Advancements and future directions in enzyme technology for biomass conversion

Enzymatic hydrolysis of pre-treated lignocellulosic biomass is an ideal alternative to acid hydrolysis for bio-ethanol production, limited primarily by pre-treatment requirements and economic considerations arising from enzyme production costs and specific activities. The quest for cheaper and better enzymes has prompted years of bio-prospecting, strain optimization through genetic engineering, enzyme characterization for simple and complex lignocellulosic feedstock, and the development of pre-treatment strategies to mitigate inhibitory effects. The recent shift to systematic characterizations of de novo mixtures of purified proteins is a promising indicator of maturation within this field of study, facilitating progression towards feedstock assay-based rapid enzyme mixture optimization. It is imperative that international standards be developed to enable meaningful comparisons between these studies and the construction of a database of enzymatic activities and kinetics, aspects of which are explored here-in. Complementary efforts to improve the economic viability of enzymatic hydrolysis through process integration and reactor design are also considered, where membrane-confinement shows significant promise despite the associated technological challenges. Significant advancements in enzyme technology towards the economic conversion of lignocellulosic biomass should be expected within the next few years as systematic research in enzyme activities conforms to that of traditional reaction engineering.     

Simultaneous saccharification and fermentation of steam-exploded corn stover at high glucan loading and high temperature

Background

Simultaneous saccharification and fermentation (SSF) is a promising process for bioconversion of lignocellulosic biomass. High glucan loading for hydrolysis and fermentation is an efficient approach to reduce the capital costs for bio-based products production. The SSF of steam-exploded corn stover (SECS) for ethanol production at high glucan loading and high temperature was investigated in this study.

Results

Glucan conversion of corn stover biomass pretreated by steam explosion was maintained at approximately 71 to 79% at an enzyme loading of 30 filter paper units (FPU)/g glucan, and 74 to 82% at an enzyme loading of 60 FPU/g glucan, with glucan loading varying from 3 to 12%. Glucan conversion decreased obviously with glucan loading beyond 15%. The results indicated that the mixture was most efficient in enzymatic hydrolysis of SECS at 3 to 12% glucan loading. The optimal SSF conditions of SECS using a novel Saccharomyces cerevisiae were inoculation optical density (OD)₆₀₀?=?4.0, initial pH 4.8, 50% nutrients added, 36 hours pre-hydrolysis time, 39°C, and 12% glucan loading (20% solid loading). With the addition of 2% Tween 20, glucan conversion, ethanol yield, final ethanol concentration reached 78.6%, 77.2%, and 59.8 g/L, respectively, under the optimal conditions. The results suggested that the solid and degradation products’ inhibitory effect on the hydrolysis and fermentation of SECS were also not obvious at high glucan loading. Additionally, glucan conversion and final ethanol concentration in SSF of SECS increased by 13.6% and 18.7%, respectively, compared with separate hydrolysis and fermentation (SHF).

Conclusions

Our research suggested that high glucan loading (6 to 12% glucan loading) and high temperature (39°C) significantly improved the SSF performance of SECS using a thermal- and ethanol-tolerant strain of S. cerevisiae due to the removal of degradation products, sugar feedback, and solid’s inhibitory effects. Furthermore, the surfactant addition obviously increased ethanol yield in SSF process of SECS.

     

A CSTR-hollow fiber system for continuous hydrolysis of proteins. Performance and kinetics

The effect of four operating variables (enzyme concentration, substrate concentration, flow rate, and reaction volume) on the performance of CSTR-hollow fiber membrane reactor was studied for the continuous hydrolysis of a soy protein isolate using Pronase. Based on a residence time distribution study, the reactor system was modeled as an ideal CSTR in combination with the Michaelis-Menten equation of enzyme kinetics. This kinetic model correlated conversion with a space-time parameter modified to include all four independent variables. An empirical model based on curvilinear regression analysis was also developed. Both models predicted conversion fairly well, although the kinetic model slightly underpredicts at high conversion.     

Modelling ethanol production from cellulose: separate hydrolysis and fermentation versus simultaneous saccharification and fermentation

In ethanol production from cellulose, enzymatic hydrolysis, and fermentative conversion may be performed sequentially (separate hydrolysis and fermentation, SHF) or in a single reaction vessel (simultaneous saccharification and fermentation, SSF). Opting for either is essentially a trade-off between optimal temperatures and inhibitory glucose concentrations on the one hand (SHF) vs. sub-optimal temperatures and ethanol-inhibited cellulolysis on the other (SSF). Although the impact of ethanol on cellobiose hydrolysis was found to be negligible, formation of glucose and cellobiose from cellulose were found to be significantly inhibited by ethanol. A previous model for the kinetics of enzymatic cellulose hydrolysis was, therefore, extended with enzyme inhibition by ethanol, thus allowing a rational evaluation of SSF and SHF. The model predicted SSF processing to be superior. The superiority of SSF over SHF (separate hydrolysis and fermentation) was confirmed experimentally, both with respect to ethanol yield on glucose (0.41 g g^?1 for SSF vs. 0.35 g g^?1 for SHF) and ethanol production rate, being 30% higher for an SSF type process. High conversion rates were found to be difficult to achieve since at a conversion rate of 52% in a SSF process the reaction rate dropped to 5% of its initial value. The model, extended with the impact of ethanol on the cellulase complex proved to predict reaction progress accurately.     

Effect of polyphenol content on the hydrolysis and fermentation of grain sorghum starch

The high polyphenol content of birdproff grain sorghum has been associated with impaired nutritional quality of the grain and with reduced brewing value of birdproof grain sorghum malt due to enzyme inhibition. In this investigation, high polyphenol grain sorghum was evaluated as a feedstock for fermentation ethanol production using NaOH pretreatment to inactivate the polyphenolic compounds prior to hydrolysis with commercial amylases. The polyphenolic inhibition of starch hydrolysis was minimal at a grain sorghum slurry concentration of 20% dry solids, but became pronounced at slurry concentrations of 28% and higher. At these high slurry concentrations the liquefaction and subsequent saccharification and fermentation were markedly improved by alkaline pretreatment. The highest ethanol concentration (12·3%, vol/vol), coupled with the best starch conversion efficiency to ethanol (83·5%), was obtained with a 28% grain sorghum slurry using a partial simultaneous saccharification and fermentation procedure. The residual fermented solids had a crude protein content of 45·4%. Tannic acid decreased yeast cell viability in synthetic media, but had no effect on the hydrolysis or fermentation of grain sorghum starch.     

Bio-ethanol from water hyacinth biomass: An evaluation of enzymatic saccharification strategy

Biomass feedstock having less competition with food crops are desirable for bio-ethanol production and such resources may not be localized geographically. A distributed production strategy is therefore more suitable for feedstock like water hyacinth with a decentralized availability. In this study, we have demonstrated the suitability of this feedstock for production of fermentable sugars using cellulases produced on site. Testing of acid and alkali pretreatment methods indicated that alkali pretreatment was more efficient in making the sample susceptible to enzyme hydrolysis. Cellulase and β-glucosidase loading and the effect of surfactants were studied and optimized to improve saccharification. Redesigning of enzyme blends resulted in an improvement of saccharification from 57% to 71%. A crude trial on fermentation of the enzymatic hydrolysate using the common baker’s yeast Saccharomyces cerevisiae yielded an ethanol concentration of 4.4 g/L.     

Kinetics of the extracellular cellulases of Clostridium thermocellum acting on pretreated mixed hardwood and Avicel

Initial hydrolysis rates were examined for mixed hardwood flour pretreated with 1% sulfuric acid for 9 s at 220 °C (PTW220) and Avicel. Linear rates were observed for fractional conversion relative to the theoretical up to 0.2 for PTW220 and 0.4 for Avicel. Initial rates were essentially unaffected by the presence of growth medium components over a range of pH values. Avicel-hydrolyzing activity was inhibited linearly by ethanol, with a 50% rate reduction at 8 wt.% ethanol. Rate saturation with either substrate or enzyme was observed in a manner qualitatively consistent with previously reported adsorption data. Although somewhat less reactive than Avicel at very low enzyme loadings, much higher reaction rates were observed for PTW220 at moderate and high enzyme loading because of its higher capacity to bind cellulase. At equal subtrate concentrations (as potential glucose) and fractional substrate coverage of 0.09, the initial rate of pretreated wood hydrolysis exceeded that of Avicel by 15-fold. For fractional substrate coverage values up to 0.09 (the maximum value achieved for PTW220), the initial rate was proportional to adsorbed enzyme for PTW220. However, the rate per adsorbed enzyme declined sharply with increasing fractional coverage for Avicel hydrolysis.     

Enhanced ethanol production from brewer's spent grain by a Fusarium oxysporum consolidated system

Background  

Brewer's spent grain (BG), a by-product of the brewing process, is attracting increasing scientific interest as a low-cost feedstock for many biotechnological applications. BG in the present study is evaluated as a substrate for lignocellulolytic enzyme production and for the production of ethanol by the mesophilic fungus Fusarium oxysporum under submerged conditions, implementing a consolidated bioconversion process. Fermentation experiments were performed with sugar mixtures simulating the carbohydrate content of BG in order to determine the utilization pattern that could be expected during the fermentation of the cellulose and hemicellulose hydrolysate of BG. The sugar mixture fermentation study focused on the effect of the initial total sugar concentration and on the effect of the aeration rate on fermenting performance of F. oxysporum. The alkali pretreatment of BG and different aeration levels during the ethanol production stage were studied for the optimization of the ethanol production by F. oxysporum.     

A mechanistic model for rational design of optimal cellulase mixtures

A model‐based framework is described that permits the optimal composition of cellulase enzyme mixtures to be found for lignocellulose hydrolysis. The rates of hydrolysis are shown to be dependent on the nature of the substrate. For bacterial microcrystalline cellulose (BMCC) hydrolyzed by a ternary cellulase mixture of EG2, CBHI, and CBHII, the optimal predicted mixture was 1:0:1 EG2:CBHI:CBHII at 24 h and 1:1:0 at 72 h, at loadings of 10 mg enzyme per g substrate. The model was validated with measurements of soluble cello‐oligosaccharide production from BMCC during both single enzyme and mixed enzyme hydrolysis. Three‐dimensional diagrams illustrating cellulose conversion were developed for mixtures of EG2, CBHI, CBHII acting on BMCC and predicted for other substrates with a range of substrate properties. Model predictions agreed well with experimental values of conversion after 24 h for a variety of enzyme mixtures. The predicted mixture performances for substrates with varying properties demonstrated the effects of initial degree of polymerization (DP) and surface area on the performance of cellulase mixtures. For substrates with a higher initial DP, endoglucanase enzymes accounted for a larger fraction of the optimal mixture. Substrates with low surface areas showed significantly reduced hydrolysis rates regardless of mixture composition. These insights, along with the quantitative predictions, demonstrate the utility of this model‐based framework for optimizing cellulase mixtures. Biotechnol. Bioeng. 2011;108: 2561–2570. © 2011 Wiley Periodicals, Inc.     

Surfactant Effects on Lipase-Catalyzed Hydrolysis of Olive Oil in AOT/ISOOCTANE Reverse Micelles

The effects of surfactant concentration on the hydrolytic activity of Candida rugosa lipase in AOT/isooctane reverse micelles with olive oil as the substrate has been investigated. A noncompetitive inhibition by the surfactant on the enzyme was observed. Strong dependences of the kinetic constants k_cat and k_M, but not k_I on the water-to-surfactant ratio (R value) have been identified. The benefits of carrying out the hydrolysis at higher surfactant and water concentrations were demonstrated from the improvement of the initial rate and time course of conversion.     

Kinetics and mechanism of hydrolysis of acetylthiocholine by butyrylcholine esterase

Kinetics and mechanism of hydrolysis of acetylthiocholine by the enzyme butyrylcholine esterase was studied. The spectrophotometric Ellman's method and potentiometric pH-stat method were used for continuous determination of the actual concentration of the products thiocholine and acetic acid in the reaction mixture. The validity of the Michaelis-Menten (Briggs-Haldane) equation in the whole course of the reaction under used conditions was proved. The corresponding kinetics parameters (Vm and KM) were calculated from the obtained dependences of concentration of thiocholine or acetic acid vs. time and compared. From this comparison the deciding kinetic role of the step producing thiocholine was derived. The values of initial molar concentration of the enzyme and of the rate constants of the kinetic model were estimated.     

Kinetic studies of enzymatic hydrolysis of insoluble cellulose: analysis of the initial rates

A study was conducted on the kinetics of enzymatic hydrolysis of pure insoluble cellulose using unpurified culture filtrate Trichoderma reesei, with the emphasis on the initial reaction period. The initial hydrolysis rate and extent of enzyme (soluble protein)adsorption, either apparent or initial, were evaluated under various experimental conditions. It has been found that the various mass-transfer steps do not control the overall hydrolysis rate and that the hydrolysis rate is mainly controlled by the surface reaction step promoted by the adsorbed enzyme. It has also been found that the initial hydrolysis rate strongly depends on the initial extent of soluble protein adsorption and the effectiveness of the adsorbed soluble protein to promote the hydrolysis. The initial extent of soluble protein adsorption, in turn, is related to the initial cellulose concentration, enzyme concentration, and specific surface area of cellulose, whereas the effectiveness of the initially adsorbed soluble protein to promote the derived to interrelate these parameters without resorting to the Michaelis-Menten kinetics. The present result appear to imply that the role of enzyme-substrate complex formation should not be ignored in deriving a mechanistic kinetic model for enzymatic hydrolysis of cellulose.     

Recycle of enzymes and substrate following enzymatic hydrolysis of steam-pretreated aspenwood

The commercial production of chemicals and fuels from lignocellulosic residues by enzymatic means still requires considerable research on both the technical and economic aspects. Two technical problems that have been identified as requiring further research are the recycle of the enzymes used in hydrolysis and the reuse of the re calcitrant cellulose remaining after incomplete hydrolysis. Enzyme recycle is required to lower the cost of the enzymes, while the reuse of the spent cellulose will lower the feedstock cost. The conversion process studied was a combined enzymatic hydrolysis and fermentation (CHF) procedure that utilized the cellulolytic enzymes derived from the fungus Trichoderma harzianum E58 and the yeast Saccharomyces cerevisiae. The rate and extent of hydrolysis and ethanol production was monitored as was the activity and hydrolytic potential of the enzymes remaining in the filtrate after the hydrolysis period. When a commercial cellulose was used as the substrate for a routine 2-day CHF process, 60% of the original treated, water-extracted aspenwood was used as the substrate, only 13% of the original filter paper activity was detected after a similar procedure. The combination of 60% spent enzymes with 40% fresh enzymes resulted in the production of 30% less reducing sugars than the original enzyme mixture. Since 100% hydrolysis of the cellulose portion is seldom accomplished in an enzymatic hydrolysis pro cess, the residual cellulose was used as a substrate for the growth of T. harzianum E58 and production of celulolytic enzymes. The residue remaining after the CHF process was used as a substrate for the production of the cellulolytic enzymes. The production of enzymes from the residue of the Solka Floc hydrolysis was greater than the production of enzymes from the original Solka Floc.     

Basis for the anomalous effect of competitive inhibitors on the kinetics of hydrolysis of short-chain phosphatidylcholines by phospholipase A2.

The effect of four specific competitive inhibitors on the kinetics of hydrolysis of short-chain diacyl-sn-glycero-3-phosphocholines below their critical micelle concentrations was examined. The kinetics of hydrolysis of short-chain substrates dispersed as solitary monomers were generally consistent with the classical Michaelis-Menten formalism; i.e., hydrolysis began without any latency period, the steady-state rate was observed at higher substrate concentrations, the steady-state initial rate showed a linear dependence on the enzyme concentration, and the hyperbolic dependence of the initial rate on the substrate concentration could be described in terms of KM and Vmax parameters. The competitive nature of the inhibitors used in this study has been established by a variety of techniques, and the equilibrium dissociation constants for the inhibitors bound to the enzyme were measured by the protection method [Jain et al. (1991) Biochemistry 30, 7306-7317]. The kinetics of hydrolysis in the presence of competitive inhibitors could be described by a single dissociation constant. However, the value of the dissociation constant obtained under the kinetic conditions was comparable to that obtained by the protection method for the inhibitor-enzyme complex bound to a neutral diluent, rather than to the value of the dissociation constant obtained with solitary monomeric inhibitors and the enzyme in the aqueous phase. Spectroscopic methods showed that the effectively lower dissociation constant of an inhibitor bound to PLA2 at the interface is due to the stabilization of the enzyme-inhibitor complex by interaction with other amphiphiles present in the reaction mixture.(ABSTRACT TRUNCATED AT 250 WORDS)     

Enzymatic hydrolysis of waste office paper using viscosity as operating parameter

Enzymatic hydrolysis of waste office (WO) paper with feeding WO paper in a reactor was investigated using apparent viscosity as operating parameter. Since the apparent viscosity was correlated with the concentration of pulping WO paper, the amount of hydrolyzed WO paper was assumed by measuring the decrease in the apparent viscosity. Then the amount of hydrolysis WO paper and the amount of enzyme corresponding to the desired ratio were fed into the reactor. When the WO paper and 1% (to the amount of WO paper) enzyme were fed to the hydrolytic reaction, 87 g/L of reducing sugar (RS) with a hydrolytic yield of 42.2% was obtained for a 24-h hydrolysis. However, when nonpulping WO paper and 5% (to the amount of WO paper) enzyme were fed to the hydrolytic reaction, 120 g/L of RS with a hydrolytic yield of 40% was obtained for a 24-h hydrolysis. Therefore, the RS concentration from this hydrolysis process feeding WO paper using apparent viscosity as operating parameter may be of sufficient concentration to serve as a carbon source in microorganism culture or chemical feedstock.     

The effect of temperature,pH, and initial glucose concentration on the kinetics of ethanol production by Zymomonas mobilis in batch fermentation

Summary Zymomonas mobilis, strain ATCC 10988, was used to evaluate the effects of pH (5.0 to 8.0), temperature (30°C to 40°C), and initial glucose concentration (75 g/l to 150 g/l) on the kinetics of ethanol production from glucose using batch fermentation. Specific ethanol production rate was maximum and nearly constant over a pH range of 6.0 to 7.5. End-of-batch ethanol yield and specific growth rate were insensitive to pH in the range of 5.0 to 7.5. End-of-batch ethanol yield was maximum and nearly constant between 30°C and 37°C but decreased by 24% between 37°C and 40°C. All other kinetic parameters are greatest at 34°C. End-of-batch ethanol yield is maximum at an initial glucose concentration of 100 g/l. Specific growth rate reaches a maximum at 75 g/l, but specific ethanol production rate decreases throughout the range. The optimum initial glucose concentration of 100 g/l gives the highest ethanol yield at a specific ethanol production rate less than 10% below the maximum observed.     

Two-stage hydrolysis of invasive algal feedstock for ethanol fermentation

The overall goal of this work was to develop a saccharification method for the production of third generation biofuel (i.e. bioethanol) using feedstock of the invasive marine macroalga Gracilaria salicornia. Under optimum conditions (120 °C and 2% sulfuric acid for 30 min), dilute acid hydrolysis of the homogenized invasive plants yielded a low concentration of glucose (4.1 mM or 4.3 g glucose/kg fresh algal biomass). However, two-stage hydrolysis of the homogenates (combination of dilute acid hydrolysis with enzymatic hydrolysis) produced 13.8 g of glucose from one kilogram of fresh algal feedstock. Batch fermentation analysis produced 79.1 g EtOH from one kilogram of dried invasive algal feedstock using the ethanologenic strain Escherichia coli KO11. Furthermore, ethanol production kinetics indicated that the invasive algal feedstock contained different types of sugar, including C(5) -sugar. This study represents the first report on third generation biofuel production from invasive macroalgae, suggesting that there is great potential for the production of renewable energy using marine invasive biomass.     

A mechanistic model for enzymatic saccharification of cellulose using continuous distribution kinetics II: cooperative enzyme action, solution kinetics, and product inhibition

The projected cost for the enzymatic hydrolysis of cellulosic biomass continues to be a barrier for the commercial production of liquid transportation fuels from renewable feedstocks. Predictive models for the kinetics of the enzymatic reactions will enable an improved understanding of current limitations, such as the slow-down of the overall conversion rate, and may point the way for more efficient utilization of the enzymes in order to achieve higher conversion yields. A mechanistically based kinetic model for the enzymatic hydrolysis of cellulose was recently reported in Griggs et al. (2011) (Part I). In this article (Part II), the enzyme system is expanded to include solution-phase kinetics, particularly cellobiose-to-glucose conversion by β-glucosidase (βG), and novel adsorption and product inhibition schemes have been incorporated, based on current structural knowledge of the component enzymes. Model results show cases of cooperative and non-cooperative hydrolysis for an enzyme system consisting of EG(I) and CBH(I). The model is used to explore various potential rate-limiting phenomena, such as substrate accessibility, product inhibition, sterically hindered enzyme adsorption, and the molecular weight of the cellulose substrate.     

Modified kit procedure for the kinetic assay of ethanol

We have examined the utility of a commercial kit procedure for the determination of ethanol (EtOH), based upon its enzymatic oxidation and the concurrent production of NADH, monitored by photometry at 340 nm. We found that the equilibrium production of NADH is not stoichiometric with respect to initial ethanol concentration, and that with this procedure, the calibration curve for end-point assay of ethanol is linear only for very dilute solutions. Likewise, the kinetic assay of ethanol using the kit procedure is limited to very dilute samples (i.e., concentration in the reaction mixture of ?2.3 mg EtOH/liter). We describe a simple modification of the kit procedure which makes it amenable to the precise kinetic assay of up to 150 mg EtOH/liter in the reaction mixture. This increase in the dynamic range for kinetic assay of ethanol results form the use of hydrazine both as a trapping agent for the acetaldehyde reaction product and as a competitive inhibitor of alcohol dehydrogenase enzyme.