Scholarly article on topic 'Thermotolerant fermenting yeasts for simultaneous saccharification and fermentation of lignocellulosic biomass'

Thermotolerant fermenting yeasts for simultaneous saccharification and fermentation of lignocellulosic biomass Academic research paper on "Industrial Biotechnology"

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Electronic Journal of Biotechnology
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{Bioethanol / Biofuel / Enzymes / "Genome shuffling" / "Lignocellulosic biomass"}

Abstract of research paper on Industrial Biotechnology, author of scientific article — Jairam Choudhary, Surender Singh, Lata Nain

Abstract Lignocellulosic biomass is the most abundant renewable source of energy that has been widely explored as second-generation biofuel feedstock. Despite more than four decades of research, the process of ethanol production from lignocellulosic (LC) biomass remains economically unfeasible. This is due to the high cost of enzymes, end-product inhibition of enzymes, and the need for cost-intensive inputs associated with a separate hydrolysis and fermentation (SHF) process. Thermotolerant yeast strains that can undergo fermentation at temperatures above 40°C are suitable alternatives for developing the simultaneous saccharification and fermentation (SSF) process to overcome the limitations of SHF. This review describes the various approaches to screen and develop thermotolerant yeasts via genetic and metabolic engineering. The advantages and limitations of SSF at high temperatures are also discussed. A critical insight into the effect of high temperatures on yeast morphology and physiology is also included. This can improve our understanding of the development of thermotolerant yeast amenable to the SSF process to make LC ethanol production commercially viable.

Academic research paper on topic "Thermotolerant fermenting yeasts for simultaneous saccharification and fermentation of lignocellulosic biomass"

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Thermotolerant fermenting yeasts for simultaneous saccharification and fermentation of lignocellulosic biomass

Jairam Choudhary, Surender Singh, Lata Nain

PII: S0717-3458(16)30016-1

DOI: doi: 10.1016/j.ejbt.2016.02.007

Reference: EJBT162

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Electronic Journal of Biotechnology

Received date: Revised date: Accepted date:

13 November 2015 6 February 2016 9 February 2016

Please cite this article as: Choudhary Jairam, Singh Surender, Nain Lata, Thermotolerant fermenting yeasts for simultaneous saccharification and fermentation of lignocellulosic biomass, Electronic Journal of Biotechnology (2016), doi: 10.1016/j.ejbt.2016.02.007

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Language revision Electronic Journal of Biotechnology EJBT-D-15-00165 R2 Review Article

Received: November 13, 2015 Accepted: February 19, 2015

Areas: Microbial Biotechnology; Industrial Biotechnology Short title: Thermotolerant yeast for SSF

Thermotolerant fermenting yeasts for simultaneous saccharification and fermentation of lignocellulosic biomass

Jairam Choudhary, Surender Singh*, Lata Nain

Division of Microbiology, ICAR - Indian Agricultural Research Institute, New Delhi, India 110012

Corresponding author:


Lignocellulosic biomass is the most abundant renewable source of energy widely explored as 2nd generation biofuels. Despite more than four decades of research, the process of ethanol production from lignocellulosic (LC) biomass remains economically unfeasible because of high cost of enzymes, end product inhibition of enzymes and the need for cost intensive inputs associated with separate hydrolysis and fermentation (SHF) process. Thermotolerant yeast strains capable of fermentation at more than 40°C are suitable alternative for developing simultaneous saccharification and fermentation (SSF) process to alleviate the problems associated with SHF. This review describes the various approaches for screening and development of thermotolerant yeasts by genetic and metabolic engineering. The benefits and limitations associated with simultaneous saccharification and fermentation at high temperature are also discussed. A critical insight into the effect of high temperature on yeast morphology and physiology is also included which can provide a better perspective for the development of thermotolerant yeast amenable to the SSF process so that LC ethanol production can become commercially viable.

Keywords: bioethanol; biofuel; enzymes; genome shuffling; lignocellulosic biomass 1. Introduction

Global pattern of increasing energy consumption, depleting fossil fuel reserves and concerns about climate change are the main driving forces responsible for exploring new renewable and environment friendly sources of energy. Plant biomass which is most abundant renewable energy source can be used for the production of second generation biofuels however it is almost wasted either by burning or by disposal in landfill sites which threatens the environment by the release of green house gases. The process of ethanol production from lignocellulosic biomass mainly comprises four steps.

i) Pretreatment of lignocellulosic biomass ii) Enzymatic saccharification of pretreated biomass to yield sugar monomers, iii) Fermentation of hydrolyzed sugars to ethanol, butanol etc. by fermenting organisms and iv) Distillation. The process is called as separate hydrolysis and fermentation (SHF) when saccharification and fermentation are performed separately. As the biomass hydrolyzing enzymes (cellulases and hemicellulases) and pretreatment methods are very costly, production of LC ethanol by SHF is not economically viable.

During enzymatic saccharification, the hydrolytic enzymes suffer the problem of feedback inhibition as the sugar monomers and cellobiose accumulate in the medium leading to reduced efficiency of these enzymes. This problem can be resolved by the process called simultaneous saccharification and fermentation (SSF) in which saccharification and fermentation is performed simultaneously, thus hydrolyzed sugars are continuously converted into ethanol thereby enhancing the efficiency of enzymatic saccharification without feedback inhibition. However the major problem encountered in SSF are the different temperature optima of biomass hydrolyzing enzymes (45-50°C) and fermenting organisms (30°C). Therefore important areas in which we need to focus are search for cold adaptive hydrolytic enzymes and thermotolerant fermenting yeasts in order to develop economically viable SSF technology. In practice, it is very difficult to bring down the optimum temperature of cellulases by protein engineering therefore identification of thermotolerant yeast with higher ethanol production efficiency can provide key breakthrough for the SSF process. The SSF by thermotolerant yeasts provide the following advantages in the bioethanol production:

• Reduction in total number of steps thereby lowering the utility requirement and hence reduction in capital investment including equipment costs

• Reduction in the chances of contamination by decreasing glucose concentration and production of ethanol

• Improvement in efficiency of saccharification by alleviating feedback inhibition of cellulase

• Reduction in cooling cost as there is no requirement of chiller unit

• Continuous ethanol evaporation from broth under reduced pressure

• Suitability for use in tropical countries where temperature is high

2. Lignocellulosic biomass as substrate for ethanol production

Lignocellulosic biomass refers to plant dry matter mainly composed of carbohydrate polymer, cellulose (38-50%), hemicellulose (23-32%) and aromatic polymer lignin (1525%) [1]. It is the most copiously available raw material on the planet for ethanol production. Worldwide, 2 x 1011 mt lignocellulosic biomass is produced annually, of which 8-20 x 109 mt is potentially accessible for processing. Structurally, cellulose and hemicellulose are tightly entangled with lignin which renders the polysaccharides inaccessible for hydrolysis by cellulases and hemicellulases. Cellulose is a linear polymer of D-glucose joined by ß(1 -4) glycosidic linkage with reducing and non-reducing ends. Cellulose fibrils are arranged in parallel stacks with hydrogen bonding and weak van der Waals forces to form cellulose microfibrils. These cellulose microfibrils

have both crystalline and amorphous regions bound together by hemicellulose and lignin to form macrofibrils. The second important fraction of lignocellulosic biomass is hemicellulose, which is a heteroplymer of pentoses (xylose, arabinose) and hexoses (glucose, galactose and mannose). Xylan, p(l -4) linked xylose homoploymer, is a major hemicellulosic component present in hardwood trees while softwood mainly contains mannans and glucomannans [2]. Lignin, which provides structural rigidity to plants, is a heteropolymer of p-hydroxyphenyl, syringyl, guaiacyl and syringyl monolignol units that form a complex network around cellulosic microfibrils. Lignocellulosic biomass can be grouped into three different categories, namely virgin biomass, energy crops and waste biomass. All terrestrial plants such as trees, bushes, grasses and crop plants are collectively called as virgin biomass. Waste biomass is the low value byproduct of virgin biomass such as corn stover, sugarcane bagasse, saw mill and paper mill wastes. Energy crops such as switch grass, elephant grass, cassava and sweet sorghum with greater production of biomass are cultivated to serve as raw material for ethanol production.

Cellulose and hemicellulose can be hydrolyzed by holocellulases to sugars which can be fermented to produce biofuel whereas lignin is non-fermentable polyphenolic compound. Agricultural crop residues, industrial and urban waste, forestry residues and dedicated energy crops like switch grass giant reed, miscanthus, poplar and willow are most popular and abundant lignocellulosic feedstocks. The proportion of constituents of lignocellulosic feedstocks varies with the type of feedstock used. The residue from cultivable land can be differentiated as agricultural crops i.e. paddy and wheat straw, groundnut shells, corn stover, sunflower stalks, cotton stalks, grass fibers and agricultural byproducts like corn cobs, sugarcane bagasse, palm mesocarp fibers, sunflower and barley hulls. Rice husks and wheat bran originating from processing of agricultural commodities can also be used as substrate for LC ethanol production [3],[4],[5],[6]. Forestry waste includes wood chips, slashes, branches of dead trees, hardwood and softwood and tree prunings [7]. Processing papers, household wastes, cotton linters, pulps, food processing waste, fruits and vegetable processing waste are categorized as industrial and urban waste [8],[9].

3. Processes of 2nd generation bioethanol production

Worldwide, scientists have invented different processes for ethanol production from lignocellulosic biomass (Table 1). These processes are namely called separate hydrolysis and fermentation (SHF), simultaneous saccharification and fermentation (SSF), simultaneous saccharification and co-fermentation (SSCF), consolidated bioprocessing (CBP) and simultaneous saccharification, filtration and fermentation (SSFF). Every process has its own advantages and limitations which are listed in Table 2.

3.1 SHF process

This is the oldest method for production of LC ethanol. In this process, externally produced enzyme cocktails are used to hydrolyse pretreated lignocellulosic biomass to yield sugar monomers and the resulting enzymatic hydrolysate is used for production of

biofuel by fermenting microorganisms. Both processes are carried out separately because of different temperature optima for hydrolytic enzymes (approximately 50°C) and fermentation (30-35°C). Currently most of LC ethanol is produced by SHF process (Figure 1).

3.2 SSF process

In this method, enzymatic saccharification of pretreated biomass and fermentation of enzymatic hydrolysate is carried out in same vessel. This method alleviates the problem of feedback inhibition of cellulases by glucose which is a limiting factor in SHF. Therefore SSF (Figure 2) improves both efficiency of enzymatic saccharification and ethanol yield. Ghosh et al. [24] reported an increase in rate of hydrolysis by 13-30% by using SSF compared to a normal enzymatic saccharification. Ohgren et al. [25] reported a 13% higher LC ethanol yield from SSF as compared to SHF process. Cold adaptive hydrolytic enzymes and thermophilic yeast are center of attraction for this process to be carried out at ambient temperature of approximately 40°C in a single vessel.

3.3. SSCF process

In this process, saccharification is performed simultaneously with the co-fermentation of hexose and pentose sugars. Traditional method of LC ethanol production with industrially important microorganism such as Saccharomyces cerevisiae does not allow the complete utilization of substrate as organism is not capable of fermenting pentose sugars. Therefore, organisms which are capable of fermenting both pentose and hexose sugars are important to carry out SSCF. SSCF can be operated at high water insoluble solid (WIS) content with fed batch fermentation which assists ease of mixing and higher ethanol yield. It also offers chances to maintain glucose concentration at low level which allows efficient co-fermentation of glucose and xylose [26].

3.4. CBP process

CBP is an integrated process in which enzyme production, biomass hydrolysis and fermentation are carried out in single step (Figure 3). It is a promising approach for economically feasible LC ethanol production as the process requires very less utilities as compared to SHF and SSF. A highly engineered microbial strain capable of producing sufficient hydrolytic enzymes with higher fermentation capacity is required for development of CBP process.

3.5. SSFF process

It is an integrated process in which a membrane filtration chamber is placed between saccharification and fermentation. Most of the engineered yeast strains have lower affinity to xylose as compared to glucose therefore, xylose utilization starts after depletion of glucose from medium. SSFF (Figure 4) overcomes the problems of both SHF and SSF and allows both hydrolytic enzymes and fermenting microorganism to be used separately at their optimum conditions. In this process, pretreated biomass slurry is exposed to hydrolytic enzymes and the mixture of pretreated biomass and enzyme is

pumped through a cross flow membrane from which, the sugar rich filtrate is pumped to the fermentation reactor and retentate and fermentate are re-circulated to saccharification chamber [27]. The fermented liquid is pumped back to the hydrolysis reactor to maintain the balance of volume in both reactors. Yeast cultures with flocculating behavior are retained by settling in the fermentation reactor [27].

4. Screening of yeast strains suitable for fermentation at high temperature

Although bioethanol production presently accounts for billions of liters of ethanol produced per annum from corn (USA) and sugarcane (Brazil), the use of thermotolerant fermenting yeasts could improve the efficiency of bioethanol production by allowing fermentation to occur at temperatures more than 40°C using SSF technology. During the last few decades, many studies reported the screening of thermotolerant yeast strains which were capable of fermentation at high temperature suitable for bioethanol production by SSF (Table 3).

Studies showed that stress conditions lead to the induction of high temperature tolerance in S. cerevisiae but if the adapted yeast is grown under normal/optimal conditions, the thermotolerance can be lost [28],[29]. In a recent study, it was revealed that thermotolerant S. cerevisiae strains isolated by physiological adaptation (adaptive evolution) to temperatures at 40°C or above had an altered sterol composition. It was believed that the modification in the sterol composition of yeast strains maintained the fluidity of cell membrane at high temperature which resulted in increased thermotolerance [36]. Suutari et al. [28] found a negative correlation between fermentation ability and temperature increase which was attributed to changes in the membrane fluidity of the S. cerevisiae at high temperature. Therefore, experiment need to be designed to isolate S. cerevisiae mutants that may be able to resist the changes in sterol composition against high temperature stress by adding inhibitors of sterol metabolism or compounds which can alter the composition of cytoplasmic membrane that stabilizes the membrane at high temperature.

Another approach includes screening of thermotolerant fermenting yeast strains, which are resistant to the glucose analogue 2-deoxy D-glucose. These S. cerevisiae mutants show improved fermentation ability at elevated temperature [37]. This increase in fermentation efficiency by the S. cerevisiae mutant strains was attributed to the absence of catabolite repression by glucose and improved uptake of glucose [38], [39]. Similarly, Candida molischiana mutant resistant to 2-deoxy D-glucose showed ethanol production capability at 45°C unlike its wild type counterparts [40].

Tropical regions are the suitable sites for the isolation of thermotolerant yeast strains. S. cerevisiae isolates from this region can tolerate temperature more than 44°C, but they grow at a slower rate as compared with growth in the mesophilic range of temperature, with reduced fermentation capability [41]. Therefore temperature appears to be an important physical parameter that limits the performance of organism in terms of ethanol production.

5. Effect of high temperature on yeast

Yeasts are mesophilic in nature. When yeasts are grown beyond optimum temperatures, their morphology and physiology is affected in several ways. Growth and metabolism of yeast at various temperatures is the function of the genetic makeup of the yeast strain, composition of culture medium and other growth parameters. Accumulation of yeast metabolites, both inside and outside the cell, may also affect temperature sensitivity of yeast. Temperature optima of yeasts vary with species. Thermotolerance is transient ability of yeast cell to survive against the higher temperature. Thermotolerant yeast strains are able to survive at temperature above 40°C. Intrinsic thermotolerance in yeast cells is observed after a heat shock (e.g. to 50°C), however induced thermotolerance occurs when cells are pre-conditioned by exposing them to a mild heat shock (e.g. 37°C for 30 min) before a more severe heat shock. Several factors, apart from a mild heat shock, such as specific chemicals, osmotic dehydration, and low external pH, composition of culture medium and phase of growth are known to influence yeast thermotolerance [29]. Regarding pH, yeast thermotolerance increases to maximum when the external pH declines to 4.0 and researchers have provided convincing evidence which tells about alterations in intracellular pH as a stimulus for thermotolerance in S. cerevisiae [42].

Growth of S. cerevisiae ScY and ScY01 at elevated temperature suppressed the expression of a large number of proteins involved in various metabolic pathways like central carbon metabolism (CCM), lipid metabolism, amino acid metabolism, vitamins and cofactor metabolism. Growth at elevated temperature also affects protein transport and vesicle organization. In contrast, sudden heat shock increased expression of many proteins having functions in carbohydrate metabolism, lipid metabolism, protein folding and degradation, and oxidative stress response. Interestingly, specific proteins such as cytochrome b2, glycogen phosphorylase, long-chain-fatty-acid--CoA ligase 1, (DL)-glycerol-3-phosphatase, catalase T, and transaminated amino acid decarboxylase were down-regulated in both ScY and ScY01 during thermotolerant response yet increased their abundances in heat shock response [43].

CCM has been drawn in to play vital roles in modulating yeast survival during lethal heat stress, although specific mechanisms of central metabolic genes in regulating thermo sensitivity remain unknown [44]. Shui et al. [43] observed variation in expression of proteins involved in CCM, like up-regulation of glycolytic enzymes and down-regulation of enzymes involved TCA cycle, glycogen and glycerol biosynthesis, pentose phosphate pathway and components of the electron transport chain such as cytochrome b/c subunits and ATP synthases in thermotolerant response.

Yeast cells exhibit a quick response at molecular level when cells experience a sudden increase in temperature. It is known as heat shock response. This regulatory phenomenon is ubiquitous in all living cells. Sub lethal/ mild heat shock treatment leads to the induction and expression of genes which are responsible for synthesis of heat shock proteins (HSPS). As S. cerevisiae is a mesophilic organism, it shows poor fermentation efficiency at elevated temperature (>35°C) due to increased fluidity of cytoplasmic membranes, to which the yeast cells respond by modifying its fatty acid composition [28]. Though, temperatures >34°C seriously affect yeast cell viability and

growth, thermotolerance in S. cerevisiae can be induced by short term exposure to nonlethal stress conditions including high osmolarity, low pH, high ethanol concentrations, and superoptimal temperatures (>37°C). The induced thermotolerance is nonheritable and is attributable to induction of various cellular responses like synthesis of heat shock proteins (HSPS) and trehalose, which can help in arresting cell cycle at G1 phase, and reduced adenosine 3',5'-monophosphate protein kinase (cAMP-PK) activity associated with low glycolytic fluxes [29],[45]. Thus, physiological adaptation of yeast is not a suitable approach for ethanol production. Multiple changes caused by high temperature in the cell ultimately affect structure and function of proteins, generate abnormal proteins, and lead to growth inhibition or cell death. These degraded and denatured proteins are mainly degraded by the proteasome pathway as a defense mechanism to make sure that cell will survive [46]. Ubiquintination is the primary signal used to target cellular proteins for destruction by 26 S proteasomes. Ubiquitin is induced by various kind of stresses in reflection of need for more extensive protein turnover in stressed cells [47] and also it is an important non proteolytic signal which regulates function of proteins by non degradative mechanisms, including modulating proteinprotein interactions in numerous biological systems [48]. Attainment of high temperature tolerance is mostly controlled through activation and regulation of specific stress related genes which play an important role in the synthesis of specific compounds that guard the organism against high temperature stress [49]. After a sub lethal heat shock, apart from heat shock proteins synthesis, yeast cells also starts accumulating other protective compounds (e.g. trehalose) along with selected enzymes (mitochondrial superoxide dismutase and catalase). Trehalose is considered as a thermo protectant as it helps in stabilizing the cytoplasmic membrane and cellular proteins [50].

6. Role of thermotolerant yeast in SSF

As we know that commercially available cellulases and hemicellulases perform hydrolysis efficiently at a temperature around 45-50°C and most fermenting microorganisms used in industry have temperature optima around 30-35°C. This necessitates the process to be carried out separately, called as SHF which is very costly and energy intensive process. Hence identification of yeasts with tolerance to high temperature can help in combining these processes, leading to SSF.

In a study carried out by Shahsavarani et al. [51], a Htg+ (high temperature growth phenotype) strain exhibited confluent growth at higher temperature (41°C) and resistance to heat shock, ethanol, osmotic, oxidative and DNA damage stresses. HTG 6, one of six genes responsible for the thermotolerant phenotype was identified to be the gene RSP5 which encodes ubiquitin ligase.

Some thermotolerant and ethanol producing yeast strains have been isolated and modified for ethanol production from lignocellulosic biomass. Candida glabrata can be a good candidate for development of SSF process, since the yeast has better tolerance to both high temperature and high acid along with superior ethanol production capability [52]. Kluyveromyces marxianus also seems to be particularly promising for production of ethanol at higher temperature. Many strains of K. marxianus grow well at temperatures as high as 45-52°C and can efficiently produce ethanol at temperatures between 38°C

and 45°C [53],[54],[55]. Moreover, K. marxianus provides additional advantages including a high growth rate and the ability to utilize a wide range of sugar substrates (e.g. galactose, arabinose, xylose and mannose) at elevated temperatures [55],[56],[57]. Because of these benefits, K. marxianus has been used for bioethanol production from various substrates such as corn silage juice, sugarcane juice, whey powder and molasses [58],[59],[60],[61]. Steam pretreated lignocellulosic material (eucalyptus, poplar, bagasse, sweet sorghum, mustard and wheat straw) were used to produce LC ethanol via SSF process by using K. marxianus [62]. The SSF experiment was performed at 42°C with 100 g/L substrate and commercial cellulase @ 15 FPU/g-substrate. After 72-82 h fermentation, 16-19 g/L of ethanol was produced from the lignocellulosic materials. Ethanol yield was 50-72% of the theoretical yield based on the glucose available in the pretreated materials. K. marxianus 1MB strains identified by Banat et al. [54] have shown favorable SSF results at temperatures between 40 and 50°C [35],[63],[64].

Some of the promising thermotolerant S. cerevisiae strains have been isolated from tropical regions (41, 65b). The S. cerevisiae TJ14 strain, had shown a high-temperature (41°C) growth optima, produced 40 g/L ethanol from 161 g/L of paper sludge organic material containing 66% (w/w) glucan in an SSF process at 42°C using a cellulase produced by the filamentous fungus Acremonium cellulolyticus [33]. Interestingly, it was investigated that RSP5, gene encoding an essential e3 ubiquitin ligase, and CDC19, gene encoding pyruvate kinase, are responsible for the high-temperature growth phenotype based on classical genetic analysis of thermotolerant S. cerevisiae strains [51],[66]. Although a number of anaerobic bacteria such as Thermoanaerobacter ethanolicus, Thermoanaerobacterium saccharolyticum and Clostridium thermotherum have been used to ferment hexose and pentose sugars to ethanol [67],[68],[69], it is difficult in practice to maintain anaerobic conditions in large-scale fermentation which restricts the use of thermophilic anaerobes.

In a study carried out by Banat et al. [54], K. marxianus IMB3 was used for industrial scale fermentation of molasses. No cooling system was used and the fermentation temperature was allowed to increase up to 42°C. It was concluded that 60-72 g/L (0.51 g/g glucose) ethanol may be produced which was similar to the yield obtained regularly by S. cerevisiae in the distillery at lower temperatures. Pessani et al. [34] carried out fermentation of pretreated switch grass (Panicum virgatum) by K. marxianus IMB3 at different temperatures and obtained ethanol yield 22.5g/L with 12% solid and enzyme loading (0.7 ml/g) of Accelerase 1500 at 45°C, achieving 86% theoretical yield.

7. Methods for developing yeast strains suitable for SSF

7.1. Site directed mutagenesis (SDM)

Using SDM also known as oligonucleotide directed mutagenesis or site specific mutagenesis, specific desired changes can be made into the DNA sequence of a particular gene resulting in altered gene products. Site directed mutagenesis play an important role in investigating structure and biological function of DNA, RNA, and proteins and also in protein engineering.

A DNA primer with desired mutation, complementary to genomic DNA around the mutation site is required to be synthesized to bring about desired changes in the genomic DNA sequence. The desired mutation may be a point mutation (single base change), multiple base changes, insertion or deletion. The single-strand primer is then extended using a DNA polymerase, which copies the rest of the gene. The gene that copied contains the desired mutation. Then gene with desired mutation is introduced into the target host with the help of suitable vector and cloned. Finally, mutants are screened and selected by DNA sequencing to check whether the gene of interest with desired mutation is present or not.

Hansenula polymorpha, a methylotrophic yeast, was mutated using SDM to produce ethanol from cellobiose, glucose and xylose at elevated temperature [70]. Dmytruk et al.

[71] improved utilization of xylose in H. polymorpha strain by site-specific mutagenesis of the endogeneous xylose reductase gene. The recombinant strain showed 7.3 folds higher ethanol productivity at 48°C as compared with the wild-type strain which makes it a suitable candidate for use in SSF.

7.2. Genome shuffling approach

This method was first introduced by Zhang and co-workers in 2002, to improve the tylosin production by Streptomyces fradiae [72]. The technology of DNA shuffling is a method used for in vitro homologous recombination of pools of selected mutant genes by random fragmentation and PCR reassembly. A similar technology for strain improvement, genome shuffling; is a way to introduce beneficial mutations in a directed evolution experiment. This method is used to increase size of DNA library. In this method, recombination between genomic DNA of different strains or species with different mutations is involved. This technique combines the benefits of multiparental crossing allowed by DNA shuffling along with the recombination of whole genomes normally associated with conventional breeding, or by protoplast fusion that increases the recombination. In addition to this, genome shuffling can accelerate directed evolution by facilitating recombination between the members of diversely selected population

[72],[73]. The procedure of genome shuffling (Figure 5) consists of following steps: construction of parental library, protoplast fusion and selection of desired phenotype.

To construct parental library, the initial strain is engineered by mutagenesis to generate more genotypes. These cells are then suspended in a buffer containing lysozyme [74],[75] or salinase [73], following which protoplast obtained are aggregated by centrifugation. Further, an equal number of protoplasts from the mutants are mixed, divided into two equal parts, and inactivated by incubating at high temperature (50-60°C) or by UV irradiation [76]. The killed protoplasts are grouped together and fused in a system containing 35% poly ethylene glycol and 0.1% calcium chloride at 35°C for 40 min. After that fused protoplast were centrifuged, washed twice and re-suspended in 10 ml buffer, serially diluted and regenerated. The strains from regenerated protoplast were pooled and used as strain library for second round and the similar process can be repeated several times. In the final step, screening is carried out for selection of desired phenotypes. This technique has been used to enhance the product yield, enhancement

of strain tolerance and improved substrate uptake. Thermotolerance, ethanol tolerance and ethanol productivity of S. cerevisiae F-34 were improved with the genome shuffling approach by a combination of protoplast fusion and UV irradiation. The strain F-34 was able to grow at 55°C and was capable of utilizing complete sugars at 20% concentration level resulting in ethanol production (9.95% w/v) and ethanol tolerance up to 25% v/v [77].

7.3. Mutagenesis

Industrially important S. cerevisiae is a good producer of ethanol with higher ethanol tolerance but lacks thermotolerance. Therefore, mutation screening for thermotolerance using a proofreading deficient DNA polymerase or ultraviolet (UV) irradiation resulted in selection of S. cerevisiae mutant that grow at temperature up to 40-42°C [78],[79]. A respiratory mutant of C. glabrata yielded 17.0 g/L ethanol from 50 g/L avicel/microcrystalline cellulose at 42°C under aerobic conditions in the presence of sufficient cellulase [80].

7.4. Metabolic engineering

Metabolic engineering deploys tools of genetic engineering to modify the metabolism of an organism. It may involve the optimization of existing biochemical pathways or the introduction of pathway components, most commonly in bacteria, yeast or plants, with the goal of high-yield production of specific metabolites for medicine or biotechnology. Understanding of yeast physiology under various stress conditions (osmotic stress, low pH, high temperature, high ethanol concentration stress etc.) experienced during fermentation might be useful to guide further improvements in large scale ethanol production via engineering of stress tolerance traits in the other strains.

Auxotrophic strains are considered important platforms for both fundamental and applied research in industrial biotechnology, mainly enabling the culture conditions to promote selective pressure on recombinant cells [81]. Uracil auxotrophic strains of S. cerevisiae CEN.PK113-5D dissimilate the carbon source mainly into ethanol and acetate under uracil limiting conditions by respiratory fermentative metabolism. Under these conditions, the yeast strains also show increased specific rates of glucose, sucrose, O2 consumption and CO2 production [82]. The uracil auxotrophy in the yeast is introduced by Ty insertion mutation within the coding region of URA3 gene. The URA 3 gene in the yeast encodes ODCase (orotidine-5-phosphatase decarboxylase) an enzyme involved in the de novo synthesis of pyrimidine nucleotides. This enzyme is responsible for decarboxylation of orotidine 5'-phosphate (OMP) to uridine 5'-phosphate (UMP). Therefore ura3-52 mutation made the cells unable to synthesize uMp [83]. Therefore, uracil auxotrophy developed by Ty insertion in URA 3 gene favors ethanol production from carbon source under uracil limiting conditions. These cells must utilize the uracil present in the medium by pyrimidine salvage pathway.

7.5. Cell encapsulation

Cell encapsulation is similar to enzyme immobilization where cell is attached to solid support such as calcium alginate or activated polyvinyl alcohol (PVA) or activated polyethylene imine (PEI). The process of cell encapsulation is described in Figure 6. Cell immobilization confers many advantages to fermentation like reuse of fermenting microorganism, achievement of high cell concentration, easier product recovery, increased substrate uptake, lesser chances of contamination, and faster sedimentation of non-flocculating cells after completion of fermentation and greater tolerance against inhibitors and high temperature [27]. Cell encapsulation is beneficial when it comes to fermenting toxic lignocellulosic hydrolysates and also for improving cell stress tolerance.

Upon immobilization, yeast cells remain in close contact and form aggregates, and cells which are grown in a small space, show modified growth pattern and metabolism. In several cases, the resultant yeast community exhibits an improved protection from harsh and inhibiting conditions [84]. Membrane composition analysis of immobilized cells showed that there is increase in fatty acids, phospholipids and sterol content which confers an improved protection against high ethanol stress [85].

Ylitervo et al. [84] reported that encapsulated S. cerevisiae CBS8066 (a non thermotolerant yeast strain) successfully fermented 30 g/L glucose and produced high ethanol yield in 5 consecutive batches of 12 h duration at 42°C, as compares to freely suspended yeast, which was completely inactivated after third batch.

7.6. Physiological adaptation or evolutionary engineering

This method follows nature's engineering principle by variation and selection. In this method, genetic diversity is created by mutagenesis and recombination, and after that continuous evolution of large populations is processed under selection pressure like high temperature, sugar/salt concentration etc. over many generations relying on the cell's inherent capacity to introduce adaptive mutations [86]. Thermotolerance in the yeast can be developed by exposing the yeast strains to gradual increase in temperature (for example 2°C at each step) for a number of generations. Thermotolerance developed by this method is not attributed permanently to the yeast and it can be lost after few generations. Hence, physiological adaptation is not a suitable approach for development of thermotolerant yeast.

8. Benefits of high temperature fermentation using thermotolerant yeast

Thermotolerant yeast which can ferment sugars at temperature around 40°C can help to minimize the cost of ethanol production as discussed below.

8.1. Cooling costs

Production of bioethanol at higher temperature has received much attention because of several advantages like decrease in cooling costs, constant evaporation of ethanol from culture broth under reduced pressure, lower chances of contamination, applicability of process in tropical countries and the improved efficiency of process [53], [87],[88]. Abdel-Banat et al. [89] calculated that a 5°C increase in fermentation

temperature can significantly reduce the overall cost of ethanol production from starchy material with a thermostable a-amylase by reducing cooling energy. The continuous evaporation of ethanol from fermentation broth during fermentation maintains the ethanol concentration at the level which is not harmful to the fermenting microorganism, thereby simplifies subsequent distillation. These researchers carried out ethanol production from starch by supplementing glucoamylase (from Bacillus licheniformis) and yeast cell together and calculated that a net increase in benefits associated with a 5°C increase in the fermentation temperature would be around US$30,000 per annum for a 30,000 kL scale ethanol plant.

8.2. Cost reduction at the SSF stage

SSF is preferred over SHF because it can reduce the requirement of equipment, which reduces the overall investment for ethanol production. The combination of saccharification and fermentation simplifies the overall process of ethanol production. Alleviation of problem of feedback inhibition of cellulases by glucose helps in improving the saccharification efficiency and ethanol yield. However, the major disadvantage of the SSF process is the reduced efficiency of saccharification carried out at lower temperature to be compatible with yeast fermentation in comparison with the SHF process. Therefore, thermotolerant fermenting microbial strains which can produce significant amount of ethanol at high temperatures are more favorable for saccharification as well as essential for the improvement of efficiency of SSF. Thermotolerant yeast strains of genera Saccharomyces, Kluyveromyces and Fabospora that can produce more than 5% (w/v) ethanol at elevated temperature (>40°C) have been identified by several workers [l2],[32],[34],[35],[90],[91].

9. Limitations associated with high temperature fermentation

During the production of bioethanol from traditional system, yeast cells experience several kinds of stress like high sugar and ethanol concentration, low nutrients, and pH change [92],[93]. In addition to these, high temperature stress creates other metabolic problems during ethanol fermentation resulting in low alcohol yield [82]. Higher than normal temperature can damage the yeast cell in many ways, with the most serious effects being membrane disruption and protein denaturation and aggregation [94]. Growth at high temperature leads to the partial or total alteration of the native secondary, and/or tertiary structure of nucleic acids and proteins because of breakage of H-bonds and hydrophobic interactions [50]. Yeast cells do not have means to regulate their internal temperature, therefore cell viability decreases rapidly when temperature is increased beyond the optimal temperature.

10. Conclusions and future perspectives

Lignocellulosic biomass offers great potential for biofuel production particularly 2nd generation bioethanol which may contributes to a cleaner environment and carbon neutral cycle. Thermotolerant and ethanolgenic yeast strains would be beneficial for production of bioethanol by simultaneous saccharification and fermentation at elevated temperature as they offer the opportunity to improve the economy of overall process.

Thermotolerant yeast can be developed by mutation, genetic engineering, metabolic engineering and physiological adaptation. Fermentation at high temperature using thermotolerant yeasts simultaneously with saccharification offers several advantages such as reduced cooling cost, reduced requirement of utilities, higher saccharification efficiency and no feedback inhibition of cellulolytic enzymes. Cold active cellulases and hemicellulases from organisms inhabiting in temperate conditions, hydrolyze the biomass at low temperature as compared to commercial enzymes but with lower efficiency. Therefore, there is need to search for thermotolerant yeast and cold active hydrolytic enzymes to develop cost effective SSF process.

Since, the traditional process suffers from various technological gaps, modern methods of genetic engineering approaches like SDM or genome shuffling along with high throughput screening techniques may be employed for the development of improved yeast strains as well as for better expression of hydrolytic enzymes to suit SSF process. Functional genomics together with metabolic engineering may aid in developing robust yeast strains capable of utilizing full sugar component of lignocellulosic biomass. However, construction of recombinant strains has been limited to a few species such as K. marxianus and P. kudriavzevii because of lack of effective genetic tools. Comparison of metabolic profiles of thermotolerant yeast and well understood mesophilic S. cerevisiae might provide us insight in the thermotolerance mechanism of yeast. Combination of cold active cellulolytic enzymes and thermotolerant yeast can overcome the problem of different temperature optima in SSF process but more research need to be carried out on sugar uptake mechanism, effects of inhibitors on yeast growth and metabolic engineering for generating co-fermenting yeasts.

Financial support

The authors acknowledge the financial help received from the Department of Science and Technology (DST) and National Agricultural Science Fund (NASF), New Delhi (India).


We are thankful to Director Indian Agricultural Research Institute (IARI) New Delhi (India) for providing necessary facilities.


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Table 1. A brief overview of conditions employed by the researchers for production of bioethanol.

Process Yeast Enzyme loading Substrate loading Temp. (°C) % Theoretical yield Reference

SHF S. cerevisiae S. cerevisiae 50 FPU 3 U/ml FPase and 9 U/ml p-glucosidase 11.25% w/v (0.651g sugars/g dry substrate 37.47 g/L sugars 30 30 41.69 96 Sindhu et al. [10] Gupta et al. [11]

SSF K. marxianus TISTR5925 Blastobotrys adeninivorans RCKP-2012 K. marxianus S. cerevisiae 22.5 FPU 96.2 g/L sugars 8% w/v 10% w/v (380mg sugars/g carrot pomace) 26% dry matter of pretreated wheat straw 40 50 42 30 92.2 46.87 92 67 Murata et al. [12] Antil et al. [13] Yu et al. [14] Paschos et al. [15]

S. cerevisiae KE6-12 S. cerevisiae TMB3400 9 FPUg-1 WIS (water insoluble solids) Cellulase 30 FPU g-1 glucan 7.9%WIS 10% WIS 30 34 77 85 Koppram et al. [16] Bertilsson et al. [17]

SSCF S. cerevisiae, SyBE005 S. cerevisiae IPE003 p-Glucosidase 60 IU g-1 glucan 15 FPUg-1 dry matter 25% dry matter of pretreated corn stover 20% steam exploded corn stover 34 30 47.2 g/L 75.3% (60.8 g/L) Zhu et al. [18] Liu et al. [19]

CBP K. marxianus DBKKU Y-102 Trichoderma reesei Rut C30, S. cerevisiae and Scheffersomyces stipitis Scheffersomyces shehatae JCM 18690 K. marxianus Y179 22.9 U/ml inulinase 250 g/L sugars 17.5 g/L Avicel 10% starch liquid 227 g/L inulin 37 28 30 30 92 67 9.2 g/L 98 g/L Charoensopharat et al. [20] Brethauer and Studer [21] Tanimura et al. [22] Gao et al. [23]

Table 2. Various processes for LC ethanol production along with advantages and disadvantages.

Process Advantages Disadvantages

SHF • Both hydrolysis and fermentation are carried out at optimum temperatures separately • High cost requirement • Time consuming process as hydrolysis and fermentation carried out separately • Hydrolytic enzymes are subjected to end product inhibition

SSF • Reduction of cooling cost as no chiller unit is required • Improved hydrolysis efficiency • Reduced contamination risk • Continuous evaporation of ethanol from culture media under reduced pressure • Suitability for use in tropical countries • Temperature optima of hydrolytic enzymes and yeast are different • Reduced hydrolytic efficiency of cellulases at lower temperature • Ethanol concentration >0.2 M, disturb the adsorption of exoglucanase on cellulose and lower the hydrolytic efficiency • Presence of cellulase enzyme cocktails in same vessel affects the yeast growth • Difficulty in recycling of fermenting microorganism since it is mixed with the biomass

SSCF • Complete utilization of substrate • Reduced capital cost • Higher bioethanol productivity • Continuous removal of end products of saccharification resolve the problem of feedback inhibition • Xylose utilization requires aerobic conditions which inhibits glucose fermentation • Higher affinity of glucose to transporters creates problem in uptake of xylose

BP • Reduction in capital investment • Elimination of utilities associated with enzyme production • Single vessel for saccharification and fermentation reduces operational complexities • Simplification of operation • Reduction of contamination risk by reducing glucose concentration produced and ethanol produced • Improvement of hydrolysis efficiency by alleviating product inhibition of cellulase • High loading rates creates problem in mixing operation • Development of new efficient microorganism capable of co-producing hydrolytic enzymes and fermentation is very difficult

SSFF • Effective in enhancing cell performance • Facilitates complete utilization of biomass • Both biomass hydrolysis and fermentation are carried out at their optimum conditions • Clogging of membrane filters with high substrate loading is the main challenge

Table 3. Ethanol production from biomass using thermotolerant yeasts.

Organism Substrate T (°C) Ethanol yield (g/L) % theoretical yield Reference

S. cerevisiae ZM1-5 Sugarcane bagasse 40 18.79 82.35 Huang et al. [30]

K. marxianus DBKKU-Y102 Jerusalem artichoke 40 97.46 92 Charoensopharat et al. [20]

K. marxianus TISTR 5925 Palm sap 40 45.4 92.2 Murata et al. [12]

Blastobotrys adeninivorans Sugarcane bagasse 50 14.05 46.87 Antil et al. [13]

RCKP 2012

Pichia kudriavzevii HOP-1 Rice straw 45 24.25 82 Oberoi et al. [31]

K. marxianus OT-1 Jerusalem artichoke 40 73.6 90 Hu et al. [32]

S. cerevisiae JZ1C Jerusalem artichoke 40 65.2 79.7 Hu et al. [32]

S. cerevisiae TJ 14 161 g/L paper sludge material 42 40 74 Prasetyo et al. [33]

K. marxianus IMB3 Kanlow switch grass 45 22.5 86 Pessani et al. [34]

S. cerevisiae D5A Switch grass ^37 21.9 92 Faga et al. [35]

SHF process

Fermenting organism (Yeast)

Externally produced hydrolytic enzymes

Yeast biomass


(Physical, chemical. -> physicochemical and biological)

Enzymatic Hydrolysate

Sacchariflcation ->

at 50 °C

Lignocellulosic biomass

Fermentation at 30 °C



Fig. 1. The process flow of separate hydrolysis and fermentation (SHF).

Physical, Chemical, physicochemical and Biological

Holocellulolytic enzymes

Thermotolerant fermenting yeast

Delignified biomass

Simultaneous saccharification and fermentation (SSF)

Lignocellulosic biomass


Yeast biomass

Fig. 2. Bioethanol production by simultaneous saccharification and fermentation (SSF) process.

Lignocellulosic biomass


(Physical, chemical, physicochemical and biological)

Engineered yeast cells

Enzyme production Saccharification Fermentation

At high temperature


Fig. 3. Schematic representation of different processes involved in CBP for direct bioethanol production from lignocellulosic biomass.


(Physical, chemical, physicochemical and biological)


Lignocellulosic biomass

Fermentation liquid

Delignified biomass

Enzymatic saccharification

of delignified biomass

Membrane filtration


Fermentation of

sugar rich hydrolysate


Fig. 4. Bioethanol production from lignocellulosic feedstock using simultaneous saccharification, filtration and fermentation (SSFF).

Creation of parental library by mutagenesis (ethyl methyl sulfonate, N- methyl- N"- nitro-

Fig. 5. Schematic representation of genome shuffling.

Fig. 6. Procedure for encapsulation of yeast cells for SSF.