Consolidated bioprocessing strategy for ethanol production from Jerusalem artichoke tubers by Kluyveromyces marxianus under high gravity conditions.

AIMS: Developing an innovative process for ethanol fermentation from Jerusalem artichoke tubers under very high gravity (VHG) conditions. METHODS AND RESULTS: A consolidated bioprocessing (CBP) strategy that integrated inulinase production, saccharification of inulin contained in Jerusalem artichoke tubers and ethanol production from sugars released from inulin by the enzyme was developed with the inulinase-producing yeast Kluyveromyces marxianus Y179 and fed-batch operation. The impact of inoculum age, aeration, the supplementation of pectinase and nutrients on the ethanol fermentation performance of the CBP system was studied. Although inulinase activities increased with the extension of the seed incubation time, its contribution to ethanol production was negligible because vigorously growing yeast cells harvested earlier carried out ethanol fermentation more efficiently. Thus, the overnight incubation that has been practised in ethanol production from starch-based feedstocks is recommended. Aeration facilitated the fermentation process, but compromised ethanol yield because of the negative Crabtree effect of the species, and increases the risk of contamination under industrial conditions. Therefore, nonaeration conditions are preferred for the CBP system. Pectinase supplementation reduced viscosity of the fermentation broth and improved ethanol production performance, particularly under high gravity conditions, but the enzyme cost should be carefully balanced. Medium optimization was performed, and ethanol concentration as high as 94·2 g l(-1) was achieved when 0·15 g l(-1) K(2) HPO(4) was supplemented, which presents a significant progress in ethanol production from Jerusalem artichoke tubers. CONCLUSIONS: A CBP system using K. marxianus is suitable for efficient ethanol production from Jerusalem artichoke tubers under VHG conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: Jerusalem artichoke tubers are an alternative to grain-based feedstocks for ethanol production. The high ethanol concentration achieved using K. marxianus with the CBP system not only saves energy consumption for ethanol distillation, but also significantly reduces the amount of waste distillage discharged from the distillation system.

Effect of the size of yeast flocs and zinc supplementation on continuous ethanol fermentation performance and metabolic flux distribution under very high concentration conditions.

Taking continuous ethanol fermentation with the self-flocculating yeast SPSC01 under very high concentration conditions as an example, the fermentation performance of the yeast flocs and their metabolic flux distribution were investigated by controlling their average sizes at 100, 200, and 300 microm using the focused beam reflectance online measurement system. In addition, the impact of zinc supplementation was evaluated for the yeast flocs at the size of 300 microm grown in presence or absence of 0.05 g L(-1) zinc sulfate. Among the yeast flocs with different sizes, the group with the average size of 300 microm exhibited highest ethanol production (110.0 g L(-1)) and glucose uptake rate (286.69 C mmol L(-1) h(-1)), which are in accordance with the increased flux from pyruvate to ethanol and decreased flux to glycerol. And in the meantime, zinc supplementation further increased ethanol production and cell viability comparing with the control. Zinc addition enhanced the carbon fluxes to the biosynthesis of ergosterol (28.6%) and trehalose (43.3%), whereas the fluxes towards glycerol, protein biosynthesis, and tricarboxylic acid cycle significantly decreased by 37.7%, 19.5%, and 27.8%, respectively. This work presents the first report on the regulation of metabolic flux by the size of yeast flocs and zinc supplementation, which provides the potential for developing engineering strategy to optimize the fermentation system.

Yeast flocculation: New story in fuel ethanol production.

Yeast flocculation has been used in the brewing industry to facilitate biomass recovery for a long time, and thus its mechanism of yeast flocculation has been intensively studied. However, the application of flocculating yeast in ethanol production garnered attention mainly in the 1980s and 1990s. In this article, updated research progress in the molecular mechanism of yeast flocculation and the impact of environmental conditions on yeast flocculation are reviewed. Construction of flocculating yeast strains by genetic approach and utilization of yeast flocculation for ethanol production from various feedstocks were presented. The concept of self-immobilized yeast cells through their flocculation is revisited through a case study of continuous ethanol fermentation with the flocculating yeast SPSC01, and their technical and economic advantages are highlighted by comparing with yeast cells immobilized with supporting materials and regular free yeast cells as well. Taking the flocculating yeast SPSC01 as an example, the ethanol tolerance of the flocculating yeast was also discussed.

An innovative consecutive batch fermentation process for very high gravity ethanol fermentation with self-flocculating yeast.

An innovative consecutive batch fermentation process was developed for very high gravity (VHG) ethanol fermentation with the self-flocculating yeast under high biomass concentration conditions. On the one hand, the high biomass concentration significantly shortened the time required to complete the VHG fermentation and the duration of yeast cells suffering from strong ethanol inhibition, preventing them from losing viability and making them suitable for being repeatedly used in the process. On the other hand, the separation of yeast cells from the fermentation broth by sedimentation instead of centrifugation, making the process economically more competitive. The VHG medium composed of 255 g L(-1) glucose and 6.75 g L(-1) each of yeast extract and peptone was fed into the fermentation system for nine consecutive batch fermentations, which were completed within 8-14 h with an average ethanol concentration of 15% (v/v) and ethanol yield of 0.464, 90.8% of its theoretical value of 0.511. The average ethanol productivity that was calculated with the inclusion of the downstream time for the yeast flocs to settle from the fermentation broth and the supernatant to be removed from the fermentation system was 8.2 g L(-1) h(-1), much higher than those previously reported for VHG ethanol fermentation and regular ethanol fermentation with ethanol concentration around 12% (v/v) as well.

Metabolic flux and cell cycle analysis indicating new mechanism underlying process oscillation in continuous ethanol fermentation with Saccharomyces cerevisiae under VHG conditions.

Process oscillation characterized by long oscillation period and large oscillation amplitude was observed in continuous ethanol fermentation with Saccharomyces cerevisiae under very high gravity conditions. Metabolic flux analysis was applied to the fermentation system, and the results indicated that carbon flux distributions at the metabolic notes oscillated, correspondingly, and the root reason for the process oscillation was the intracellular metabolism of yeast cells. Cell cycle analysis with the flow cytometry showed that no cell-cycle-dependent synchronization of the daughter and mother cells occurred within the duration of the oscillation, and thus different mechanism existed compared with the oscillation observed in the continuous culture of Saccharomyces cerevisiae and triggered by the synchronization of the daughter and mother cells under specific conditions. Furthermore, the overall metabolic activity of the yeast cells was examined, which was found not exactly out of phase but lag behind ethanol concentration that accumulated within the fermentation system and its inhibition on the yeast cells as well, which supported the mechanistic speculation for the process oscillation: the lag response of yeast cells to ethanol inhibition.

Mechanisms of yeast stress tolerance and its manipulation for efficient fuel ethanol production.

Yeast strains of Saccharomyces cerevisiae have been extensively studied in recent years for fuel ethanol production, in which yeast cells are exposed to various stresses such as high temperature, ethanol inhibition, and osmotic pressure from product and substrate sugars as well as the inhibitory substances released from the pretreatment of lignocellulosic biomass. An in-depth understanding of the mechanism of yeast stress tolerance contributes to breeding more robust strains for ethanol production, especially under very high gravity conditions. Taking advantage of the "omics" technology, the stress response and defense mechanism of yeast cells during ethanol fermentation were further explored, and the newly emerged tools such as genome shuffling and global transcription machinery engineering have been applied to breed stress resistant yeast strains for ethanol production. In this review, the latest development of stress tolerance mechanisms was focused, and improvement of yeast stress tolerance by both random and rational tools was presented.

Parameter oscillation attenuation and mechanism exploration for continuous VHG ethanol fermentation.

A bioreactor system composed of a stirred tank and three tubular bioreactors in series was established, and continuous ethanol fermentation was carried out using a general Saccharomyces cerevisiae strain and a very high gravity medium containing 280 g L(-1) glucose, supplemented with 5 g L(-1) yeast extract and 3 g L(-1) peptone. Sustainable oscillations of glucose, ethanol, and biomass were observed when the tank was operated at the dilution rate of 0.027 h(-1), which significantly affected ethanol fermentation performance of the system. After the tubular bioreactors were packed with 1/2'' Intalox ceramic saddles, the oscillations were attenuated and quasi-steady states were achieved. Residence time distributions were studied for the packed bioreactors by the step input response technique using xylose as a tracer, which was added into the medium at a concentration of 20 g L(-1), indicating that the backmixing alleviation assumed for the packed tubular bioreactors could not be established, and its contribution to the oscillation attenuation could not be verified. Furthermore, the role of the packing's yeast cell immobilization in the oscillation attenuation was investigated by packing the tubular bioreactors with packings with significant difference in yeast cell immobilization effects, and the experimental results revealed that only the Intalox ceramic saddles and wood chips with moderate yeast cell immobilization effects could attenuate the oscillations, and correspondingly, improved the ethanol fermentation performance of the system, while the porous polyurethane particles with good yeast cell immobilization effect could not. And the viability analysis for the immobilized yeast cells illustrated that the extremely lower yeast cell viability within the tubular bioreactors packed with the porous polyurethane particles could be the reason for their inefficiency, while the yeast cells loosely immobilized onto the surfaces of the Intalox ceramic saddles and wood chips could be renewed during the fermentation, guaranteeing their viability and making them more efficient in attenuating the oscillations. The packing Raschig rings without yeast cell immobilization effect did not affect the oscillatory behavior of the tubular bioreactors, further supporting the role of the yeast cell immobilization in the oscillation attenuation.

Impact of zinc supplementation on the improvement of ethanol tolerance and yield of self-flocculating yeast in continuous ethanol fermentation.

The effects of zinc supplementation were investigated in the continuous ethanol fermentation using self-flocculating yeast. Zinc sulfate was added at the concentrations of 0.01, 0.05 and 0.1 g l(-1), respectively. Reduced average floc sizes were observed in all the zinc-supplemented cultures. Both the ethanol tolerance and thermal tolerance were significantly improved by zinc supplements, which correlated well with the increased ergosterol and trehalose contents in the yeast flocs. The highest ethanol concentration by 0.05 g l(-1) zinc sulfate supplementation attained 114.5 g l(-1), in contrast to 104.1 g l(-1) in the control culture. Glycerol production was decreased by zinc supplementations, with the lowest level 3.21 g l(-1), about 58% of the control. Zinc content in yeast cells was about 1.4 microMol g(-1) dry cell weight, about sixfold higher than that of control in all the zinc-supplemented cultures, and close correlation of zinc content in yeast cells with the cell viability against ethanol and heat shock treatment was observed. These studies suggest that exogenous zinc addition led to a reprogramming of cellular metabolic network, resulting in enhanced ethanol tolerance and ethanol production.

Ethanol fermentation with Kluyveromyces marxianus from Jerusalem artichoke grown in salina and irrigated with a mixture of seawater and freshwater.

AIMS: To study fuel ethanol fermentation with Kluyveromyces marxianus ATCC8554 from Jerusalem artichoke (Helianthus tuberosus) grown in salina and irrigated with a mixture of seawater and freshwater. METHODS AND RESULTS: The growth and ethanol fermentation of K. marxianus ATCC8554 were studied using inulin as substrate. The activity of inulinase, which attributes to the hydrolysis of inulin, the main carbohydrate in Jerusalem artichoke, was monitored. The optimum temperatures were 38 degrees C for growth and inulinase production, and 35 degrees C for ethanol fermentation. Aeration was not necessary for ethanol fermentation with the K. marxianus from inulin. Then, the fresh Jerusalem artichoke tubers grown in salina and irrigated with 25% and 50% seawater were further examined for ethanol fermentation with the K. marxianus, and a higher ethanol yield was achieved for the Jerusalem artichoke tuber irrigated with 25% seawater. Furthermore, the dry meal of the Jerusalem artichoke tubers irrigated with 25% seawater was examined for ethanol fermentation at three solid concentrations of 200, 225 and 250 g l(-1), and the highest ethanol yield of 0.467, or 91.5% of the theoretical value of 0.511, was achieved for the slurry with a solid concentration of 200 g l(-1). CONCLUSIONS: Halophilic Jerusalem artichoke can be used for fuel ethanol production. SIGNIFICANCE AND IMPACT OF THE STUDY: Halophilic Jerusalem artichoke, not competing with grain crops for arable land, is a sustainable feedstock for fuel ethanol production.

Ethanol fermentation technologies from sugar and starch feedstocks.

This article critically reviews some ethanol fermentation technologies from sugar and starch feedstocks, particularly those key aspects that have been neglected or misunderstood. Compared with Saccharomyces cerevisiae, the ethanol yield and productivity of Zymomonas mobilis are higher, because less biomass is produced and a higher metabolic rate of glucose is maintained through its special Entner-Doudoroff pathway. However, due to its specific substrate spectrum as well as the undesirability of its biomass to be used as animal feed, this species cannot readily replace S. cerevisiae in ethanol production. The steady state kinetic models developed for continuous ethanol fermentations show some discrepancies, making them unsuitable for predicting and optimizing the industrial processes. The dynamic behavior of the continuous ethanol fermentation under high gravity or very high gravity conditions has been neglected, which needs to be addressed in order to further increase the final ethanol concentration and save the energy consumption. Ethanol is a typical primary metabolite whose production is tightly coupled with the growth of yeast cells, indicating yeast must be produced as a co-product. Technically, the immobilization of yeast cells by supporting materials, particularly by gel entrapments, is not desirable for ethanol production, because not only is the growth of the yeast cells restrained, but also the slowly growing yeast cells are difficult to be removed from the systems. Moreover, the additional cost from the consumption of the supporting materials, the potential contamination of some supporting materials to the quality of the co-product animal feed, and the difficulty in the microbial contamination control all make the immobilized yeast cells economically unacceptable. In contrast, the self-immobilization of yeast cells through their flocculation can effectively overcome these drawbacks.

Intrinsic kinetics of continuous growth and ethanol production of a flocculating fusant yeast strain SPSC01.

The intrinsic kinetics of continuous yeast cell growth and ethanol production for a self-flocculating fusant yeast strain SPSC01 was investigated by means of mechanically dispersing the flocs and correspondingly established floc size distribution on-line monitoring technique using the focused beam reflectance measurement system, through which the floc intra-particle mass transfer limitation was effectively eliminated, but its ethanol formation metabolism was not affected. Modified kinetic models were developed, which can be used to predict the continuous kinetic behaviors of SPSC01, especially when low dilution rates are applied and limiting substrate concentrations are undetectable and almost all kinetic models developed previously are failed in predicting corresponding kinetic behaviors. Both substrate and product inhibitions reported for freely suspended yeast cell ethanol production were also observed for SPSC01 when high gravity media were fed and relatively high levels of residual sugar and ethanol presented. Model parameters were evaluated through numerical calculation method and validated by experimental data mu = 0.584C(s)/0.155 + C(s) + C(2)(s)/160.7(1 -P/125)(3.68) + 0.004 for growth, nu = 1.998C(s)/0.427 + C(s) + C(2)(s)/366.7(1- P/125)(1.72) + 0.060 for ethanol production These intrinsic kinetic models can be further used to develop the observed kinetic models that quantitatively correlate the impact of the self-flocculating yeast cell size distributions on their apparent rates for yeast cell growth, substrate uptake and ethanol production and optimize the ethanol production process.

Online monitoring and characterization of flocculating yeast cell flocs during continuous ethanol fermentation.

Both intrinsic and observed kinetic investigations for those ethanol fermentations using self-flocculated yeast strains have been hindered by the lack of real online monitoring techniques and proper characterization methods for the flocs. An optical detecting technique, the focused beam reflectance measurement probe developed by Lasentec (Redmond, WA) was inserted into a fermentor to monitor the floc chord length distributions. Using a simulating system composed of the floc-buffer suspensions, the total floc chord length counts per second were directly correlated with the floc biomass concentrations so that the floc biomass concentrations can be in situ detected. Furthermore, a characterization method of the flocs was established by properly weighted treatments of the detected floc chord length distributions. When a real yeast floc ethanol fermentation system was detected during its intrinsic kinetic investigations in which the floc size needed to be controlled at a level of micrometer scale to eliminate inner mass transfer limitations, it was found and validated that CO(2) produced during fermentation exerted significant disturbances. By applying 1/length-weighted treatment, these disturbances were effectively overcome.

Parameter oscillations in a very high gravity medium continuous ethanol fermentation and their attenuation on a multistage packed column bioreactor system.

The quasi-steady-states, marked by small fluctuations of residual glucose, ethanol, and biomass concentrations, and sustainable oscillations marked by big fluctuations of these monitored fermentation parameters were observed during the continuous ethanol fermentation of Saccharomyces cerevisiae when very high gravity media were fed and correspondingly high ethanol concentrations reached. A high ethanol concentration was shown to be one of the main factors that incited these oscillations, although the residual glucose level affected the patterns of these oscillations to some extent. The lag response of S. cerevisiae to high ethanol stress that causes the shifts of morphology, viability loss, and death of yeast cells is assumed to be one of the probable mechanisms behind these oscillations. It was predicted that the longer the delay of this response was, the longer the oscillation periods would be, which was validated by the experimental data and the comparison with the oscillatory behaviors reported for the ethanologen bacterium, Zymomonas mobilis. Furthermore, three tubular bioreactors in series were arranged to follow a stirred tank bioreactor to attenuate these oscillations. However, exaggerated oscillations were observed for the residual glucose, ethanol, and biomass concentrations measured in the broth from these tubular bioreactors. After the tubular reactors were packed with Intalox ceramic saddle packing, these oscillations were effectively attenuated and quasi-steady-states were observed during which there were very small fluctuations of residual glucose, ethanol, and biomass within the entire experimental run.

Continuous ethanol production and evaluation of yeast cell lysis and viability loss under very high gravity medium conditions.

A combined bioreactor system, composed of a stirred tank and a three-stage tubular bioreactor in series and with a total working volume of 3260 ml, was established. Continuous ethanol production was carried out using Saccharomyces cerevisiae and a very high gravity (VHG) medium containing 280 g l(-1) glucose. An average ethanol concentration of 124.6 g l(-1) or 15.8% (v) was produced when the bioreactor system was operated at a dilution rate of 0.012 h(-1). The yield of ethanol to glucose consumed was calculated to be 0.484 or 94.7% of its theoretical value of 0.511 when ethanol entrapped in the exhaust gas was incorporated. Meanwhile, quasi-steady states and non-steady oscillations were observed for residual glucose, ethanol and biomass concentrations for all of these bioreactors during their operations. Models that can be used to predict yeast cell lysis and viability loss were developed.

Selection of phage-display peptides that bind specifically to the outer coat protein of Rice black streaked dwarf virus.

Several peptides that could bind specifically to the outer coat protein encoded by the S10 gene of Rice black streaked virus (RBSDV) were isolated from a phage-display random 12-mer peptide library. The sequence analysis showed that the amino acid motif (K)K**(*)P, the asterisk denoting any amino acid, might be the core sequence by which the peptides bind to the target protein. The peptide 1 that had a high affinity to RBSDV outer coat protein was synthesized by a chemical method and its fusion protein with glutathione-S-transferase (GST) was produced in an Escherichia coli expression system. The dot and Western blot analyses indicated that RBSDV could be detected with a high sensitivity in crude extracts of diseased plant leaves using a purified GST fusion protein. The circular dichroism (CD) spectroscopy revealed that the synthesized binding peptide but not a nonbinding peptide could bring about a marked change in the conformation of outer coat RBSDV protein. Since the protein functions only when it has correct conformation, the peptides binding specifically to it could possibly disturb the function of the virus outer coat protein and might be used to block the transmission pathway of the virus. Summing up, as these peptides showed a high specificity and sensitivity and diagnostic potential for RBSDV, they may represent the basis of a novel strategy for development of resistance to RBSDV.

Selection and expression of peptides which can change the conformation of p20 protein of rice stripe virus.

Phages with high affinity to the P20 protein of rice stripe virus (RSV) were enriched from phage-displayed random 12-mer peptide library after three rounds of phage display screening. Nine different peptides from the enriched library were selected by enzyme-linked immunosorbent assay (ELISA). The P20 protein from raw extracts of rice leaves infected with RSV could be detected by those 9 peptides displayed on the phage, which suggested that a peptide could be an effective tool for diagnosis of RSV in rice and planthopper. Circular dichroism (CD) spectra of P20 fusion proteins with the binding phages and non-binding phages showed that the conformation of P20 protein was changed after binding to each of the 9 selected 12-mer peptides, which suggested that these peptides might disrupt the function of the P20 protein. Thereafter, those peptides might be used to develop plant resistance and disrupt virus transmission. Three of the 12-mer peptide genes were fused with the glutathione-S-transferase (GST) gene in the vector pGEX 3X. The fusion proteins were obtained from an Escherichia coli expression system and purified. The fusion proteins might have a potential to develop a plant peptide-based resistance to its pathogens and virus diagnosis. It also provided a tool (i) to confirm the inhibition of the function of P20 protein by the fusion peptides in vivo, and (ii) to detect the function of P20 protein and the interaction between the virus and its vector.

Identification of rice black streaked dwarf virus in different cereal crops with dwarfing symptoms in China.

This report describes isolation of virus particles from plants of rice, maize, wheat and sorghum with symptoms of dwarfing collected from two provinces of China, purification of double-stranded RNA (dsRNA) from the virus particles, and synthesis of full-length cDNAs of genome segments 9 (S9) and 10 (S10) by reverse transcription-polymerase chain reaction (RT-PCR). Sequence analysis showed that the S9 sequences of the Chinese isolates and a Japanese rice black-streaked disease virus (RBSDV) isolate were very similar (89.1-89.6% homology at nucleotide level and 92.3-92.9% and 95.8-98.6% homology at amino acid level for ORF1 and ORF2, respectively). Analogical similarity was found also for the S10 sequences of the isolates under comparison: 93.0-95.4% homology at nucleotide level and 96.2-97.0% homology at amino acid level. However, there was a relatively lower similarity for S9 and S10 segments ofthe Chinese isolates and an Italian maize rough dwarf virus (MRDV) isolate. The phylogenetic analysis indicated that the Chinese isolates that infect rice, maize, wheat and sorghum and cause similar symptoms could represent the same virus species, RBSDV.