Simultaneous Saccharification and Fermentation

A major problem with fermentative ethanol production is the formation of large amounts of numerous organic pollutants. In an industrial distillery, stillage, fermenter and condenser cooling water are the main sources of wastewater. However, the selection of a proper technology makes it possible to almost completely avoid emissions of such kind of wastewater to the environment. This study examines the effect of stillage recirculation on fuel ethanol production. It is based on the use of Saccharomyces cerevisiae and a granular starch hydrolyzing enzyme in a simultaneous saccharification and fermentation process using a native starch obtained from corn flour. It was shown that the yield of the ethanol production was not influenced by the recycled stillage, a mean yield being 83.38% of the theoretical value. No significant trend for change in the ethanol concentration or in the residual starch was observed during any particular run, even after the 75% of fresh water was replaced with stillage. Thus, by applying this new clean technology it is possible to significantly reduce the rate of water consumption and in this way the production of by-products such as stillage.

Lignocellulosic ethanol has been proposed as a green alternative to fossil fuels for many decades. However, commercialization of lignocellulosic ethanol faces major hurdles including pretreatment, efficient sugar release and fermentation. Several processes were developed to overcome these challenges e.g. simultaneous saccharification and fermentation (SSF). This review highlights the various ethanol production processes with their advantages and shortcomings. Recent technologies such as singlepot biorefineries, combined bioprocessing, and bioenergy systems with carbon capture are promising. However, these technologies have a lower technology readiness level (TRL), implying that additional efforts are necessary before being evaluated for commercial availability. Solving energy needs is not only a technological solution and interlinkage of various factors needs to be assessed beyond technology development.

Two samples of low-grade wheat flour, namely low-grade 1 (LG1) and low-grade 2 (LG2), with different carbohydrate and fibrous content, were used as substrates. The samples were liquefied using various concentrations of a- or b-amylase, in order to optimize the production of fermentable sugars; the enzyme a-amylase revealed higher performance. After liquefaction, the simultaneous saccharification and fermentation was conducted in a jar fermentor. Amyloglucosidase was used for saccharification, and dry baker's yeast, S. cerevisiae, for fermentation simultaneously. Glucose was consumed promptly in both cases, LG1 and LG2; ethanol production was considerably higher in LG1 (38.6 g/L), compared to LG2 (24.9 g/L). The maximum ATP production was observed early in the SSF process. LG1 revealed higher potential as substrate for ethanol production.

Processes that produce only ethanol from lignocellulosics display poor economics. This is generally overcome by constructing large facilities having satisfactory economies of scale, thus making financing onerous and hindering the development of suitable technologies. Lignol Innovations has developed a biorefining technology that employs an ethanol-based organosolv step to separate lignin, hemicellulose components, and extractives from the cellulosic fraction of woody biomass. The resultant cellulosic fraction is highly susceptible to enzymatic hydrolysis, generating very high yields of glucose (>90% in 12–24 h) with typical enzyme loadings of 10–20 FPU (filter paper units)/g. This glucose is readily converted to ethanol, or possibly other sugar platform chemicals, either by sequential or simultaneous saccharification and fermentation. The liquor from the organosolv step is processed by well-established unit operations to recover lignin, furfural, xylose, acetic acid, and a lipophylic extractives fraction. The process ethanol is recovered and recycled back to the process. The resulting recycled process water is of a very high quality, low BOD5 and suitable for overall system process closure. Significant benefits can be attained in greenhouse gas (GHG) emission reductions, as per the Kyoto Protocol. Revenues from the multiple products, particularly the lignin, ethanol and xylose fractions, ensure excellent economics for the process even in plants as small as 100 mrpd (metric tonnes per day) dry woody biomass input—a scale suitable for processing wood residues produced by a single large sawmill.

Conversion of food wastes into lactic acid by simultaneous saccharification and fermentation (SSF) was investigated. The process involves saccharification of the starch component in food wastes by a commercial amylolytic enzyme preparation (a mixture of amyloglucosidase, α-amylase, and protease) and fermentation by Lactobacillus delbrueckii. The highest observed overall yield of lactic acid in the SSF was 91% of theoretical. Lactic acid concentration as high as 80 g/L was attainable in 48 h of the SSF. The optimum operating conditions for the maximum productivity were found to be 42°C and pH 6.0. Without supplementation of nitrogen-containing nutrients, the lactic acid yield in the SSF decreased to 60%: 27 g/L of lactic acid from 60 g/L of food waste. The overall performance of the SSF, however, was not significantly affected by the elimination of mineral supplements.

Glycopeptides produced by Streptomyces species are the drugs used against β-lactam drug-resistant staphylococcal infections, and vancomycin is important among them. Increased prevalence of resistant strains increased the usage of vancomycin worldwide and also promoted attempts for indigenous production. The optimum process conditions pH, temperature, inoculum size, agitation, and aeration for vancomycin production by Amycolatopsis orientalis were evaluated, statistically analyzed, and the response surface curves were constructed. The optimum process conditions were a pH of 7.6, a temperature of 29°C, an inoculum size of 4.5%, an agitation of 255 rpm, and an aeration of less than 1:10 medium-to-air ratio.

Whole treechips obtained from softwood forest thinnings were pretreated via single-and two-stage dilute-sulfuric acid pretreatment. Whole-tree chips were impregnated with dilute sulfuric acid and steam treated in a 4-L steam explosion reactor. In single-stage pretreatment, wood chips were treated using a wide range of severity. In two-stage pretreatment, the first stage was carried out at low severity tomaximize hemicellulose recovery. Solubilized sugars were recovered from the first-stage prehydrolysate by washing with water. In the second stage, water-insoluble solids from first-stage prehydrolysate were impregnated with dilute sulfuric acid, then steam treated at more severe conditions to hydrolyze a portion of the remaining cellulose to glucose and to improve the enzyme digestibility. The total sugar yields obtained after enzymatic hydrolysis of two-stage dilute acid-pretreated samples were compared with sugar yields from single-stage pretreatment. The overall sugar yield from two-stage dilute-acid pretreatment was approx 10% higher, and the net enzyme requirement was reduced by about 50%. Simultaneous saccharification and fermentation using an adapted Saccharomyces cerevisiae yeast strain further improved cellulose conversion yield and lowered the enzyme requirement.

Whole treechips obtained from softwood forest thinnings were pretreated via single-and two-stage dilute-sulfuric acid pretreatment. Whole-tree chips were impregnated with dilute sulfuric acid and steam treated in a 4-L steam explosion reactor. In single-stage pretreatment, wood chips were treated using a wide range of severity. In two-stage pretreatment, the first stage was carried out at low severity tomaximize hemicellulose recovery. Solubilized sugars were recovered from the first-stage prehydrolysate by washing with water. In the second stage, water-insoluble solids from first-stage prehydrolysate were impregnated with dilute sulfuric acid, then steam treated at more severe conditions to hydrolyze a portion of the remaining cellulose to glucose and to improve the enzyme digestibility. The total sugar yields obtained after enzymatic hydrolysis of two-stage dilute acid-pretreated samples were compared with sugar yields from single-stage pretreatment. The overall sugar yield from two-stage dilute-acid pretreatment was approx 10% higher, and the net enzyme requirement was reduced by about 50%. Simultaneous saccharification and fermentation using an adapted Saccharomyces cerevisiae yeast strain further improved cellulose conversion yield and lowered the enzyme requirement.

We have used liquid waste obtained from a beer brewery process to produce ethanol. To increase the productivity, genetically modified organism, Escherichia coli KO11, was used for ethanol fermentation. Yeast was also used to produce ethanol from the same feed stock, and the ethanol production rates and resulting concentrations of sugars and ethanol were compared with those of KO11. In the experiments, first the raw wastewater was directly fermented using two strains with no saccharification enzymes added. Then, commercial enzymes, α-amylase, pectinase, or a combination of both, were used for simultaneous saccharification and fermentation, and the results were compared with those of the no-enzyme experiments for KO11 and yeast. Under the given conditions with or without the enzymes, yeast produced ethanol more rapidly than E. coli KO11, but the final ethanol concentrations were almost the same. For both yeast and KO11, the enzymes were observed to enhance the ethanol yields by 61–84%...

The technology is available to produce fuel ethanol from renewable lignocellulosic biomass. The current challenge is to assemble the various process options into a commercial venture and begin the task of incremental improvement. Current process designs for lignocellulose are far more complex than grain to ethanol processes. This complexity results in part from the complexity of the substrate and the biological limitations of the catalyst. Our work at the University of Florida has focused primarily on the genetic engineering of Enteric bacteria using genes encoding Zymomonasmobilis pyruvate decarboxylase and alcohol dehydrogenase. These two genes have been assembled into a portable ethanol production cassette, the PET operon, and integrated into the chromosome of Escherichia coli B for use with hemicellulose-derived syrups. The resulting strain, KO11, produces ethanol efficiently from all hexose and pentose sugars present in the polymers of hemicellulose. By using the same approach, we integrated the PET operon into the chromosome of Klebsiella oxytoca to produce strain P2 for use in the simultaneous saccharification and fermentation (SSF) process for cellulose. Strain P2 has the native ability to ferment cellobiose and cellotriose, eliminating the need for one class of cellulase enzymes. Recently, the ability to produce and secrete high levels of endoglucanase has also been added to strain P2, further reducing the requirement for fungal cellulase. The general approach for the genetic engineering of new biocatalysts using the PET operon has been most successful with Enteric bacteria but was also extended to Gram positive bacteria, which have other useful traits for lignocellulose conversion. Many opportunities remain for further improvements in these biocatalysts as we proceed toward the development of single organisms that can be used for the efficient fermentation of both hemicellulosic and cellulosic substrates.

The effect of process stream recirculation on ethanol production from steam- pretreated softwood based on simultaneous saccharification and fermentation (SSF) was investigated for two process configurations. In the first configuration, a part of the stillage stream after distillation was recycled and, in the second configuration, the liquid after SSF was recycled. The aim was to minimize the energy consumption in the distillation of the fermentation broth and in the evaporation of the stillage, as well as the use of fresh water. However, recirculation leads to an increased concentration of nonvolatiles in the first configuration, and of both volatiles and nonvolatiles in the second configuration. These substances might be inhibitory to the enzymes and the yeast in SSF. When 60% of the fresh water was replaced by stillage, the ethanol yield and the productivity were the same as for the configuration without recirculation. The ethanol production cost was reduced by 17%. In the second configuration, up to 40% of the fresh water could be replaced without affecting the final ethanol yield, although the initial ethanol productivity decreased. The ethanol production cost was reduced by 12%. At higher degrees of recirculation, fermentation was clearly inhibited, resulting in a decrease in ethanol yield while hydrolysis seemed unaffected.

In this work, the effect of the addition of different concentrations of Tween-80 and three different zeolite-like products on enzymatic hydrolysis, ethanol fermentation, and simultaneous saccharification and fermentation (SSF) process has been investigated. The ability of these products to enhance the effectiveness of the SSF process to ethanol of steam-exploded poplar biomass using the thermotolerant strainKluyveromyces marxianus EMS-26 has been tested. Tween-80 (0.4 g/L) increased enzymatic hydrolysis yield by 20% when compared to results obtained in hydrolysis in absence of the additive. Zeolite-like products (ZESEP-56 and ZECER-56) (2.5 g/L) improved rates of conversion and ethanol yields in the fermentation of liquid fraction recovered from steam-exploded poplar. The periods required for the completion of fermentation were approx 10 h in the presence of zeolite-like products and 24 h in the absence of additives. The probable mode of action is through lowered levels of inhibitory substances because of adsorption by the additive.

Sugarcane bagasse is an agroindustrial residue generated in large amounts in Brazil. This biomass can be used for the production of cellulases, aiming at their use in second-generation processes for bioethanol production. Therefore, this work reports the ability of a fungal strain, Trichoderma harzianum IOC-4038, to produce cellulases on a novel material, xylan free and cellulose rich, generated from sugarcane bagasse, named partially delignified cellulignin. The extract produced by T. harzianum under submerged conditions reached 745, 97, and 559 U L−1 of β-glucosidase, FPase, and endoglucanase activities, respectively. The partial characterization of this enzyme complex indicated, using a dual analysis, that the optimal pH values for the biocatalysis ranged from 4.9 to 5.2 and optimal temperatures were between 47 and 54 °C, depending on the activity studied. Thermal stability analyses revealed no significant decrease in activity at 37 °C during 23 h of incubation. When compared to model strains, Aspergillus niger ATCC-16404 and Trichoderma reesei RutC30, T. harzianum fermentation was faster and its extract showed a better balanced enzyme complex, with adequate characteristics for its application in simultaneous saccharification and fermentation processes.