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Improving Fermentation Performance of Recombinant Zymomonas in Acetic Acid-Containing Media
Abstract
In the production of ethanol from lignocellulosic biomass, the hydrolysis of the acetylated pentosans in hemicellulose during pretreatment produces acetic acid in the prehydrolysate. The National Renewable Energy Laboratory (NREL) is currently investigating a simultaneous saccharification and cofermentation (SSCF) process that uses a proprietary metabolically engineered strain of Zymomonas mobilis that can coferment glucose and xylose. Acetic acid toxicity represents a major limitation to bioconversion, and cost-effective means of reducing the inhibitory effects of acetic acid represent an opportunity for significant increased productivity and reduced cost of producing fermentation fuel ethanol from biomass. In this study, the fermentation performance of recombinant Z. mobilis 39676:pZB4L, using a synthetic hardwood prehydrolysate containing 1% (w/v) yeast extract, 0.2% KH2PO4, 4% (w/v) xylose, and 0.8% (w/v) glucose, with varying amounts of acetic acid was examine. To minimize the concentration of the inhibitory undissociated form of acetic acid, the pH was controlled at 6.0. The final cell mass concentration decreased linearly with increasing level of acetic acid over the range 0-0.75% (w/v), with a 50% reduction at about 0.5% (w/v) acetic acid. The conversion efficiency was relatively unaffected, decreasing from 98 to 92%. In the absence of acetic acid, batch fermentations were complete at 24 h. In a batch fermentation with 0.75% (w/v) acetic acid, about two-thirds of the xylose was not metabolized after 48 h. In batch fermentations with 0.75% (w/v) acetic acid, increasing the initial glucose concentration did not have an enhancing effect on the rate of xylose fermentation. However, nearly complete xylose fermentation was achieved in 48h when the bioreactor was fed glucose. In the fed-batch system, the rate of glucose feeding (0.5 g/h) was designed to simulate the rate of cellulolytic digestion that had been observed in a modeled SSCF process with recombinant Zymomonas. In the absence of acetic acid, this rate of glucose feeding did not inhibit xylose utilization. It is concluded that the inhibitory effect of acetic acid on xylose utilization in the SSCF biomass-to-ethanol process will be partially ameliorated because of the simultaneous saccharification of the cellulose.
... Different conditions of liquid/solid ratio (8, 10 and 12 g g −1 ), sulfuric acid concentration (100, 120 and 140 mg g −1 dry matter) and reaction time (17,27 and 37 min) were employed in the hydrolysis process. Reactions were carried out at 120 • C in a 1.5 dm 3 stainless steel batch reactor (made in the Faculty of Chemical Engineering of Lorena), which was filled with 90 g of BSG (containing 90% dry matter) and the required amount of acid solution. ...
... Experimental design Experiments were carried out according to a 2 3 statistical experimental design to evaluate the effects of the variables liquid/solid ratio (8, 10 and 12 g g −1 ), sulfuric acid concentration (100, 120 and 140 mg g −1 dry matter) and reaction time (17,27 and 37 min) on BSG hemicellulose hydrolysis. Temperature was fixed at 120 • C. The experimental error was estimated at the centre point (10 g g −1 , 120 mg g −1 , 27 min), which was replicated fourfold. ...
... Acetic acid is a compound that, when present in the hydrolysates in concentrations higher than 3 g dm −3 , acts as a potent micro-organism inhibitor if the hydrolysate is employed as a fermentation medium. 16 According to Lawford and Rousseau,17 this acid can pass through the cellular membranes and decrease the intracellular pH, thus affecting the metabolism. In the present work the acetic acid concentration in the hydrolysates varied from 0.76 to 1.39 g dm −3 (Table 1), the maximum concentration being reached under the most drastic condition studied (8 g g −1 liquid/solid ratio, 140 mg H 2 SO 4 g −1 dry matter, 37 min reaction time, i.e. assay 7). ...
The hemicellulosic fraction of brewer's spent grain (BSG) was hydrolysed with diluted acid under different conditions of liquid/solid ratio (8–12 g g−1), sulfuric acid concentration (100–140 mg g−1 dry matter) and reaction time (17–37 min) in order to produce a liquor with a large amount of xylose and good fermentability to produce xylitol. Results showed that all the evaluated reaction conditions were able to hydrolyse xylan and arabinan with efficiencies higher than 85.8 and 95.7% respectively, and even under the mildest reaction condition a considerable amount (92.7%) of the hemicellulosic fraction could be extracted. The hydrolysates presented different fermentabilities when used as fermentation media for xylitol production by Candida guilliermondii yeast, owing to the differences in their composition. Based on statistical analysis, the best condition for BSG acid hydrolysis was the use of a liquid/solid ratio of 8 g g−1, 100 mg H2SO4 g−1 dry matter and a reaction time of 17 min. Under this condition a high extraction efficiency of hemicellulosic sugars (92.7%) and good fermentation results (YP/S = 0.70 g g−1 and QP = 0.45 g dm−3 h−1) were attained. Copyright
... a Mango peels, b seed kernel, c mango peels and de-oiled seed kernel (MP + SK) in 2:1 ratio, d mango peels and de-oiled seed kernel in 1:1 ratio, e mango peels and de-oiled seed kernel in 1:4 ratio. The methane yields are shown only during pseudo steady state (days [31][32][33][34][35] (p < 0.05) with 50%, 39.4%, and 32.1% increases, respectively, compared with the calculated corresponding methane yields (Fig. 3b). The results show that as the C/N ratio increases from 20:1 to 39:1, the normalized methane yield increases, consistent with previous studies [11,12,23]. ...
... With acid hydrolysis, hemicellulose releases sugars which are sometimes subsequently converted to inhibitory products such as furfural, hydroxymethyl furfural, and acetic acid, which can be inhibitory to microorganisms. In addition, the subsequent conversion of sugars to these inhibitors also causes sugar loss which leads to decrease in biogas yield [25,30,31]. The high concentration of lignin (49.4% content in the seed coat) may also be the reason for the low biogas yield, since lignin prevents microbial access to cellulose and sugars [32]. ...
Jatropha curcas seeds, as an abundant lignocellulosic biomass, offer a highly promising and ideal alternative for producing energy in the form of methane. Use of J. curcas seeds has the potential to significantly bolster the biofuel sector, fostering a more sustainable circular economy. In the current study, different fractions of pro- cessed J. curcas seeds were investigated for biogas production. J. curcas seed pressed cake, a by-product of biodiesel production, was subjected to methanolic extraction. The remaining solids, referred to as methanolic residues, yielded more biogas in batch experiments than pressed cake and residues from aqueous and n-hexane extractions. The compounds extracted with methanol inhibited hydrolysis and reduced biogas production by 35.5% compared to the same setup without extracts. In continuous reactors fed with methanolic residues, the highest biogas yield occurred at an organic loading rate (OLR) of 1 g VS L− 1 day− 1 and a hydraulic retention time (HRT) of 20 days. The relative abundance of acetogenic bacteria was higher in reactors fed with methanolic residues than in those fed with seed pressed cake, seed oil, and whole seed. Jatropha seed oil and whole seed did not inhibit methanogens. A higher relative abundance of methanogenic communities was observed in all reactors at HRT of 20 days compared to those at HRTs at 15 and 10 days. These findings can be used to increase biogas production during anaerobic digestion of J. curcas seed components and suggests a zero-waste biorefinery pro- duction route for value added compounds derived from the removal of biogas-inhibiting components.
... a Mango peels, b seed kernel, c mango peels and de-oiled seed kernel (MP + SK) in 2:1 ratio, d mango peels and de-oiled seed kernel in 1:1 ratio, e mango peels and de-oiled seed kernel in 1:4 ratio. The methane yields are shown only during pseudo steady state (days [31][32][33][34][35] (p < 0.05) with 50%, 39.4%, and 32.1% increases, respectively, compared with the calculated corresponding methane yields (Fig. 3b). The results show that as the C/N ratio increases from 20:1 to 39:1, the normalized methane yield increases, consistent with previous studies [11,12,23]. ...
... With acid hydrolysis, hemicellulose releases sugars which are sometimes subsequently converted to inhibitory products such as furfural, hydroxymethyl furfural, and acetic acid, which can be inhibitory to microorganisms. In addition, the subsequent conversion of sugars to these inhibitors also causes sugar loss which leads to decrease in biogas yield [25,30,31]. The high concentration of lignin (49.4% content in the seed coat) may also be the reason for the low biogas yield, since lignin prevents microbial access to cellulose and sugars [32]. ...
The combustion of fossil fuels is accompanied with a number of alarming problems such as fossil fuel depletion, increase in their prices, and emission of greenhouse gases. Thus, the need for the alternative renewable biofuels was increased to replace non-renewable fossil fuels. The sustainable use of non-edible feedstocks and waste for production of biofuels is a potential approach for reducing dependency on fossil fuels and mitigating environmental pollution. In the current study, the effects of carbon to nitrogen (C/N) ratios on methane yield during anaerobic co-digestion of Jatropha curcas de-oiled seed kernel and mango peels were evaluated in continuous reactors. The biogas potential and effects of acid pretreatment on J. curcas fruit were also evaluated during anaerobic batch digestion. The methane yield of co-digested mango peels and seed kernel (1:4 weight ratio based on volatile solids) was 61%, 50%, 36%, and 25% higher compared with the methane yields of mango peels, seed kernel, mango peels/seed kernel (2:1 w/w), and mango peels/seed kernel (1:1 w/w), respectively. The methane yields of the co-digestion of mango peels and seed kernel at 1:4, 1:1, and 2:1 ratios were 52%, 39%, and 32% of the theoretical yields, respectively, illustrating the importance of adjusting C/N ratio with the right amounts of co-substrate. The biogas yield of pretreated fruit coat was 7%, 22%, 34%, 50%, and 74% higher than that of the seed kernel, fruit coat (non-pretreated), de-oiled kernel plus seed coat (pretreated) (1.7:1, by weight), seed coat (pretreated), and seed coat (non-pretreated), respectively. Additionally, pretreatment of fruit coat and seed coat resulted in 22% and 47% higher biogas yields compared with their non-pretreated counterparts. This study revealed key substrate selection and pretreatment methods for increasing methane production from common seed oil production and agricultural wastes.
Graphical abstract
... Our results that the growth of ZM481 was decreased corresponding to increasing concentration of acetate were in consistent with previous studies. pH value has been shown to have an impact on inhibitory effect of acetic acid on the growth of ethanologen [29][30][31]. In the present experimental setting, the percent dissociation of acetic acid in the growth medium at pH 5 was 64%, resulting in 36% uncharged, undisscoated acetic acid that across cell membrane freely and compromise the biological balance. ...
... Several strains of Z. mobilis have been generated for tolerance to inhibitors by transposon mutagenesis [33][34][35][36]. Evolutionary adaptation has been employed to improve fermentation capability and resistance to inhibitory stress [29]. In this study, we obtained two mutants, ZMA-142 and ZMA-167 generated from ZM481, which exhibited improved acetate tolerance with favorable ethanol production at low pH. ...
Background
Acetic acid is a predominant by-product of lignocellulosic biofuel process, which inhibits microbial biocatalysts. Development of bacterial strains that are tolerant to acetic acid is challenging due to poor understanding of the underlying molecular mechanisms.
Results
In this study, we generated and characterized two acetic acid-tolerant strains of Zymomonas mobilis using N-methyl-N′-nitro-N-nitrosoguanidine (NTG)-acetate adaptive breeding. Two mutants, ZMA-142 and ZMA-167, were obtained, showing a significant growth rate at a concentration of 244 mM sodium acetate, while the growth of Z. mobilis ATCC 31823 were completely inhibited in presence of 195 mM sodium acetate. Our data showed that acetate-tolerance of ZMA-167 was attributed to a co-transcription of nhaA from ZMO0117, whereas the co-transcription was absent in ATCC 31823 and ZMA-142. Moreover, ZMA-142 and ZMA-167 exhibited a converstion rate (practical ethanol yield to theorical ethanol yield) of 90.16% and 86% at 195 mM acetate-pH 5 stress condition, respectively. We showed that acid adaptation of ZMA-142 and ZMA-167 to 146 mM acetate increased ZMA-142 and ZMA-167 resulted in an increase in ethanol yield by 32.21% and 21.16% under 195 mM acetate-pH 5 stress condition, respectively.
Conclusion
The results indicate the acetate-adaptive seed culture of acetate-tolerant strains, ZMA-142 and ZMA-167, could enhance the ethanol production during fermentation.
... The maximum concentrations varied between 2 g/l (experiment 1) and 4 g/l (experiment 9). Acetic acid can be an inhibitor of microbial growth when present from 4–10 g/l (Ferrari, Neirotti, Albornoz, & Saucedo, 1992; Lawford & Rousseau, 1998) because it enters the cell membrane and decreases intracellular pH, thus affecting the metabolism of the microorganism (Maiorella, Blanch, & Wilke, 1983; van Zyl, Prior, & Du Preez, 1991). However, it has been reported that acetic acid concentration of 9–10 g/l can stimulate the growth of microorganism (Palmqvist, Almeida, & Hahn- H€ a agerdal, 1999). ...
... Some hemicellulosic monomers like xylose are linked to acetyl groups (Ac), which can be hydrolysed to acetic acid in acid media. Consistently with this knowledge, our results showed that the acetic acid concentration increased until a constant value, according to the simple model (Garrote et al., 2001c; Lawford & Rousseau, 1998; Maloney et al., 1985): ...
Sugar cane bagasse was hydrolysed using nitric acid at variable concentration (2–6%), reaction time (0–300 min) and temperature (100–128 °C). The concentration of sugars released (xylose, glucose and arabinose) and degradation products (acetic acid and furfural) were determined and the kinetic parameters of mathematical models for predicting them in the hydrolysates were obtained. The influence of temperature was also studied using the Arrhenius equation. Applying the kinetic models obtained, the optimal conditions selected were: 122 °C, 6% HNO3 and 9.3 min. Using these conditions, 18.6 g xylose/l; 2.04 g arabinose/l; 2.87 g glucose/l; 0.9 g acetic acid/l and 1.32 g furfural/l were obtained. Comparison of these results with those obtained using sulphuric and hydrochloric acids demonstrated that the nitric acid was the most efficient catalyst for hydrolysis.
... Using concentrations of phosphoric acid of 2% or 6%, the trend was the same than using 4%, but the maximum values were close to 3 g acetic acid/l. Acetic acid can be an inhibitor of microbial growth from 4 to 10 g/l (Lawford & Rousseau, 1998) because it goes through the cellular membranes and decrease intracellular pH, which affect to the metabolism of the microorganism (Maiorella, Blanch, & Wilke, 1983; van Zyl, Prior, & Du Preez, 1991 ). In our study, the maximum acetic acid concentrations were in the lower limit of the toxic effect. ...
... The results showed that the acetic acid concentration increased until a constant value. The following simple model, according with the literature , was applied (Garrote, Domínguez, & Parajó , 2001b; Lawford & Rousseau, 1998; Maloney et al., 1985): ...
In the present work, samples of sugar cane bagasse were hydrolysed with phosphoric acid under mild conditions (H3PO4 2–6%, time 0–300 min and 122 °C) to study the feasibility of using the liquid phase as fermentation media. Solid yield, sugar concentrations and decomposition product concentrations were measured. The composition of hydrolysates, their purity and the ratio sugars/inhibitors were analyzed. Kinetic models were developed to describe the course of products of the acid hydrolysis. The course of xylose, glucose, arabinose, acetic acid and furfural were satisfactorily described by the models. The optimal conditions selected were 122 °C, 4% H3PO4 and 300 min. Using these conditions, 17.6 g of xylose/l; 2.6 g of arabinose/l; 3.0 g of glucose/l, 1.2 g furfural/l and 4.0 g acetic acid/l were obtained. The efficiency in these conditions was 4.46 g sugars/g inhibitors and the mass fraction of sugars in dissolved solids in liquid phase was superior to 55%.
... Acetic acid is also known as antimicrobial compound and the mechanism of inhibition is well-understood. At low pH, in the undissociated form, it can diffuse across the cell membrane, promoting the decrease of the cytoplasmatic cell activity and even causing cell death (Lawford et al. 1998; Mussatto et al. 2004). It has been reported that acetic acid inhibition degree depends not only on its concentration, but also on oxygen concentration and on pH of fermentation medium (Vanzyl et al. 1991). ...
... It was reported that HSSL content higher than 40% (v/v) was critical for bioethanol production (Table 3). Acetic acid has been appointed as the main inhibitor of P. stipitis and other microorganisms (Schneider 1996; Lawford et al. 1998). After the removal of acetic acid, ethanol fermentations were still unsuccessful, meaning that other compounds present had a toxic effect (Xavier et al. 2010). ...
... The concept of metabolic engineering has been used recently for an efficient fermentation of mixed sugars. Certain recombinant bacteria and yeasts, such as Z. mobilis [129,131,135], E. coli [136], and S. cerevisiae [110,128,137,138] etc., have shown promising results and they are being considered for commercial scaling up. ...
Algeria is facing two serious constraints, energy shortage and environment pollution. To overcome these two problems, renewable energies are the best sustainable alternatives. In this context, Algeria Government has established in 2011 a program for the development of renewable energies. The prospective results show a substantial contribution to supplying national energy demand as well as some significant environmental benefits, namely through major greenhouse gas savings. In fact, lignocellulosic sources as Algerian Alfa, olive pomace and cereal straw could provide up to 0.67 Mtoe which represents 4.37% of the energy consumption of transport sector in Algeria. In the same vein, introducing energy crops and dedicated cereal crop technologies allows Algeria to progressively increase its renewable energy supply. Up to 73.5 Mtoe and 57.9 Mtoe can be produced from the two cited resources respectively. That is more than the national energy consumption which reached 60.96 MToe in 2018.
Graphic abstract
... Thus, the internal pH drops and cell inhibition occurs, and may even cause the cell to die. Accordingly, before fermentation, AA should be removed from the hydrolysates through detoxification processes, as it is an inhibitory component, or its amount in the hydrolysate should be reduced [19,32]. Interestingly, in this study, AA was not detected when RB was used for acidic pretreatment. ...
The production of value-added products from renewable resources by fermentation is very attractive. In this study, the effect of pretreatment time on the chemical composition of acid-pretreated tea processing waste (TPW), spent tea waste (STW), barley husk (BH), and rye bran (RB) was examined. After the pretreatment processes, reducing sugar concentration (RSC), phenolics (PHs), glucose (Glc), xylose (Xyl), arabinose (Ara), acetic acid (AA), D-glucuronic acid (D-GA), 5-hydroxymethyl furfural (HMF), 2-furaldehyde (2-F) and catalytic efficiency (E, g sugars/g inhibitors) were determined. Results indicated that maximum E values were 3.79, 4.57, 21.28, and 33.46 g/g when the pretreatment times were 50, 6, 30, and 1 min for TPW, STW, BH, and RB, respectively. At these E values, the levels of RSC, PHs, Glc, Xyl, Ara, AA, D-GA, and HMF were 22.85, 1.60, 2.76, 6.98, 4.26, 1.28, 0.62, and 0.81 g/L for TPW; 17.67, 1.12, 1.55, 4.80, 4.05, 0.91, 0.98, and 0.25 g/L for STW; 64.30, 1.25, 30.14, 16.05, 3.57, 0.85, 0.00, and 0.23 g/L for BH; and 66.62, 0.79, 35.21, 13.08, 4.44, 0.00, 0.00, and 0.80 g/L for RB, respectively. Consequently, the hydrolysates obtained after suitable pretreatment times can be evaluated as substrate after removal of the inhibitors formed when necessary.
... Phenolic compounds may affect xylose consumption, cell growth and xylitol production by C. guilliermondii at concentrations between 0.5 and 2.0 g L −1 [42][43][44]. The toxic effect of acetic acid is associated with its ability to traverse the plasma membrane in its undissociated form, causing cytoplasm acidification [45]. However, for C. guilliermondii, it has already been reported that the presence of this acid at low concentrations (less than 3.0 g L −1 ) may improve xylose bioconversion into xylitol because it directs to the citric acid cycle resulting into ATP formation [46,47]. ...
Purpose
Barley straw from brewing process is an attractive and renewable raw material for the production of biofuel and useful chemicals, such as xylitol. It is necessary to determine the best conditions of biomass hydrolysis and fermentation for boosting the incorporation of this biomass in a biorefinery.
Methods
We optimized the conditions for acid hydrolysis of barley straw to obtain a hemicellulosic hydrolysate rich in xylose with low energy consumption. Moreover, the energy consumption was simulated per quantity of xylose extracted. In order to obtain a hydrolysate with the highest xylose extraction efficiency (99%), low inhibitors concentration and energy consumption (8.41 KW/Kg Xylose), we used 1.0% H2SO4 (w/v) at 120 °C, with 1:10 dry-weight/acid solution for 40 min. We also optimized the medium composition to improve xylitol production by Candida guilliermondii.
Results
The hemicellulosic hydrolysate was used as a fermentation medium and the best condition showing the highest xylitol volumetric productivity (0.69 g L⁻¹ h⁻¹) by C. guilliermondii was found to be 60 g L⁻¹ initial xylose supplemented with 1.5 g L⁻¹ (NH4)2SO4, 0.75 CaCl2 and 8.75 g L⁻¹ rice bran extract.
Conclusions
It can be concluded that barley straw can be used in biorefinery, wherein the hemicellulose fraction would be utilized to produce xylitol and the cellulosic fraction (more accessible to enzymatic hydrolysis after pre-treatment) would be used for the production of cellulosic ethanol.
Graphical Abstract
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... The bacterium Z. mobilis represents an empirical example of the long gestation period in the cellulosic ethanol innovation chain, with early commercial scale demonstration studies dating back to the 1990s and early 2000s by Prof. Lawford at the University of Toronto (Dept. of Biochemistry), and focusing on fermentation processes/industrial bioconversion technologies [133][134][135][136][137][138][139][140]. Their work [137] involving a partnership with Iogen Canada (an early pioneer of commercial cellulosic ethanol production and the "Iogen Process") evaluated fermentation performance characteristics of metabolically engineered Zymomonas mobilis strain AX101 developed at the US National Renewable Energy Laboratory. ...
Canada's cellulosic ethanol biorefinery concept is supported by federal and provincial government legislative ethanol mandates as well as enabling science and innovation policies for technology development to support the economic and sustainable production of cellulosic ethanol and co-products from Canada's abundant supply of lignocellulosic agricultural and forestry biomass. In particular, the development of pretreatment, hydrolysis, and fermentation technologies is regarded as a critical integrating step for the commercialization of cellulosic ethanol biorefinery business concepts. These critical steps are necessitated by the chemical structure of lignocellulosic biomass comprising carbohydrate polymers and lignin which constrains the ability of enzymes to convert these polymers into fermentable sugars without expensive and highly capital intensive pretreatment processes. This paper reviews science and innovation efforts by Canadian researchers in finding solutions to these constraints, in particular the development of hydrolysis and fermentation technologies. This paper also highlights the role of multi-institutional science and innovation collaborative approaches for advancing Canada's cellulosic ethanol biorefinery concept further downstream. While highlighting Canada's scientific progress, this review also outlines technology commercialization lags between basic research and full scale commercialization of a Canadian cellulosic ethanol biorefinery concept. Although this paper focuses on the near-term goal of cellulosic ethanol production, it nevertheless recognizes that ethanol is only the first step in the longer-term goal aimed at a full integrated bioconversion of lignocellulosic biomass into biofuels and a wide range of value-added biochemicals and biomaterials, consistent with the cellulosic biorefinery concept.
... Z. mobilis CP4/pZB5 fermented with an ethanol yield of 93% of the theoretical on alkali peracetic acid pretreated poplar wood [272]. The particular advantage with this pretreatment was the reduction of the amount of acetic acid during the pretreatment, as it has been demonstrated that the growth yield in Z. mobilis 39676:pZB4L fermentation is reduced by 50%, when the acetic acid concentration increased from 0 to 5 g L À1 [277]. Furthermore, the authors demonstrate that during conditions prevailing under SSF conditions, i.e., slow release of glucose, the detrimental effects of acetic acid on xylose utilization could be diminished. ...
Presently, bioethanol production receives tremendous attention,
as bioethanol used as fuel in the transportation
sector is sustainable and carbon dioxide neutral in contrast
to fossil fuels. Furthermore, being produced from lignocellulosic
material, it can represent a domestic energy
source that is renewable. Bioethanol production is getting
close to commercialization and presently large resources
are put into solving the bottlenecks in the process. The use
of ethanol is forecastedtogrowrapidly inthe coming years,
and the first plant producing fuel ethanol fromlignocellulosic
material is expected to be running in the near future.
This chapter describes and evaluates the process for
fuel ethanol production. The rawmaterial, hydrolysis, and
fermentation are described in detail, and different possibilities
to perform these process steps in various process
designs are discussed. The bottlenecks for the process and
possible improvements in the future are assessed.
... Besides, during fermentation of hemicellulosic hydrolysate, a decrease in pH due to acetic acid formation was observed, but pH values remained higher than the pKa (4.75). Such behavior was favorable, since according to Lawford and Rousseau (1998), the acetic acid toxicity is related to the ability of undissociated (protonated) weak acid to act as a membrane protonophore, causing acidification of the cytoplasm. ...
This paper evaluates the fermentative potential of Kluyveromyces marxianus grown in sugarcane bagasse cellulosic and hemicellulosic hydrolysates obtained by acid hydrolysis. Ethanol was obtained from a single glucose fermentation product, whereas xylose assimilation resulted in xylitol as the main product and ethanol as a by-product derived from the metabolism of this pentose. Fermentation performed in a simulated hydrolysate medium with a glucose concentration similar to that of the hydrolysate resulted in ethanol productivity (Qp = 0.86 g L−1 h−1) that was tenfold higher than the one observed in the cellulosic hydrolysate. However, the use of hemicellulosic hydrolysate favored xylose assimilation in comparison with simulated medium with xylose and glucose concentrations similar to those found in this hydrolysate, without toxic compounds such as acetic acid and phenols. Under this condition, xylitol yield was 53.8 % higher in relation to simulated medium. Thus, the total removal of toxic compounds from the hydrolysate is not necessary to obtain bioproducts from lignocellulosic hydrolysates.
... Undissociated acetic acid (pKa 4.75) freely passes through cellular membranes, where it can then reach a new equilibrium with dissociated acetic acid at the intracellular pH. The net result is both uncoupling of the transmembrane pH gradient and an accumulation of acetate anion in the cytoplasm [9,10]. ...
The liberation of acetate from hemicellulose negatively impacts fermentations of cellulosic biomass, limiting the concentrations of substrate that can be effectively processed. Solvent-producing bacteria have the capacity to convert acetate to the less toxic product acetone, but to the best of our knowledge, this trait has not been transferred to an organism that produces ethanol at high yield.
We have engineered a five-step metabolic pathway to convert acetic acid to acetone in the thermophilic anaerobe Thermoanaerobacterium saccharolyticum. The first steps of the pathway, a reversible conversion of acetate to acetyl-CoA, are catalyzed by the native T. saccharolyticum enzymes acetate kinase and phosphotransacetylase. ack and pta normally divert 30% of catabolic carbon flux to acetic acid; however, their re-introduction in evolved ethanologen strains resulted in virtually no acetic acid production. Conversion between acetic acid and acetyl-CoA remained active, as evidenced by rapid 13C label transfer from exogenous acetate to ethanol. Genomic re-sequencing of six independently evolved ethanologen strains showed convergent mutations in the hfs hydrogenase gene cluster, which when transferred to wildtype T. saccharolyticum conferred a low acid production phenotype. Thus, the mutated hfs genes effectively separate acetic acid production and consumption from central metabolism, despite their intersecting at the common intermediate acetyl-CoA. To drive acetic acid conversion to a less inhibitory product, the enzymes thiolase, acetoacetate:acetate CoA-transferase, and acetoacetate decarboxylase were assembled in T. saccharolyticum with genes from thermophilic donor organisms that do not natively produce acetone. The resultant strain converted acetic acid to acetone and ethanol while maintaining a metabolic yield of 0.50 g ethanol per gram carbohydrate.
Conversion of acetic acid to acetone results in improved ethanol productivity and titer and is an attractive low-cost solution to acetic acid inhibition.
... It has been shown that utilization of a membrane process such as ultrafiltration, nanofiltration, or reverse osmosis can also obtain lignosulphonates from spent liquor (Bhattacharya 2005). Several reports have been published concerning simultaneous saccharification and fermentation processes (Gauss et al. 1976;Hamelinck et al. 2005;Wu et al. 1998), and simultaneous conversion of D-glucose and D-xylose into ethanol has been achieved by utilizing recombinant saccharomyces yeast (Lawford and Rousseau 1998). In addition, the detoxification of dilute-acid hydrolyzate by application of Ca(OH) 2 (overliming) and its effect on fermentation inhibitors removal and on fermentability of the detoxified hydrolyzates was also investigated (Millati 2002;Purwadi 2000Purwadi , 2004. ...
Precipitation of lignosulphonates from the liquor for sulfite pretreatment to overcome recalcitrance of lignocellulose (SPORL) by addition of Ca(OH) 2 was investigated in this work. The experiment was conducted in a reaction temperature range of 20 to 75°C for 90 minutes with Ca(OH) 2 charge varying from 20 to 90 g/L and a range of liquid enrichment ratio of 1 to 5. It was found that increased Ca(OH)2 charge, duration time, reaction temperature, and liquor concentration each tended to improve lignosulphonates precipitation, but tended to hurt fermentable sugars conservation. Application of Ca(OH) 2 20 g/L to SPORL liquid without enrichment at 30oC for 90 minutes could be an optimal condition. Under this condition, 25.95% of the lignosulphonates was precipitated for further utilization, while calculated amounts of 106.46% of glucose and 60.25% of xylose were conserved for further fermentation.
... However, Z. mobilis is not capable of fermenting mannose, galactose or pentoses (Olsson and Hahn-Hägerdal, 1993), and these sugars are present in a lignocellulosic hydrolysate. Furthermore, Z. mobilis appears to be sensitive to acetic acid present in the lignocellulosic hydrolysate (Lawford and Rousseau, 1998;Kim et al., 2000) and no large-scale process employing Z. mobilis has yet been established. ...
Akademisk avhandling för avläggande av teknologie doktorsexamen vid tekniska fakulteten vid Lunds universitet. Avhandlingen kommer att försvaras på svenska vid en offentlig disputation på Kemicentrum, Sölvegatan 39, Lund, hörsal C, fredagen den 14 juni 2002, kl 10.15.
... Their inhibitory effects on microbial xylitol production and their inhibitory mechanisms have been reported (Iwaki et al. 2013;Zhang et al. 2012). Non-ionized acetic acid passes through the cellular membranes and decreases the intracellular pH thus affecting the metabolism (Lawford and Rousseau 1998). Allen et al. (2010) pointed out that furfural causes oxidative stress and deteriorates the activities of different dehydrogenases in yeasts. ...
Xylose reductase (XR) is an oxidoreductase having potential applications in the production of various specialty products, mainly xylitol. It is important to screen for compounds that can decrease XR activity and consequently can decrease xylitol production. We have identified the byproducts in the hemicellulosic hydrolysate that inhibit XR from Candida tropicalis and measured their effects. XR inhibitory activities of byproducts, glucose, acetic acid, arabinose, lignin-degradation products (LDPs), furfural and hydroxymethylfurfural (HMF), were evaluated by measuring the MIC and IC50 values. XR activity was 11.2 U/ml. Acetic acid, LDPs, furfural and HMF significantly inhibited XR with IC50 values of 11, 6.4, 2.3 and 0.4 g/l, respectively. This is the first report on the inhibitory activities of several byproducts for XR.
... Different strategies were developed to improve the tolerance of acetic acid and xylose utlization. For example, Lawford and Rousseau et al. developed a process via addition of extra glucose in acetic acid-containing media for improving fermentation performance of recombinant Zymomonas [82]. Recombinant plasmid pZB5 was also transferred into an acetic acid-tolerant strain (ZM4/Ac R ) [47], and a mutant recombinant Z. mobilis ZM4/Ac R (pZB5) strain with increased acetate resistance was obtained [48]. ...
Biosynthesis of liquid fuels and biomass-based building block chemicals from microorganisms have been regarded as a competitive alternative route to traditional. Zymomonas mobilis possesses a number of desirable characteristics for its special Entner-Doudoroff pathway, which makes it an ideal platform for both metabolic engineering and commercial-scale production of desirable bio-products as the same as Escherichia coli and Saccharomyces cerevisiae based on consideration of future biomass biorefinery. Z. mobilis has been studied extensively on both fundamental and applied level, which will provide a basis for industrial biotechnology in the future. Furthermore, metabolic engineering of Z. mobilis for enhancing bio-ethanol production from biomass resources has been significantly promoted by different methods (i.e. mutagenesis, adaptive laboratory evolution, specific gene knock-out, and metabolic engineering). In addition, the feasibility of representative metabolites, i.e. sorbitol, bionic acid, levan, succinic acid, isobutanol, and isobutanol produced by Z. mobilis and the strategies for strain improvements are also discussed or highlighted in this paper. Moreover, this review will present some guidelines for future developments in the bio-based chemical production using Z. mobilis as a novel industrial platform for future biofineries.
... However, dilute acid pretreatment and enzymatic hydrolysis of rice straw release acidic inhibitors into hydrolysate. The sensitivity of the organism to acetic acid depends on the culture condi- tions [7]. Formic acid at the concentration less than 1 mM reduced fermentation efficiency of Clostridium acetobutylicum [8]. ...
Growth inhibition kinetics of a novel non-acetone forming butanol producer, Clostridium sporogenes BE01, was studied under varying concentrations of acetic and formic acids in rice straw hydrolysate medium. Both the organic acids were considered as inhibitors as they could inhibit the growth of the bacterium, and the inhibition constants were determined to be 1.6 and 0.76 g/L, respectively, for acetic acid and formic acid. Amberlite resins—XAD 4, XAD 7, XAD 16, and an anion exchange resin—Seralite 400 were tested for the efficient removal of these acidic inhibitors along with minimal adsorption of sugars and essential minerals present in the hydrolysate. Seralite 400 was an efficient adsorbent of acids, with minimal affinity towards minerals and sugars. Butanol production was evaluated to emphasize the effect of minerals loss and acids removal by the resins during detoxification.
... The profile of acetic acid was found to be similar to that of arabinose, establishing a maximum concentration of 1.42 g/L at 10:1 LSR. Acetic acid is an inhibitor for the microbial growth because it enters through the cellular membranes and decreases the intracellular pH, which affects the cellular metabolism [57,58]. However, it has been reported that acetic acid concentration of 9-10 g/L stimulates the growth of the microorganism [59]. ...
This study presents the sono-assisted pretreatment and enzymatic saccharification of sugarcane bagasse (SCB) for the production of bioethanol. The effect of sono-assisted alkali (NaOH) pretreatment on the removal of hemicellulose and lignin from SCB was studied and the results showed 80.8% of hemicellulose and 90.6% of lignin removal. Sono-assisted enzymatic saccharification was performed with Cellulomonas flavigena (MTCC 7450) and the yield was found to be affected by liquid-to-solid ratio (LSR), cell mass and pH. The optimum reaction time, LSR, cell mass and pH were found to be 360 min, 15:1, 15 g/L and 6.0 respectively. At optimum conditions, the maximum glucose yield obtained was 91.28% of the theoretical yield and the maximum amount of glucose obtained was 38.4 g/L. The enhancement in performance may be correlated with the swelling of cellulose and accelerated enzymatic saccharification due to the application of ultrasound. The hydrolyzate obtained was fermented using Zymomonas mobilis (MTCC 89) and about 91.22% of the theoretical ethanol yield was observed in 36 h of fermentation.
... Therefore, only a single fermentation step is required to process hydrolyzed and solid fractions of the pretreated lignocellulose (McMillan 1997). Lawford and Rousseau (1998) used a metabolically engineered strain of Zymomonas mobilis that can coferment glucose and xylose, developed in the National Renewable Energy Laboratory (NREL) for ethanol production by SSCF from a synthetic hardwood prehydrolyzate and glucose. McMillan et al. (1999) used an adapted variant of the NREL xylose-fermenting Z. mobilis for ethanol production from dilute-acidpretreated yellow poplar by SSCF. ...
This article reviews developments in the technology for ethanol produc- tion from lignocellulosic materials by "enzymatic" processes. Several methods of pretreatment of lignocelluloses are discussed, where the crystalline structure of lignocelluloses is opened up, making them more accessible to the cellulase enzymes. The characteristics of these enzymes and important factors in enzymatic hydrolysis of the cellulose and hemicellulose to cellobiose, glucose, and other sugars are discussed. Different strategies are then described for enzymatic hydrolysis and fermentation, including separate enzymatic hydrolysis and fermentation (SHF), simultaneous saccharification and fermentation (SSF), non-isothermal simultaneous saccharification and fermentation (NSSF), simultaneous saccharification and co-fermentation (SSCF), and consolidated bioprocessing (CBP). Furthermore, the by-products in ethanol from lignocellulosic materials, wastewater treatment, commercial status, and energy production and integration are reviewed.
... Wild-type Z. mobilis strains can only utilize a limited range of carbon sources, namely, glucose, fructose, and sucrose. To overcome this limitation, recombinant strains have been engineered to ferment hexose and pentose sugars such as xylose, arabinose, and other substrates with high yields (Deanda et al. 1996;Zhang et al. 1995), but a low tolerance to acetic acid and a decreased tolerance to ethanol have been reported (Dien et al. 2003;Lawford and Rousseau 1998;Lawford et al. 2001;Ranatunga et al. 1997). In addition, recent achievements to improve transformation efficiency by modifying the DNA restriction-modification systems (Kerr et al. 2010), cellulase expression and secretion (Linger et al. 2010), as well as the genome-scale modeling and in silico analysis (Widiastuti et al. 2010), will aid future metabolic engineering and synthetic biology endeavors greatly. ...
The development and use of robust ethanologenic microorganisms resistant to industrially relevant pretreatment inhibitors
will be a critical component in the successful generation of biofuel on the industrial scale. Recent progress to understand
the genetic basis of pretreatment inhibitor tolerance using genomics and systems biology tools for metabolic engineering for
the model ethanologenic bacterium Zymomonas mobilis is reviewed in this chapter. The importance of accurate genome annotations and the integration of systems biology data for
annotation improvement are highlighted, and case studies that describe the identification and characterization of the Z. mobilis nhaA, hfq, and himA inhibitor tolerance related gene targets are presented.
... Once in the cell interior, where pH is 7.4, this acid dissociates and accumulates in the cytoplasm, discharging protons. As a consequence, the internal pH drops, inhibiting cell activity and even causing death [7]. Xylitol production was also higher in buffered media than in nonbuffered media (Fig. 1b). ...
The inhibitory action of acetic acid, ferulic acid, and syringaldehyde on metabolism of Candida guilliermondii yeast during xylose to xylitol bioconversion was evaluated. Assays were performed in buffered and nonbuffered semidefined medium containing xylose as main sugar (80.0 g/l), supplemented or not with acetic acid (0.8-2.6 g/l), ferulic acid (0.2-0.6 g/l), and/or syringaldehyde (0.3-0.8 g/l), according to a 2(3) full factorial design. Since only individual effects of the variables were observed, assays were performed in a next step in semidefined medium containing different concentrations of each toxic compound individually, for better understanding of their maximum concentration that can be present in the fermentation medium without affecting yeast metabolism. It was concluded that acetic acid, ferulic acid, and syringaldehyde are compounds that may affect Candida guilliermondii metabolism (mainly cell growth) during bioconversion of xylose to xylitol. Such results are of interest and reveal that complete removal of toxic compounds from the fermentation medium is not necessary to obtain efficient conversion of xylose to xylitol by Candida guilliermondii. Fermentation in buffered medium was also considered as an alternative to overcome the inhibition caused by these toxic compounds, mainly by acetic acid.
... Z. mobilis are Gram-negative facultative anaerobic bacteria with a number of desirable industrial characteristics, such as high-specific productivity and ethanol yield, unique anaerobic use of the Entner-Doudoroff pathway that results in low cell mass formation, high ethanol tolerance (12%), pH 3.5-7.5 range for ethanol production and has a generally regarded as safe (GRAS) status456789. Z. mobilis strains have been engineered to ferment pentose sugars such as xylose, arabinose and other substrates with high yields, but a low tolerance to acetic acid and decreased ethanol tolerance have been reported in recombinant strains [4,101112. Z. mobilis mutant strains tolerant to a pretreatment inhibitor such as acetate have been generated by chemical mutagenesis with N-methyl N'-nitro N-nitrosoguanidine and selection in continuous culture with a progressively increasing concentration of sodium acetate in the medium feed [13]. AcR is capable of efficient ethanol production in the presence of 20 g/L NaAc, while the parent ZM4 is inhibited significantly above 12 g/L NaAc under the same conditions [13]. ...
Zymomonas mobilis produces near theoretical yields of ethanol and recombinant strains are candidate industrial microorganisms. To date, few studies have examined its responses to various stresses at the gene level. Hfq is a conserved bacterial member of the Sm-like family of RNA-binding proteins, coordinating a broad array of responses including multiple stress responses. In a previous study, we observed Z. mobilis ZM4 gene ZMO0347 showed higher expression under anaerobic, stationary phase compared to that of aerobic, stationary conditions.
We generated a Z. mobilis hfq insertion mutant AcRIM0347 in an acetate tolerant strain (AcR) background and investigated its role in model lignocellulosic pretreatment inhibitors including acetate, vanillin, furfural and hydroxymethylfurfural (HMF). Saccharomyces cerevisiae Lsm protein (Hfq homologue) mutants and Lsm protein overexpression strains were also assayed for their inhibitor phenotypes. Our results indicated that all the pretreatment inhibitors tested in this study had a detrimental effect on both Z. mobilis and S. cerevisiae, and vanillin had the most inhibitory effect followed by furfural and then HMF for both Z. mobilis and S. cerevisiae. AcRIM0347 was more sensitive than the parental strain to the inhibitors and had an increased lag phase duration and/or slower growth depending upon the conditions. The hfq mutation in AcRIM0347 was complemented partially by trans-acting hfq gene expression. We also assayed growth phenotypes for S. cerevisiae Lsm protein mutant and overexpression phenotypes. Lsm1, 6, and 7 mutants showed reduced tolerance to acetate and other pretreatment inhibitors. S. cerevisiae Lsm protein overexpression strains showed increased acetate and HMF resistance as compared to the wild-type, while the overexpression strains showed greater inhibition under vanillin stress conditions.
We have shown the utility of the pKNOCK suicide plasmid for mutant construction in Z. mobilis, and constructed a Gateway compatible expression plasmid for use in Z. mobilis for the first time. We have also used genetics to show Z. mobilis Hfq and S. cerevisiae Lsm proteins play important roles in resisting multiple, important industrially relevant inhibitors. The conserved nature of this global regulator offers the potential to apply insights from these fundamental studies for further industrial strain development.
... HMFs and furfurals are formed during degradation of hexose and pentose sugars, respectively ( Table 3). Acetic acid, a known inhibitor to yeast growth and metabolism, is generally formed due to break down of the acetyl bonds in hemicellulose, when subjected to high pressure and temperature under acidic conditions (20). Most of the pentose sugars are concentrated in the hemicellulosic fraction of the cell wall (4). ...
Orange peels were evaluated as a fermentation feedstock, and process conditions for enhanced ethanol production were determined. Primary hydrolysis of orange peel powder (OPP) was carried out at acid concentrations from 0 to 1.0% (w/v) at 121 degrees C and 15 psi for 15 min. High-performance liquid chromatography analysis of sugars and inhibitory compounds showed a higher production of hydroxymethyfurfural and acetic acid and a decrease in sugar concentration when the acid level was beyond 0.5% (w/v). Secondary hydrolysis of pretreated biomass obtained from primary hydrolysis was carried out at 0.5% (w/v) acid. Response surface methodology using three factors and a two-level central composite design was employed to optimize the effect of pH, temperature, and fermentation time on ethanol production from OPP hydrolysate at the shake flask level. On the basis of results obtained from the optimization experiment and numerical optimization software, a validation study was carried out in a 2 L batch fermenter at pH 5.4 and a temperature of 34 degrees C for 15 h. The hydrolysate obtained from primary and secondary hydrolysis processes was fermented separately employing parameters optimized through RSM. Ethanol yields of 0.25 g/g on a biomass basis (YP/X) and 0.46 g/g on a substrate-consumed basis (YP/S) and a promising volumetric ethanol productivity of 3.37 g/L/h were attained using this process at the fermenter level, which shows promise for further scale-up studies.
... Therefore, only a single fermentation step is required to process hydrolyzed and solid fractions of the pretreated lignocellulose (McMillan 1997). Lawford and Rousseau (1998) used a metabolically engineered strain of Zymomonas mobilis that can coferment glucose and xylose, developed in the National Renewable Energy Laboratory (NREL) for ethanol production by SSCF from a synthetic hardwood prehydrolyzate and glucose. McMillan et al. (1999) used an adapted variant of the NREL xylose-fermenting Z. mobilis for ethanol production from dilute-acidpretreated yellow poplar by SSCF. ...
This article reviews developments in the technology for ethanol produc-tion from lignocellulosic materials by “enzymatic” processes. Several methods of pretreatment of lignocelluloses are discussed, where the crystalline structure of lignocelluloses is opened up, making them more accessible to the cellulase enzymes. The characteristics of these enzymes and important factors in enzymatic hydrolysis of the cellulose and hemicellulose to cellobiose, glucose, and other sugars are discussed. Different strategies are then described for enzymatic hydrolysis and fermentation, including separate enzymatic hydrolysis and fermentation (SHF), simultaneous saccharification and fermentation (SSF), non-isothermal simultaneous saccharification and fermentation (NSSF), simultaneous saccharification and co-fermentation (SSCF), and consolidated bioprocessing (CBP). Furthermore, the by-products in ethanol from lignocellulosic materials, wastewater treatment, commercial status, and energy production and integration are reviewed.
... . Therefore, two bioreactors and two biomass production setup is required in SSF. In SSCF process, it is suggested to ferment both hexoses and pentoses in a single bioreactor with a single microorganism. Therefore, only a single fermentation step is required to process hydrolyzed and solid fractions of the pretreated lignocellulose (McMillan 1997). Lawford and Rousseau (1998) used a metabolically engineered strain of Zymomonas mobilis that can coferment glucose and xylose, developed in the National Renewable Energy Laboratory (NREL) for ethanol production by SSCF from a synthetic hardwood prehydrolyzate and glucose. McMillan et al. (1999) used an adapted variant of the NREL xylose-fermenting Z. mobilis for e ...
Bioethanol is nowadays one of the main actors in the fuel market. It is currently produced from sugars and starchy materials, but lignocelluloses can be expected to be major feedstocks for ethanol production in the future. Two processes are being developed in parallel for conversion of lignocelluloses to ethanol, “acid-based” and “enzyme-based” processes. The current article is dedicated to review of progress in the “acid-based-hydrolysis” process. This process was used industrially in the 1940s, during wartime, but was not economically competitive afterward. However, intensive research and development on its technology during the last three decades, in addition to the expanding ethanol market, may revive the process in large scale once again. In this paper the ethanol market, the composition of lignocellulosic materials, concentrated- and dilute-acid pretreatment and hydrolysis, plug-flow, percolation, counter-current and shrinking-bed hydrolysis reactors, fermentation of hexoses and pentoses, effects of fermentation inhibitors, downstream processing, wastewater treatment, analytical methods used, and the current commercial status of the acid-based ethanol processes are reviewed.
... The most frequently used microorganism for fermenting ethanol in industrial processes is S. cerevisiae, which has proved to be very robust and well suited to the fermentation of lignocellulosic hydrolysates (Olsson and Hahn-Hägerdal 1993). Zymomonas mobilis can ferment glucose to ethanol with higher yields, due to the production of less biomass, but is less robust (Rogers et al. 1979; Lawford and Rousseau 1998 ). Since lignocellulosic hydrolysates contain pentoses, which are not readily fermented by these microorganisms, several attempts to genetically engineer S. cerevisiae (Walfridsson 1996; Tonn et al. 1997), Z. mobilis (Lawford et al. 1997) and the bacteria Escherichia coli (Ingram et al. 1997) have been performed. ...
Ethanol produced from various lignocellulosic materials such as wood, agricultural and forest residues has the potential to be a valuable substitute for, or complement to, gasoline. One of the major resources in the Northern hemisphere is softwood. This paper reviews the current status of the technology for ethanol production from softwood, with focus on hemicellulose and cellulose hydrolysis, which is the major problem in the overall process. Other issues of importance, e.g. overall process configurations and process economics are also considered.
... According to McMillan (1994), these extractives produce less inhibition of microbial growth than lignin derivatives or acetic acid. Lawford and Rousseau (1998) reported that when the pH of the medium is low, acetic acid (pK a ¼ 4:75) appears in the undissociated form, is liposoluble, and diffuses across the plasma membrane. Once in the cell interior, where pH is 7.4, this acid dissociates and accumulates in the cytoplasm, discharging protons. ...
Acid hydrolysis of lignocellulosic materials produces several inhibitory compounds, such as sugar and lignin degradation products, compounds derived from the lignocellulosic structure, and heavy metal ions. Their toxicity is a major factor limiting bioconversion processes that utilize hydrolyzates. The identification of these compounds and the choice of the best hydrolyzate detoxification method are important for improving the efficiency of the fermentative processes. A variety of biological, physical, and chemical techniques have been proposed to reduce the concentration of these compounds in lignocellulose hydrolyzates. However, the efficiency of any detoxification method depends both on the hydrolyzate composition, which differs according to the raw material used, and on the hydrolysis conditions employed. This review describes the effects of the inhibitory compounds on fermentation yield and productivity, as well as various detoxification methods for treating the hydrolyzates.
A potential method by which society's reliance on fossil fuels can be lessened is via the large-scale utilization of biofuels derived from the secondary cell walls of woody plants; however, there remain a number of technical challenges to the large-scale production of biofuels. Many of these challenges emerge from the underlying complexity of the secondary cell wall. The challenges associated with lignin have been well explored elsewhere, but the dicot cell wall components of hemicellulose and pectin also present a number of difficulties. Here, we provide an overview of the research wherein pectin and xylan biosynthesis has been altered, along with investigations on the function of irregular xylem 8 (IRX8) and glycosyltransferase 8D (GT8D), genes putatively involved in xylan and pectin synthesis. Additionally, we provide an analysis of the evidence in support of two hypotheses regarding GT8D and conclude that while there is evidence to lend credence to these hypotheses, there are still questions that require further research and examination.
Biomassas lignocelulósicas, como resíduos agrícolas e florestais, podem servir de matriz carbônica para a fermentação e obtenção do etanol de segunda geração (2G). Porém, os açúcares fermentescíveis não estão prontamente disponíveis, precisando ser liberados por operações adicionais de pré-tratamento e hidrólise. Paralelamente, substâncias químicas com potencial efeito inibitório ao metabolismo microbiano também são formadas e podem comprometer a produtividade e o rendimento global do processo. Esse estudo teve como objetivo avaliar a influência de vanilina, ácido acético, ácido vanílico e ácido 4-hidroxibenzoico na fermentação de glicose pela levedura Saccharomyces cerevisiae JP1. O cultivo ocorreu em meio sintético composto por nutrientes e 40 g.L-1 de glicose, inibidor em diferentes concentrações e 3% (v/v) de inóculo obtido em meio YPD líquido. O experimento foi conduzido a 30 °C e 150 rpm por 22 h. Amostras foram coletadas periodicamente para monitoramento da multiplicação celular e consumo de substrato, bem como quantificação de etanol no final do procedimento. Os resultados indicaram que vanilina (0,1; 0,5; 1,0 e 1,5 g.L-1) e ácido vanílico (0,1; 0,5 e 1,0 g.L-1) inibiram o crescimento da levedura em uma extensão diretamente proporcional à concentração inicial destes no meio fermentativo. O ácido 4-hidroxibenzoico apresentou, em média, 25% de toxicidade em relação ao crescimento celular, independente das quantidades estudadas (0,1; 0,5 e 1,0 g.L-1). Total inibição do crescimento celular, consumo de glicose e produção de etanol foi observada no meio com ácido acético em concentrações iguais ou superiores a 3,5 g.L-1.
Um modelo cinético semimecanístico multirreacional foi desenvolvido para a modelagem da reação de hidrólise enzimática de material lignocelulósico. Além das reações usadas para representar a hidrólise da celulose a glicose, o modelo incorporou uma reação para considerar a hidrólise da hemicelulose a xilose. A adsorção das enzimas no material sólido foi considerada na modelagem e foi descrita pela isoterma de Langmuir. A concentração das enzimas celulase e celobiase foi mantida numa razão constante e foram representadas por uma única variável. A inibição causada pela celobiose, glicose e xilose nestas enzimas foi considerada, bem como a variação da reatividade do substrato durante a reação. Os parâmetros do modelo foram estimados pelo método de Marquardt por meio de ajuste aos dados experimentais de hidrólise enzimática de bagaço de cana-de-açúcar (48,9% de celulose, 22,5% de hemicelulose e 16,87% de lignina) obtidos a 50 °C, pH 4,8 (tampão de citrato) e agitação de 150 rpm na condição de referência (concentração de enzimas de 15 FPU/g-celulose e concentração de sólidos de 5% m/m, base seca). A mistura enzimática utilizada na obtenção dos dados de hidrólise foi formada por Celluclast 1,5L e Novozym 188 na proporção
1 FPU : 2 CBU. A biomassa foi pré-tratada por peróxido de hidrogênio alcalino 11% v/v (15,65% m/m), pH 11,5 (corrigido com NaOH), a 25 °C durante 1 h. O modelo foi validado com relação à concentração enzimática (5, 30 e 60 FPU/g-celulose), concentração de sólidos (3%, 6%, 9% e 12%) e inibição pelos açúcares (celobiose 20 mg/mL, glicose 20 e 40 mg/mL e xilose 20 mg/mL). O modelo reproduziu os resultados com razoável precisão, mas apresentou desvios para a maior parte das condições avaliadas e a principal causa possível para os desvios observados foi a subestimação da adsorção improdutiva das enzimas na lignina.
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A semimecanistic multi-reaction kinetic model was developed for the modeling of the enzymatic hydrolysis of lignocellulosic material. In addition to the reactions used to represent the enzymatic hydrolysis of cellulose to glucose, the model incorporated a reaction to consider the hydrolysis of hemicellulose to xylose. The adsorption of enzymes onto the solid material was considered and was described by the Langmuir isotherm. The concentration of the enzymes cellulase and celobiase were kept at a constant ratio and described by a single variable. The inhibition caused by cellobiose, glucose and xylose on these enzymes was accounted, and also the variation of substrate reactivity during reaction. The model parameters were estimated using the Marquardt method by fitting the experimental data of enzymatic hydrolysis of sugarcane bagasse (48.9% cellulose, 22.5% hemicellulose, and 16.87% lignin) obtained at 50 °C, pH 4,8 (citrate buffer), and 150 rpm under standard conditions (15 FPU/g-cellulose enzyme concentration and 5% w/w, dry basis, solid loading). The enzymatic mixture used in the acquisition of the experimental data was formed by Celluclast 1.5L and Novozym 188 in the ratio 1 FPU : 2 CBU. The biomass was pretreated by 11% v/v (15.65% w/w) alkaline hydrogen peroxide, pH 11.5 (adjusted with NaOH), at 25 °C during 1 h. The model was validated with respect to enzyme concentration (5, 30, and 60 FPU/g-cellulose), solids loading (3%, 6%, 9%, and 12%), and sugar inhibition (20 mg/mL cellobiose, 20 and 40 mg/mL glucose, and 20 mg/mL xylose). The model predicted the results with reasonable accuracy but showed deviations for most of the conditions assessed and the main possible cause for the observed deviations was the underestimation of the nonproductive enzyme adsorption on lignin.
Sugarcane bagasse hydrolysate (initial acetic acid concentration = 3.5g/L), was used as a fermentation medium for conversion of xylose into xylitol by the yeast Candida guilliermondii FTI 20037. Acetic acid (2.0g/L) was added to the medium at different times of fermentation, with the aim of evaluating its effects on the bioconversion process. The addition of acetic acid to the medium after 12h of fermentation resulted in the strongest inhibition of the yeast metabolism. In this case, the xylose consumption and cell growth were, respectively, 23.22 and 11.24% lower than when acid was added to the medium at the beginning of fermentation. As a consequence of the inhibitory effect, lower values of the xylitol yield (0.39g/g) and productivity (0.22g/L.h) were observed, corresponding to a reduction of 36 and 48%, respectively, in relation to the values obtained with the addition of acetic acid after other fermentation times. The results obtained allowed to conclude that, under the experimental conditions employed in this work, the inhibitory effect of acetic acid on the xylose-xylitol bioconversion depends on the fermentation time when this acid was added, and not only on its concentration in the medium.
The application of systems biology tools holds promise for rational industrial microbial strain development. Here, we characterize a Zymomonas mobilis mutant (AcR) demonstrating sodium acetate tolerance that has potential importance in biofuel development. The genome changes associated with AcR are determined using microarray comparative genome sequencing (CGS) and 454-pyrosequencing. Sanger sequencing analysis is employed to validate genomic differences and to investigate CGS and 454-pyrosequencing limitations. Transcriptomics, genetic data and growth studies indicate that over-expression of the sodium-proton antiporter gene nhaA confers the elevated AcR sodium acetate tolerance phenotype. nhaA over-expression mostly confers enhanced sodium (Na(+)) tolerance and not acetate (Ac(-)) tolerance, unless both ions are present in sufficient quantities. NaAc is more inhibitory than potassium and ammonium acetate for Z. mobilis and the combination of elevated Na(+) and Ac(-) ions exerts a synergistic inhibitory effect for strain ZM4. A structural model for the NhaA sodium-proton antiporter is constructed to provide mechanistic insights. We demonstrate that Saccharomyces cerevisiae sodium-proton antiporter genes also contribute to sodium acetate, potassium acetate, and ammonium acetate tolerances. The present combination of classical and systems biology tools is a paradigm for accelerated industrial strain improvement and combines benefits of few a priori assumptions with detailed, rapid, mechanistic studies.
Fermentation-based bioprocesses rely extensively on strain improvement for commercialization. Whole-cell biocatalysts are commonly limited by low tolerance of extreme process conditions such as temperature, pH, and solute concentration. Rational approaches to improving such complex phenotypes lack good models and are especially difficult to implement without genetic tools. Here we describe the use of genome shuffling to improve the acid tolerance of a poorly characterized industrial strain of Lactobacillus. We used classical strain-improvement methods to generate populations with subtle improvements in pH tolerance, and then shuffled these populations by recursive pool-wise protoplast fusion. We identified new shuffled lactobacilli that grow at substantially lower pH than does the wild-type strain on both liquid and solid media. In addition, we identified shuffled strains that produced threefold more lactic acid than the wild type at pH 4.0. Genome shuffling seems broadly useful for the rapid evolution of tolerance and other complex phenotypes in industrial microorganisms.
Sugar cane bagasse, a renewable and cheap bioresource, was hydrolyzed at 100 degrees C using phosphoric acid at different concentrations (2, 4, or 6%) and reaction times (0-300 min) to obtain fermentable sugar solutions, which have a high concentration of sugars (carbon source for microorganism growth) and a low concentration of growth inhibitors (acetic acid and furfural). Xylose, glucose, arabinose, acetic acid, and furfural were determined following the hydrolysis. Kinetic parameters of mathematical models for predicting these compounds in the hydrolysates were obtained. Derived parameters such as efficiency of hydrolysis or purity of hydrolysates were considered to select as optimal conditions 6% phosphoric acid at 100 degrees C for 300 min. Using these conditions, 21.4 g of sugars L(-)(1) and <4 g of inhibitors L(-)(1) were obtained from the hydrolysis with a water/solid ratio of 8 g of water g(-)(1) of sugar cane bagasse on a dry basis.
Efficient utilization of the pentosan fraction of hemicellulose from lignocellulosic feedstocks offers an opportunity to increase
the yield and to reduce the cost of producing fuel ethanol. The patented, genetically engineered, ethanologenEscherichia coli B (pLOI297) exhibits high-performance characteristics with respect to both yield and productivity in xylose-rich lab media.
In addition to producing monomer sugar residues, thermochemical processing of biomass is known to produce substances that
are inhibitory to both yeast and bacteria. During prehydrolysis, acetic acid is formed as a consequence of the deacetylation
of the acetylated pentosan. Our investigations have shown that the acetic acid content of hemicellulose hydrolysates from
a variety of biomass/waste materials was in the range 2–10 g/L (33–166 mM). Increasing the reducing sugar concentration by evaporation did not alter the acetic acid concentration. Acetic acid toxicity
is pH dependent. By virtue of its ability to traverse the cell membrane freely, the undissociated (protonated) form of acetic
acid (HAc) acts as a membrane protonophore and causes its inhibitory effect by bringing about the acidification of the cytoplasm.
With recombinantE. coli B, the pH range for optimal growth with glucose and xylose was 6.4–6.8. With glucose, the pH optimum for ethanol yield and
volumetric productivity was 6.5, and for xylose it was 6.0 and 6.5, respectively. However, the decrease in growth and fermentation
efficiency at pH 7 is not significant. At pH 7, only 0.56% of acetic acid is undissociated, and at 10 g/L, neither the ethanol
yield nor the maximum volumetric productivity, with glucose or xylose, is significantly decreased. The “uncoupling” effect
of HAc is more pronounced with xylose and the potency of HAc is potentiated in a minimal salts medium. Controlling the pH
at 7 provided an effective means of circumventing acetic acid toxicity without significant loss in fermentation performance
of the recombinant biocatalyst.
Acetic acid acts to promote uncoupling withZymomonas. At pH 5, 36% of acetic acid is in the uncharged and undissociated form (HAc), which is able to permeate the plasma membrane.
The transmembrane ΔpH drives the accumulation of acetic acid, which results in the acidification of the cytoplasm. The consequential
increase in maintenance metabolism represents a diversion of energy that would otherwise be available for growth. At pH 5,
the growth ofZ. mobilis (ATCC 29191) was 50% inhibited with 8.3 g/L acetic acid (50 mM HAc) and completely inhibited by 11 g/L. Addition of 6 g/L acetic acid caused the glucose-to-ethanol conversion efficiency
to decrease from 98 to 90% of theoretical maximum. The growth yield coefficient for glucose was 50% decreased by 2.3 g/L acetic
acid (13.5 mM HAc) from 0.036 to 0.018 g cell/g glucose. However, the specific (ethanol) productivity of batch cultures was enhanced by
<5 g/L acetic acid (<30 mM HAc). For continuous cultures, the acetic acid sensitivity depends on the growth rate (dilution rate), but an increase in
specific productivity can be achieved at proportionately lower concentrations of acetic acid. At a growth rate of 0.112/h,
the addition of 1.7 g/L acetic acid to the 5% glucose feed resulted in an increase in specific productivity from 2.68 to 5.87
g ethanol/g cell/h. The uncoupling effect of acetic acid could be beneficial in terms of improving the productivity in closed,
continuous fermentations, such as cell recycle or immobilized cell reactors.
Efficient utilization of the pentosan fraction of hemicellulose from lignocellulosic feedstocks offers an opportunity to increase
the yield and to reduce the cost of producing fuel ethanol. During prehydrolysis (acid hydrolysis or autohydrolysis of hemicellulose),
acetic acid is formed as a consequence of the deacetylation of the acetylated moiety of hemicellulose. Recombinant Escherichia
coli B (ATCC 11303), carrying the plasmid pLO1297 with pyruvate decarboxylase and alcohol dehydrogenase II genes from Zymomonas
mobilis (CP4), converts xylose to ethanol with a product yield that approaches theoretical maximum. Although other pentose-utilizing
microorganisms are inhibited by acetic acid, the recombinant E. coli displays a high tolerance for acetic acid. In xylose
fermentations with a synthetic medium (Luria broth), where the pH was controlled at 7, neither yield nor productivity was
affected by the addition of 10.7 g/L acetic acid. Nutrient-supplemented, hardwood (aspen) hemicellulose hydrolysate (40.7
g/L xylose) was completely fermented to ethanol (16.3 g/L) in 98 h. When the acetic acid concentration was reduced from 5.6
to 0.8 g/L, the fermentation time decreased to 58 h. Overliming, with Ca(OH)2 to pH 10, followed by neutralization to pH 7 with sulfuric acid and removal of insolubles, resulted in a twofold increase
in volumetric productivity. The maximum productivity was 0.93 g/L/h. The xylose-to-ethanol conversion efficiency and productivity
in Ca(OH)2-treated hardwood prehydrolysate, fortified with only mineral salts, were 94% and 0.26 g/L/h, respectively. The recombinant
E. coli exhibits a xylose-to-ethanol conversion efficiency that is superior to that of other pentose-utilizing yeasts currently
being investigated for the production of fuel ethanol from lignocellulosic materials.
pH-dependent energetic uncoupling ofZymomonas by acetic acid occurs by virtue of the permeability of the plasma membrane to the undissociated form of acetic acid (HAc)
and the acidification of the cytoplasm resulting from the uptake of HAc and the consequential diversion of energy away from
biosynthetic processes (growth) in order to maintain constant intracellular pH. Energetic uncoupling is manifested by an increase
in specific productivity. The degree of uncoupling caused by HAc depends on a rather complex interaction between several different
variables including membrane permeability, the transmembrane δpH and the concentraiton of undissociated form of acetic acid
in the medium. Within the pH range of 5.0–5.5, maximal energic uncoupling is produced by 30–38 mM HAc. For practical purposes,
in terms of the concentration of acetic acid, this corresponds to about 5 and 15 g/L at pH 5.0 and 5.5, respectively. Assuming
any upper limit concentration of acetic acid in hydrolysate fermentation media of about 12 g/L, inhibition of Z.mobilis in terms of both ethanol yield and productivity is avoided by controlling the pH in the range of 5.5–6.0.
The economic impact of conversion of xylose to ethanol for a wood-to-ethanol plant was examined, and the maximum potential
reduction in the price of ethanol from utilization of xylose is estimated to be $0.42 per gallon from a base case price of
$0.42 per gallon from a base case price of
1.65. The sensitivity of the price of ethanol to the yield, ethanol concentration and rate of the xylose fermentation was
also examined, and the price of ethanol is most affected by changes in yield and ethanol concentration, with rate of lesser
importance. Current performances of various xylose conversion biocatalysts were analyzed, andC. shehatae andP. stipitis appear to be the best yeasts.
The five-carbon sugar D-xylose is a major component of hemicellulose and accounts for roughly one-third of the carbohydrate content of many lignocellulosic materials. The efficient fermentation of xylose-rich hemicellulose hydrolyzates (prehydrolyzates) represents an opportunity to improve significantly the economics of large-scale fuel ethanol production from lignocellulosic feedstocks. The National Renewable Energy Laboratory (NREL) is currently investigating a simultaneous saccharification and cofermentation (SSCF) process for ethanol production from biomass that uses a dilute-acid pretreatment and a metabolically engineered strain of Zymomonas mobilis that can coferment glucose and xylose. The objective of this study was to establish optimal conditions for cost-effective seed production that are compatible with the SSCF process design.
Summary Escherichia coli B (ATCC 11303) carrying the PET operon on plasmid pLOI 297 converted hemicellulose hydrolysate to ethanol at an efficiency of 94% theoretical maximum, which is 15% better than the highest efficiency reported for pentose utilizing yeasts in a comparable system. Aspen prehydrolysate (APH), that had been produced by theBio-Hol Process using a Wenger extruder with SO2 as catalyst, was used as feedstock. The fermentation medium contained predominantly xylose (35g/L) with acetic acid present at about 6g/L. With the pH controlled at 7.0, this concentration of acetic acid was not inhibitory for growth or xylose fermentation. When the APH was fortified with nutrients (tryptone and yeast extract), the recombinant (inoculated at 0.5 g dry wt/L) converted 100% of the xylose to ethanol with a volumetric productivity of 0.29 g/L/hr. Overliming the APH with Ca(OH)2, followed by neutralization to pH 7 with sulphuric acid and removal of the insolubles, resulted in a 2-fold increase in productivity. The max. productivity was 0.76 g/L/hr. The productivity in Ca(OH)2-treated APH, fortified with only mineral salts, was 0.26 g/L/hr.
Although lignocellulosic biomass and wastes are targeted as an attractive alternative fermentation feedstock for the production of fuel ethanol, cellulosic ethanol is not yet an industrial reality because of problems in bioconversion technologies relating both to depolymerization and fermentation. In the production of wood pulp by the sulfite process, about 50% of the wood (hemicellulose and lignin) is dissolved to produce cellulose pulp, and the pulp mill effluent (“spent sulfite liquor” SSL) represents the only lignocellulosic hydrolysate available today in large quantities (about 90 billion liters annually worldwide). Although softwoods have been the traditional feedstock for pulping operations, hardwood pulping is becoming more popular, and the pentose sugars in hardwood SSL (principally xylose) are not fermented by the yeasts currently being used in the production of ethanol from softwood SSL.
This study assessed the fermentation performance characteristics of a patented (US Pat. 5,000,000), recombinantEscherichia coli B (ATCC 11303 pLOI297) in anaerobic batch fermentations of both nutrient-supplemented soft and hardwood SSL (30–35 g/L total reducing sugars). The pH was controlled at 7.0 to maximize tolerance to acetic acid. In contrast to the high-performance characteristics exhibited in synthetic media, formulated to mimic the composition of softwood and hardwood SSL (yield approaching theoretical maximum), performance in SSL media was variable with conversion efficiencies in the range of 67–84% for hardwood SSL and 53–76% for softwood SSL. Overlimiting treatment of HSSL, using Ca(OH)2, improved overall volumetric productivity two- to sevenfold to a max of 0.42 g/L/h at an initial cell loading of 0.5 g dry wt/L. A conversion efficiency of 92% (6.1 g/L ethanol) was achieved using diluted Ca(OH)2-treated hardwood SSL. The variable behavior of this particular genetic construct is viewed as a major detractant regarding its candidacy as a biocatalyst for SSL fermentations.
The ethanol-producing bacterium Zymomonas mobilis was metabolically engineered to broaden its range of fermentable substrates to include the pentose sugar xylose. Two operons
encoding xylose assimilation and pentose phosphate pathway enzymes were constructed and transformed into Z. mobilis in order to generate a strain that grew on xylose and efficiently fermented it to ethanol. Thus, anaerobic fermentation of
a pentose sugar to ethanol was achieved through a combination of the pentose phosphate and Entner-Doudoroff pathways. Furthermore,
this strain efficiently fermented both glucose and xylose, which is essential for economical conversion of lignocellulosic
biomass to ethanol.
Summary In a mineral salts medium containing yeast extract, NH4Cl and glucose (50g/L), the pH range producing the fastest growth ofZ. mobilis was 5.5–6.5 with an apparent optimum at 6.5. At constant growth rate of 0.15hr-1, the specific rates of glucose utilization (qs) and ethanol production (qp) were relatively unaffected by pH over the range 7.0–5.5 but increased sharply as the pH was further decreased below 5.5 to 4.0. Under these conditions the ethanol yield was unaffected by pH over the range 4.0–6.5 but decreased markedly at pH of 7.
Hard and softwoods were pretreated by mild acid hydrolysis and their pore size distribution determined. Regardless of the substrate, the initial rate of hydrolysis using cellulase from Trichoderma reesei is linearly correlated with the pore volume of the substrate accessible to a nominal diameter of 51 Å representative of the size of the cellulase. In contrast, crystallinity index has no relationship to the rate of hydrolysis.
An economical biomass-to-ethanol process depends on the efficient conversion of both its cellulose and hemicellulose components. On a dry weight basis, the typical feedstock contains approx 25-50% (w/w) glucose, 10-30% (w/w) xylose, 15-30% (w/w) lignin, and 1-5% (w/w) of other minor pentose and hexose sugars. Although many microorganisms can ferment the glucose component in cellulose to ethanol, conversion of pentose sugars in the hemicellulose fraction, particularly xylose, has been hindered by the lack of a suitable biocatalyst. Despite the development of recombinant strains with improved fermentation performance, increased ethanol yields and concentrations and shorter fermentation times are key targets that have yet to be achieved from lignocellulosic hydrolyzates. Our objective is to develop biocatalysts for the rapid and efficient conversion of xylose by engineering key metabolic pathways in selected organisms. To identify promising biocatalysts for these efforts, we have surveyed several industrial microorganisms according to several primary traits considered to be essential, as well as a number of secondary traits considered to be desirable, in a commercial biomass-to-ethanol process.
Ethanol produced from lignocellulose is considered as a largescale transportation fuel in the United States. Five key issues
are identified and considered in relation to the status of current and foreseeable technology. These are: conversion and production
energy balances, suitability of ethanol as a transportation fuel, air quality impacts, raw material supply, and cost. Energy
balances and fuel characteristics appear to be consistent with large-scale transportation fuel use of ethanol produced from
lignocellulose. Local and global air-quality benefits are expected to accompany use of lignocellulose ethanol. Raw material
availability is examined for wastes and for trees and grasses grown as energy crops. Ethanol production levels appear unlikely
to be limited by raw material availability as long as economic and other factors are sufficiently favorable to justify allocation
of land for this use. Projected ethanol production costs based on current directions of research would allow neat ethanol
to become competitive with gasoline by the year 2000 according to current oil price predictions. Biological process steps
have the largest contribution to overall costs, are among the least developed aspects of the technology, and appear to have
the greatest potential for improvement. Research priorities are discussed.
Large-scale production of ethanol or other transportation fuels by biological conversion of lignocellulosic biomass will eventually
require integration with large-scale production of biomass substrates. The most promising candidates for efficient production
of biomass in the US are short rotation hardwoods and herbaceous crops. The following samples of short rotation hardwoods
were provided by the Biomass Production Program managed for the Department of Energy Biofuels and Municipal Waste Technology
Division by the Oak Ridge National Laboratory: Poplar hybrid NE388(Populus, maximowiczii xP. trichocarpa), Poplar hybrid Nil (Populus. trichocarpa xP. deltoides), and Sweetgum(Liquidambar styraciflua). Samples of two short rotation grasses (weeping lovegrass and switchgrass) and one herbaceous legume (sericea lespedeza) were
also obtained from the same source. Milled and debarked hardwoods and herbaceous samples were subjected to prehydrolysis with
dilute sulfuric acid at 140 and 160 °C for reaction times ranging from 5 to 60 min. The dilute sulfuric acid hydrolyzed all
hemicelluloses at longer reaction times (30–60 min at 140 °C and 5–10 min at 160 °C), but solubilized very small amounts (<
15%) of lignin and cellulose. Cellulose in all three pretreated hardwoods became highly digestible by cellulase enzyme fromTrichoderma reesei. The two grasses also responded to dilute acid pretreatment very well.
The Solar Energy Research Institute (SERI) recently completed a detailed engineering and economic analysis of the simultaneous
saccharification and fermentation (SSF) based wood-to-ethanol process. The reference-case design was based on a plant capacity
of 1920 dry t/d and a wood cost of 42/dry t. For this case, the preliminary estimate of the production cost of the ethanol product is about42/dry t. For this case, the preliminary estimate of the production cost of the ethanol
product is about 1.22/gal. The combined effects of optimizing SSF enzyme loading, increasing plant capacity to 10,000 dry
t/d, and reducing wood cost to 34/dry t are to reduce the preliminary estimate of the production cost to about34/dry t are to reduce the preliminary estimate of the production cost to about 0.95/gal.
Other technological improvements may further reduce the production cost. Certain technical assumptions, inherent in the analysis,
are being investigated further.
Aspen wood substrates with varying degrees of deacetylation, xylan, and lignin removal have been prepared and submitted to
enzymatic hydrolysis with a cellulase/hemicellulase preparation for an extended constant period of hydrolysis. Controlled
deacetylation has been achieved by treating wood with various alkali metal hydroxide solutions, at various alkali/wood ratios.
It has been found that samples with the same extent of deacetylation produce the same sugar yields upon enzymatic hydrolysis.
Increased degree of deacetylation increases the yield of sugars obtained from enzymatic hydrolysis, all other compositional
parameters held constant. The acetyl group removal is proportional to the stoichiometric relation between added base and wood
acetyl content, i.e., the same number of milliequivalents of base/weight of wood remove the same extent of acetyl groups,
regardless of the concentration of the base solution. No cation effects are found among Li, Na, and K alkali hydroxide solutions,
suggesting that swelling is not as important a parameter as is the removal of the acetyl groups from the xylan backbone in
determining the extent of hydrolyzability of the resulting sample.
Most antimicrobial food additives are lipophilic acids which prevent growth by inhibiting the transport of substrate molecules into cells. The inhibition of transport was also observed in membrane vesicles, even when they were derived from organisms for which growth and transport are not affected by the larger inhibitors.
A minimal medium which allowed the sustained, rapid growth of Zymomonas mobilis and the isolation of a range of auxotrophic mutants was developed.
This chapter discusses wood hemicelluloses. The three principal portions of a tree are the wood or xylem, the inner bark or phloem, and the outer bark. Hemicelluloses from terrestrial plants are built up of a relatively limited number of sugar residues, the principal of which are D-xylose, D-mannose, D-glucose, D-galactose, L-arabinose, 4-O-methyl-D-glucuronic acid, D-galacturonic acid, D-glucuronic acid, and to a lesser extent L-rhamnose, L-fucose, and various O-methylated neutral sugars. The wood of the angiosperms of the temperate zones is chemically distinguished from that of the gymnosperms by its lower content of lignin and glucomannan and higher content of xylan. Partial hydrolysis gave a mixture of oligosaccharides, which is resolved into a neutral and an acidic portion, each of which is further resolved by a combination of carbon chromatography and paper chromatography.
The properties of the d-glucose transport system of Zymomonas mobilis were determined by measuring the uptake of nonmetabolizable analogs (2-deoxy-d-glucose and d-xylose) by wild-type cells and the uptake of d-glucose itself by a mutant lacking glucokinase. d-Glucose was transported by a constitutive, stereospecific, carrier-mediated facilitated diffusion system, whereby its intracellular concentration quickly reached a plateau close to but not above the external concentration. d-Xylose was transported by the d-glucose system, as evidenced by inhibition of its uptake by d-glucose. d-Fructose was not an efficient competitive inhibitor of d-glucose uptake, indicating that it has a low affinity for the d-glucose transport system. The apparent K(m) of d-glucose transport was in the range of 5 to 15 mM, with a V(max) of 200 to 300 nmol min mg of protein. The K(m) of Z. mobilis glucokinase (0.25 to 0.4 mM) was 1 order of magnitude lower than the K(m) for d-glucose transport, although the V(max) values for transport and phosphorylation were similar. Thus, glucose transport cannot be expected to be rate limiting at concentrations of extracellular glucose normally used in fermentation processes, which greatly exceed the K(m) for the transport system. The low-affinity, high-velocity, nonconcentrative system for d-glucose transport described here is consistent with the natural occurrence of Z. mobilis in high-sugar environments and with the capacity of Z. mobilis for rapid conversion of glucose to metabolic products with low energetic yield.
Removal of hemicellulose by acid pretreatment in a flow reactor followed by enzymatic hydrolysis of the neutralized slurry has resulted in glucose yields as high as 95% for mixed hardwood. For white pine, however, the maximum glucose yield is 65%. Although pine has a higher extractives content, removal of the extractives prior to enzymatic hydrolysis does not increases the glucose yield. Pore size measurements reveal that the increase in pore volume, in the size range of the cellulase molecule, following pretreatment for pine is only about one-half the value obtained with mixed hardwood. This suggests that pore volume is an important determinant of substrate-enzyme reactivity.