 49.5 g/l. With the unwashed steam-exploded corn stover, though a cellulose conversion of 70.9% was obtained in hydrolysis at 30% solid concentration (27.9% WIS), its hydrolysate did not ferment at all, and the hydrolysate of 20% solid loading containing 3.3 g/l acetic acid and 145 mg/l furfural already exerted a strong inhibition on the fermentation and ethanol production.     

Continuous Ethanol Production from Cassava Through Simultaneous Saccharification and Fermentation by Self-Flocculating Yeast Saccharomyces Cerevisiae CHFY0321

In this study, a fermentor consisting of four linked stirred towers that can be used for simultaneous saccharification and fermentation (SSF) and for the accumulation of cell mass was applied to the continuous production of ethanol using cassava as the starchy material. For the continuous process with SSF, the pretreated cassava liquor and saccharification enzyme at total sugar concentrations of 175 g/L and 195 g/L were continuously fed to the fermentor with dilution rates of 0.014, 0.021, 0.031, 0.042, and 0.05 h⁻¹. Considering the maximum saccharification time, the highest volumetric productivity and ethanol yield were observed at a dilution rate of 0.042 h⁻¹. At dilution rates in the range of 0.014 h⁻¹ to 0.042 h⁻¹, high production rates were observed, and the yeast in the first to fourth fermentor showed long-term stability for 2 months with good performance. Under the optimal culture conditions with a feed sugar concentration of 195 g/L and dilution rate of 0.042 h⁻¹, the ethanol volumetric productivity and ethanol yield were 3.58 g/L∙h and 86.2%, respectively. The cell concentrations in the first to fourth stirred tower fermentors were 74.3, 71.5, 71.2, and 70.1 g dry cell/L, respectively. The self-flocculating yeast, Saccharomyces cerevisiae CHFY0321, developed by our group showed excellent fermentation results under continuous ethanol production.     

Ethanol Production from H<Subscript>2</Subscript>SO<Subscript>3</Subscript>-Steam-Pretreated Fresh Sweet Sorghum Stem by Simultaneous Saccharification and Fermentation

The present work presents an alternative approach to ethanol production from sweet sorghum: without detoxification, acid-impregnated fresh sweet sorghum stem which contains soluble (glucose and sucrose) and insoluble carbohydrates (cellulose and hemicellulose) was steam pretreated under mild temperature of 100 °C. Simultaneous saccharification and fermentation experiments were performed on the pretreated slurries using Saccharomyces cerevisiae. Experimentally, ground fresh sweet sorghum stem was combined with H₂SO₃ at dosages of 0.25, 0.50, and 0.75 g/g dry matter (DM) and steam pretreated by varying the residence time (60, 120, or 240 min). According to enzymatic hydrolysis results and ethanol yields, H₂SO₃ was a powerful and mild acid for improving enzymatic digestibility of sorghum stem. At a solid loading of 10% (w/v) and acid dosage of 0.25 g/g DM H₂SO₃ at 100 °C for 120 min, 44.5 g/L ethanol was obtained after 48 ± 4 h of simultaneous saccharification and fermentation. This corresponded to an overall ethanol yield of 110% of the theoretical one, based on the soluble carbohydrates in the fresh sweet sorghum stem. The concentrations of hydroxymethylfurfural and furfural of the sulfurous acid pretreated samples were below 0.4 g/L. Ethanol would not inhibit the cellulase activity, at least under the concentration of 34 g/L.     

Statistical Optimization of Recycled-Paper Enzymatic Hydrolysis for Simultaneous Saccharification and Fermentation Via Central Composite Design

A central composite design of the response surface methodology (RSM) was employed to study the effects of temperature, enzyme concentration, and stirring rate on recycled-paper enzymatic hydrolysis. Among the three variables, temperature and enzyme concentration significantly affected the conversion efficiency of substrate, whereas stirring rate was not effective. A quadratic polynomial equation was obtained for enzymatic hydrolysis by multiple regression analysis using RSM. The results of validation experiments were coincident with the predicted model. The optimum conditions for enzymatic hydrolysis were temperature, enzyme concentration, and stirring rate of 43.1 °C, 20 FPU g⁻¹ substrate, and 145 rpm, respectively. In the subsequent simultaneous saccharification and fermentation (SSF) experiment under the optimum conditions, the highest 28.7 g ethanol l⁻¹ was reached in the fed-batch SSF when 5% (w/v) substrate concentration was used initially, and another 5% added after 12 h fermentation. This ethanol output corresponded to 77.7% of the theoretical yield based on the glucose content in the raw material.     

Ethanol Production from Wet-Exploded Wheat Straw Hydrolysate by Thermophilic Anaerobic Bacterium Thermoanaerobacter BG1L1 in a Continuous Immobilized Reactor

Thermophilic ethanol fermentation of wet-exploded wheat straw hydrolysate was investigated in a continuous immobilized reactor system. The experiments were carried out in a lab-scale fluidized bed reactor (FBR) at 70°C. Undetoxified wheat straw hydrolysate was used (3–12% dry matter), corresponding to sugar mixtures of glucose and xylose ranging from 12 to 41 g/l. The organism, thermophilic anaerobic bacterium Thermoanaerobacter BG1L1, exhibited significant resistance to high levels of acetic acid (up to 10 g/l) and other metabolic inhibitors present in the hydrolysate. Although the hydrolysate was not detoxified, ethanol yield in a range of 0.39–0.42 g/g was obtained. Overall, sugar efficiency to ethanol was 68–76%. The reactor was operated continuously for approximately 143 days, and no contamination was seen without the use of any agent for preventing bacterial infections. The tested microorganism has considerable potential to be a novel candidate for lignocellulose bioconversion into ethanol. The work reported here also demonstrates that the use of FBR configuration might be a viable approach for thermophilic anaerobic ethanol fermentation.     

Fermentation performance assessment of a genomically integrated xylose-utilizing recombinant of Zymomonas mobilis 39676

In pH-controlled batch fermentations with pure sugar synthetic hardwood hemicellulose (1% [w/v] glucose and 4% xylose) and corn stover hydrolysate (8% glucose and 3.5% xylose) lacking acetic acid, the xyloseutilizing, tetracycline (Tc)-sensitive, genomically integrated variant of Zymomonas mobilis ATCC 39676 (designated strain C25) exhibited growth and fermentation performance that was inferior to National Renewable Energy Laboratory's first-generation, Tc-resistant, plasmid-bearing Zymomonas recombinants. With C25, xylose fermentation following glucose exhaustion wasmarkellyslower, and the ethanol yield (based on sugars consumed) was lower, owing primarily to an increase in lactic acid formation. There was an apparent increased sensitivity to acetic acid inhibition with C25 compared with recombinants 39676:pZB4L, CP4:pZB5, and ZM4:pZB5. However, strain C25 performed well in continous ferm entation with nutrient-rich synthetic corn stover medium over the dilution range 0.03–0.06/h, with a maximum provess ethanol yield at D=0.03/h of 0.46 g/g and a maximum ethanol productivity of 3 g/(L·h). With 0.35% (w/v) acetic acid in the medium, the process yield at D=0.04/h dropped to 0.32 g/g, and the maximum productivity decreased by 50% to 1.5 g/(L·h). Under the same operating conditions, rec Zm Zm 4:pZB5 performed better; however, the medium contained 20 mg/L of Tc to constantly maintain selective pressure. The absence of any need for antibiotics and antiboitic resistance genes makes the chromosomal integrant C25 more com patible with current regulatory specifications for biocatalysts in large-scale commercial operations.     

Novel Isolates for Biological Detoxification of Lignocellulosic Hydrolysate

In this paper, two new strians, Issatchenkia occidentalis (Lj-3, CCTCC M 2006097) and Issatchenkia orienalis (S-7, CCTCC M 2006098), isolated from different environments on solid media, were used in the detoxification process of the hemicellulosic hydrolysate of sugarcane bagasse. High-pressure liquid chromatography elution curve of UV-absorption compounds represented by acetic acid, furfural, and guaiacol (toxic compounds found in the hemicellulosic hydrolysate) showed that several chromatographic peaks were evidently diminished for the case of detoxified hydrolysate with isolate strains compared to the high peaks resulted for no detoxified hydrolysate. It was clear that these inhibitors were degraded by the two new isolates during their cultivation process. Fermentation results for the biodetoxified hydrolysate showed an increase in xylitol productivity (Q _p) by 1.97 and 1.95 times (2.03 and 2.01 g l⁻¹ h⁻¹) and in xylitol yield (Y _p) by 1.72 and 1.65 times (0.93 and 0.89 g xylitol per gram xylose) for hydrolysate treated with S-7 and Lj-3, respectively, in comparison with no detoxified hydrolysate (1.03 g l⁻¹ h⁻¹ and 0.54 g xylitol per gram xylose). This present work demonstrated the importance of Issatchenkia yeast in providing an effective biological detoxification approach to remove inhibitors and improve hydrolysate fermentability, leading to a high xylitol productivity and yield.     

Lactic Acid Production from a Whole Slurry of Acid-Pretreated Spent Coffee Grounds by Engineered Saccharomyces cerevisiae

Spent coffee grounds (SCG) generated after coffee extraction are the main byproduct of the coffee industry. Valorization of the SCG has been increasingly focused following considerable attention in coffee consumption. Lactic acid bacteria fermentation is the primary source of generation of lactic acid, a monomer of polylactic acid that has various industrial applications; however, because of the low tolerance of lactic acid bacteria to toxic compounds, it is necessary to apply Saccharomyces cerevisiae to produce lactic acid whose tolerance to toxic compounds is higher. In this study, we evaluated the feasibility of using SCG as substrate for the production of lactic acid by S. cerevisiae strain expressing heterologous lactate dehydrogenase. The fermentation profiles of the engineered yeast showed that lactic acid production was promoted by xylose addition. From simultaneous saccharification and fermentation (SSF) using a whole slurry of acid-pretreated SCG, containing high amounts of hemicellulose fractions, lactic acid (0.11 g) and ethanol (0.10 g) per g SCG were obtained after 24 h of SSF, of which yields were 413% and 221% higher, respectively, than those of washed pretreated SCG. Thus, fermentation of whole slurry SCG by engineered S. cerevisiae is a suitable way of lactic acid production, selectively.

     

Fumaric Acid Production from Alkali-Pretreated Corncob by Fed-Batch Simultaneous Saccharification and Fermentation Combined with Separated Hydrolysis and Fermentation at High Solids Loading

Production of fumaric acid from alkali-pretreated corncob (APC) at high solids loading was investigated using a combination of separated hydrolysis and fermentation (SHF) and fed-batch simultaneous saccharification and fermentation (SSF) by Rhizopus oryzae. Four different fermentation modes were tested to maximize fumaric acid concentration at high solids loading. The highest concentration of 41.32 g/L fumaric acid was obtained from 20 % (w/v) APC at 38 °C in the combined SHF and fed-batch SSF process, compared with 19.13 g/L fumaric acid in batch SSF alone. The results indicated that a combination of SHF and fed-batch SSF significantly improved production of fumaric acid from lignocellulose by R. oryzae than that achieved with batch SSF at high solids loading.     

Comparative ethanol productivities of different Zymomonas recombinants fermenting oat hull hydrolysate

Iogen Corporation of Ottawa, Canada, has recently built a 50 t/d biomass-to-ethanol demonstration plant adjacent to its enzyme production facility. Iogen has partnered with the University of Toronto to test the C6/C5 cofermentation performance characteristics of National Renewable Energy Laboratory's metabolically engineered Zymomonas mobilis using its biomass hydrolysates. In this study, the biomass feedstock was an agricultural waste, namely oat hulls, which was hydrolyzed in a proprietary two-stage process involving pretreatment with dilute sulfuric acid at 200–250°C, followed by cellulase hydrolysis. The oat hull hydrolysate (OHH) contained glucose, xylose, and arabinose in a mass ratio of about 8:3:0.5. Fermentation media, prepared from diluted hydrolysate, were nutritionally amended with 2.5 mL/L of corn steep liquor (50% solids) and 1.2 g/L of diammonium phosphate. The estimated cost for large-scale ethanol production using this minimal level of nutrient supplementation was 4.4c/gal of ethanol. This work examined the growth and fermentation performance of xyloseutilizing, tetracycline-resistant, plasmid-bearing, patented, recombinant Z. mobilis cultures: CP4:pZB5, ZM4:pZB5, 39676:pZB4L, and a hardwood prehydrolysate-adapted variant of 39676:pZB4L (designated asthe “adapted” strain). In pH-stat batch fermentations with unconditioned OHH containing 6% (w/v) glucose, 3% xylose, and 0.75% acetic acid, rec Zm ZM4:pZB5 gave the best performance with a fermentation time of 30h, followed by CP4:pZB5 at 48h, with corresponding volumetric productivities of 1.4 and 0.89 g/(L·h), respectively. Based on the available glucose and xylose, the process ethanol yield for both strains was 0.47 g/g (92% conversion efficiency). At 48 h, the process yield for rec Zm 39676:pZB4L and the adapted strain was 0.32 and 0.34 g/g, respectively. None of the test strains was able to fermentarabinose. Acetic acid tolerance appeared to be a major determining factor in cofermentation performance.     

Butanol Production from Cane Molasses by <Emphasis Type="Italic">Clostridium saccharobutylicum</Emphasis> DSM 13864: Batch and Semicontinuous Fermentation

Clostridium acetobutylicum strains used in most Chinese ABE (acetone–butanol–ethanol) plants favorably ferment starchy materials like corn, cassava, etc., rather than sugar materials. This is one major problem of ABE industry in China and significantly limits the exploitation of cheap waste sugar materials. In this work, cane molasses were utilized as substrate in ABE production by Clostridium saccharobutylicum DSM 13864. Under optimum conditions, total solvent of 19.80 g/L (13.40 g/L butanol) was reached after 72 h of fermentation in an Erlenmeyer flask. In a 5-L bioreactor, total solvent of 17.88 g/L was attained after 36 h of fermentation, and the productivity and yield were 0.50 g/L/h and 0.33 g ABE/g sugar consumption, respectively. To further enhance the productivity, a two-stage semicontinuous fermentation process was steadily operated for over 8 days (205 h, 26 cycles) with average productivity (stage II) of 1.05 g/L/h and cell concentration (stage I) of 7.43 OD₆₆₀, respectively. The average batch fermentation time (stage I and II) was reduced to 21−25 h with average solvent of 15.27 g/L. This study provides valuable process data for the development of industrial ABE fermentation process using cane molasses as substrate.     

Sugarcane Bagasse Mild Alkaline/Oxidative Pretreatment for Ethanol Production by Alkaline Recycle Process

In order to decrease the alkali and water consumptions in the sugarcane bagasse alkaline/oxidative pretreatment for ethanol production, an alkaline recycle process was carried out. Two recycles of NaOH/H₂O₂ pretreatment did not decrease the pretreatment and enzymatic hydrolysis efficiencies and the consumptions of NaOH and water would be saved by 26% and 40%, respectively. A simultaneous saccharification and fermentation (SSF) culture with pretreated bagasse as substrate was developed giving 25 g ethanol l⁻¹ with a yield of 0.2 g g⁻¹ bagasse and productivity of 0.52 g l⁻¹ h⁻¹.     

Optimization of the simultaneous saccharification and fermentation process using thermotolerant yeasts

Different treatments to improve the thermotolerance of fermenting yeasts for simultaneous ethanol saccharification and fermentation process of cellulosic materials have been examined. Yeasts of the generaSaccharomyces andKluyveromyces were tested for growth and fermentation at progressively higher temperatures in the range of 42–47°C. The best results were obtained withK. marxianus LG, which was then submitted to different treatments in order to achieve thermotolerant clones. A total of 35 new clones were obtained that dramatically improved the SSF of 10% Solka-floc substrate at 45°C when compared to the original strain, some with ethanol concentrations as high as 33 g/L.     

The Effect of Different Ethoxylations for Sorbitan Monolaurate on Enhancing Simultaneous Saccharification and Fermentation (SSF) of Wheat Straw to Ethanol

In this paper, four nonionic surfactants with different hydrophilic–lipophilic balance (HLB) based on sorbitan monolaurate were synthesized by introducing ethylene oxide gas (n = 20, 40, 60, and 80 ethylene oxide units). The chemical structure of the prepared ethoxylated surfactants was confirmed using Fourier transform-infrared and ¹H NMR spectroscopes. The surface tension and thermodynamic properties of the prepared surfactants have been studied. The simultaneous saccharification and fermentation (SSF) process for ethanol production from microwave/alkali pretreated wheat straw has been assayed using nonionic surfactants have different ethylene oxide units. Ethanol yield was 82% and 61% for Kluyveromyces marxianus and Saccharomyces cerevisiae, respectively, with the addition of 2.5 g/l of the prepared nonionic surfactant (HLB = 18.2). Results show that the production of ethanol from microwave/alkali pretreated wheat straw increased with increasing the (HLB) value of the nonionic surfactant.