Yen-Han Lin

Redox potential control and applications in microaerobic and anaerobic fermentations

Many fermentation products are produced under microaerobic or anaerobic conditions, in which oxygen is undetectable by dissolved oxygen probe, presenting a challenge for process monitoring and control. Extracellular redox potentials that can be detected conveniently affect intracellular redox homeostasis and metabolism, and consequently control profiles of fermentation products, which provide an alternative for monitoring and control of these fermentation processes. This article reviews updated progress in the impact of redox potentials on gene expression, protein biosynthesis and metabolism as well as redox potential control strategies for more efficient production of fermentation products, taking ethanol fermentation by the yeast Saccharomyces under microaerobic conditions and butanol production by the bacterium Clostridium under anaerobic conditions as examples.

Development of redox potential-controlled schemes for very-high-gravity ethanol fermentation.

Fermentation redox potential reflects the momentary physiological status of organisms. Controlling redox potential can modulate the redistribution of intracellular metabolic flux to favor the formation of the desired metabolite. Accordingly, we have developed three redox potential-controlled schemes to maximize their effects on the very-high-gravity (VHG) ethanol fermentation. They are aeration-controlled scheme (ACS), glucose-controlled feeding scheme (GCFS), and combined chemostat and aeration-controlled scheme (CCACS). These schemes can maintain fermentation redox potential at a prescribed level (i.e., -50, -100, and -150 mV) by supplementing sterile air, fresh glucose media, or a combination of sterile air and fresh glucose media into a fermenter to counteract the decline of redox potential due to yeast growth. When ACS was employed, the fermentation efficiency at -150 mV is superior to the other two redox potential levels especially when the initial glucose concentration is higher than 250 g/l. The redox potential-controlled period for ACS, GCFS, and CCACS at -150 mV under the same 200 g glucose/l condition was 2.5, 21.7 and 64.6h and the corresponding fermentation efficiency was 85.9,89.3 and 92.7%, respectively.

Very high gravity ethanol fermentation by flocculating yeast under redox potential-controlled conditions

BackgroundVery high gravity (VHG) fermentation using medium in excess of 250 g/L sugars for more than 15% (v) ethanol can save energy consumption, not only for ethanol distillation, but also for distillage treatment; however, stuck fermentation with prolonged fermentation time and more sugars unfermented is the biggest challenge. Controlling redox potential (ORP) during VHG fermentation benefits biomass accumulation and improvement of yeast cell viability that is affected by osmotic pressure and ethanol inhibition, enhancing ethanol productivity and yield, the most important techno-economic aspect of fuel ethanol production.ResultsBatch fermentation was performed under different ORP conditions using the flocculating yeast and media containing glucose of 201 ± 3.1, 252 ± 2.9 and 298 ± 3.8 g/L. Compared with ethanol fermentation by non-flocculating yeast, different ORP profiles were observed with the flocculating yeast due to the morphological change associated with the flocculation of yeast cells. When ORP was controlled at −100 mV, ethanol fermentation with the high gravity (HG) media containing glucose of 201 ± 3.1 and 252 ± 2.9 g/L was completed at 32 and 56 h, respectively, producing 93.0 ± 1.3 and 120.0 ± 1.8 g/L ethanol, correspondingly. In contrast, there were 24.0 ± 0.4 and 17.0 ± 0.3 g/L glucose remained unfermented without ORP control. As high as 131.0 ± 1.8 g/L ethanol was produced at 72 h when ORP was controlled at −150 mV for the VHG fermentation with medium containing 298 ± 3.8 g/L glucose, since yeast cell viability was improved more significantly.ConclusionsNo lag phase was observed during ethanol fermentation with the flocculating yeast, and the implementation of ORP control improved ethanol productivity and yield. When ORP was controlled at −150 mV, more reducing power was available for yeast cells to survive, which in turn improved their viability and VHG ethanol fermentation performance. On the other hand, controlling ORP at −100 mV stimulated yeast growth and enhanced ethanol production under the HG conditions. Moreover, the ORP profile detected during ethanol fermentation with the flocculating yeast was less fluctuated, indicating that yeast flocculation could attenuate the ORP fluctuation observed during ethanol fermentation with non-flocculating yeast.

Ageing vessel configuration for continuous redox potential-controlled very-high-gravity fermentation

The development of continuous very-high-gravity (VHG) fermentation is hindered by ineffective glucose uptake in order to result in zero discharge in the effluent stream. To overcome the problem, we proposed a continuous redox potential-controlled fermentation configuration, consisting of a Chemostat vessel connected with two ageing vessels installed in parallel, and the relevant design criteria are also specified. The Chemostat vessel is subjected to redox potential control to maintain yeast viability, and the ageing vessels are used to completely utilize glucose before discharging to next process unit. Two ageing vessels are scheduled alternatively, resulting in continuously-like operation. The size of ageing vessel is governed by the Chemostat size, dilution rate and filling time. The guideline to choose proper dilution rate is provided and the selection criterion of the proposed continuous configuration over batch fermentation is derived. The excess ethanol produced by the proposed continuous configuration over batch fermenter is quantified. As an illustration, a bio-ethanol plant is typically operated 8000 h per annum and the downtime between batches is 6h. Given that the fermenter size of 100 m(3) for both batch fermenter and Chemostat vessel, and glucose fed at 300 g/l, if the proposed continuous redox potential-controlled fermentation configuration (operated at 0.028 h(-1) and controlled at -50 mV) is selected, it will take 191 h for this configuration to outperform the batch counterpart, and the excess amount of ethanol being produced will be 1142 t.

Global gene expression analysis of Saccharomyces cerevisiae grown under redox potential‐controlled very‐high‐gravity conditions

Redox potential (ORP) plays a pivotal role in yeast viability and ethanol production during very-high-gravity (VHG) ethanol fermentation. In order to identify the correlation between redox potential profiles and gene expression patterns, global gene expression of Saccharomyces cerevisiae was investigated. Results indicated that significant changes in gene expression occurred at the periods of 0 - 6 h and 30 - 36 h, respectively. Changes noted in the period of 0 - 6 h were mainly related to carbohydrate metabolism. In contrast, gene expression variation at 30 - 36 h could be attributed primarily to stress response. Although CDC19 was down-regulated, expression of PYK2, PDC6 and ADH2 correlated inversely with ORP. Meanwhile, expression of GPD1 decreased due to the depletion of dissolved oxygen in the fermentation broth, but expression of GPD2 correlated with ORP. Transcription of genes encoding heat shock proteins was characterized by uphill, downhill, valley and plateau expression profiles, accordingly to specific function in stress response. These results highlight the role of ORP in modulating yeast physiology and metabolism under VHG conditions.

Ethanol from Starch-Based Feedstocks

The escalation of global warming has forced the public to rethink how policies of energy utilization can ever sustain human activities. Over 40% of fossil energy use is linked to the transportation sector, which is deemed as the major culprit that continues to worsen our earthly environment. Many measures have been proposed and taken in order to minimize the emission of greenhouse gases. Ethanol, made by Saccharomyces yeasts through fermentation, is currently the world’s largest fermented liquid fuel. The discharge of carbon as CO2 during its production and use is essentially zero when the fixation of carbon dioxide by the plant is considered, and ethanol is therefore a green and sustainable alternative energy to partially replace or augment petroleum. In this article, the biochemistry of yeast and ethanol production is detailed. The relevant unit operations including grain processing, starch hydrolysis, ethanol fermentation, and postprocessing of ethanol are described. Particularly, guidelines for successful operation of very-high-gravity (VHG) fermentation technology are provided. Factors including temperature, pH, and aeration as they affect fermentation efficacy are illustrated, and approaches to reduce their impacts on ethanol yield are given. Aeration schemes to accelerate VHG fermentation rates are described. Techniques used to quantify fermentation efficiency are mentioned. Guidelines for improving overall performance of an ethanol plant are given. This article concludes by proposing a possible cooperative business scheme to increase the total profit gain for fuel alcohol biorefineries.

Whole-Cell Protein Identification Using the Concept of Unique Peptides

A concept of unique peptides (CUP) was proposed and implemented to identify whole-cell proteins from tandem mass spectrometry (MS/MS) ion spectra. A unique peptide is defined as a peptide, irrespective of its length, that exists only in one protein of a proteome of interest, despite the fact that this peptide may appear more than once in the same protein. Integrating CUP, a two-step whole-cell protein identification strategy was developed to further increase the confidence of identified proteins. A dataset containing 40,243 MS/MS ion spectra of Saccharomyces cerevisiae and protein identification tools including Mascot and SEQUEST were used to illustrate the proposed concept and strategy. Without implementing CUP, the proteins identified by SEQUEST are 2.26 fold of those identified by Mascot. When CUP was applied, the proteins bearing unique peptides identified by SEQUEST are 3.89 fold of those identified by Mascot. By cross-comparing two sets of identified proteins, only 89 common proteins derived from CUP were found. The key discrepancy between identified proteins was resulted from the filtering criteria employed by each protein identification tool. According to the origin of peptides classified by CUP and the commonality of proteins recognized by protein identification tools, all identified proteins were cross-compared, resulting in four groups of proteins possessing different levels of assigned confidence.

Prediction of Protein-Protein Interactions Using Protein Signature Profiling

Protein domains are conserved and functionally independent structures that play an important role in interactions among related proteins. Domain-domain interactions have been recently used to predict protein-protein interactions (PPI). In general, the interaction probability of a pair of domains is scored using a trained scoring function. Satisfying a threshold, the protein pairs carrying those domains are regarded as “interacting”. In this study, the signature contents of proteins were utilized to predict PPI pairs in Saccharomyces cerevisiae, Caenorhabditis elegans, and Homo sapiens. Similarity between protein signature patterns was scored and PPI predictions were drawn based on the binary similarity scoring function. Results show that the true positive rate of prediction by the proposed approach is approximately 32% higher than that using the maximum likelihood estimation method when compared with a test set, resulting in 22% increase in the area under the receiver operating characteristic (ROC) curve. When proteins containing one or two signatures were removed, the sensitivity of the predicted PPI pairs increased significantly. The predicted PPI pairs are on average 11 times more likely to interact than the random selection at a confidence level of 0.95, and on average 4 times better than those predicted by either phylogenetic profiling or gene expression profiling.

Redox potential driven aeration during very-high-gravity ethanol fermentation by using flocculating yeast

Ethanol fermentation requires oxygen to maintain high biomass and cell viability, especially under very-high-gravity (VHG) condition. In this work, fermentation redox potential (ORP) was applied to drive the aeration process at low dissolved oxygen (DO) levels, which is infeasible to be regulated by a DO sensor. The performance and characteristics of flocculating yeast grown under 300 and 260 g glucose/L conditions were subjected to various aeration strategies including: no aeration; controlled aeration at −150, −100 and −50 mV levels; and constant aeration at 0.05 and 0.2 vvm. The results showed that anaerobic fermentation produced the least ethanol and had the highest residual glucose after 72 h of fermentation. Controlled aerations, depending on the real-time oxygen demand, led to higher cell viability than the no-aeration counterpart. Constant aeration triggered a quick biomass formation, and fast glucose utilization. However, over aeration at 0.2 vvm caused a reduction of final ethanol concentration. The controlled aeration driven by ORP under VHG conditions resulted in the best fermentation performance. Moreover, the controlled aeration could enhance yeast flocculating activity, promote an increase of flocs size, and accelerate yeast separation near the end of fermentation.

Flux distribution and partitioning in Corynebacterium glutamicum grown at different specific growth rates

Corynebacterium glutamicum (ATCC 21253) was cultivated in a pH-auxostat. The flux partitioning at key branch nodes in the central metabolism train of C. glutamicum was used to illustrate the physiological responses subject to the influence of specific growth rates. In glycolysis, the flux partitioning at the G6P point (51.94±0.7%) was insensitive to variations of specific growth rate, whereas the partitioning decreased approximately 30% from PEP to PYR. In the TCA cycle, the specific growth rates had negligible effects on the flux partitioning at both OAA (76.80±1.19%) and FUM (70.46±2.42%) nodes, but had a significant effect on the AKG node (63.2, 43.26, and 30.65% corresponding to specific growth rates of 0.23, 0.45, and 0.51 h−1, respectively). The flux partitioning at the anaplerotic pathway (from PEP to OAA) was maintained at 25.46±2.26% for all the specific growth rates tested. The flexibility of PYR and AKG nodes shows a potential gateway for altering amino acid distribution, whereas the rigidity of G6P, SUC, FUM and OAA nodes implies that enzymes that associate with these nodes are insensitive to the manipulation of specific growth rates.