Biotechnological production of ethanol: Biochemistry, processes and technologies

Abstract

The majority of environmental problems arise from the use of conventional energy sources. The liability of such problems along with the reduction of fossil energy resources has led to the global need for alternative renewable energy sources. Using renewable biofuels as energy sources is of remarkable and continuously growing importance. Producing bioethanol through conversion of waste and residual biomass can be a viable and important perspective. In the first part of this review, general concepts, approaches and considerations concerning the utilization of the most important liquid biofuels, namely biodiesel and bioethanol, are presented. Unlike biodiesel (specifically first generation biodiesel), the production of bioethanol is exclusively based on the utilization of microbial technology and fermentation engineering. In the second part of this review, the biochemistry of ethanol production, with regards to the use of hexoses, pentoses or glycerol as carbon sources, is presented and critically discussed. Differences in the glycolytic pathways between the major ethanol‐producing strains (Saccharomyces cerevisiae and Zymomonas mobilis) are presented. Regulation between respiration and fermentation in ethanol‐producing yeasts, viz. effects “Pasteur”, “Crabtree”, “Kluyver” and “Custers”, is discussed. Xylose and glycerol catabolism related with bioethanol production is also depicted and commented. The technology of the fermentation is presented along with a detailed illustration of the substrates used in the process and in pretreatment of lignocellulosic biomass, and the various fermentation configurations employed (separate hydrolysis and fermentation, simultaneous saccharification and fermentation, simultaneous saccharification and co‐fermentation and consolidated bioprocessing). Finally, the production of bioethanol under non‐aseptic conditions is presented and discussed.

Abbreviations
• Adh
• aldehyde dehydrogenase
• AFEX
• Ammonia fiber explosion
• 1,3-BGP
• 1,3-biphosphoglycerate
• CBP
• consolidated bioprocess
• DHA-K
• DHA kinase
• DHAP
• dihydroxyacetone phosphate
• EMP
• Embdem-Meyenhorf-Parnas pathway
• EtOH
• ethanol
• FAD
• flavin-adenine dinucleotide
• FFV
• flexible-fuel vehicle
• GDH
• glycerol dehydrogenase
• G3P
• glyceraldehyde-3-phosphate
• GK
• glycerol kinase
• 3-HPA
• 3-hydroxypropionaldehyde
• OMW
• olive mill waste water
• 1,3-PD
• 1,3-propanediol
• Pdc
• pyruvate decarboxylase
• PEP
• phosphoenolpyruvate
• PFK
• phosphofructokinase
• PP
• pentose-phosphate pathway
• SCO
• single cell oil
• SHF
• separate hydrolysis and fermentation
• SSCF
• simultaneous saccharification and co-fermentation
• SSF
• simultaneous saccharification and fermentation
• TPP
• thiamine pyrophosphate
• TS
• total sugars
• μ_max
• maximum specific growth rate
• Y_EtOH/Gly
• conversion yield of ethanol produced per glycerol consumed
• Y_EtOH/TS
• EtOH yield per unit of TS consumed

1 General approaches related with the production of liquid biofuels (biodiesel and bioethanol)

The current significant expansion of the agricultural production, the subsequent numerous agro-industrial applications as well as the continuous and rapid increase of the world's population have led to an enormous need and utilization of all types of energy sources, both renewable or non-renewable ones. Furthermore, the decrease of petroleum stock, which up to date remains the main energy source that practically is a non-renewable one as well as its instability in price, have led to the worldwide rise both of the need and the of interest in alternative energy sources . On the other hand, it has been demonstrated that due to the utilization of the non-renewable energy sources, the greenhouse gas emissions have increased to ∼70% from 1970 to 2004 , while in 2012, these emissions have presented an increment of 2.6 and 58% compared with the emissions in 2011 and 1990 respectively . From the above-mentioned analysis it can easily be understood that utilization of various renewable biofuels as energy sources in engines and heating systems has become of remarkable and continuously growing importance . Two of the most important types of liquid renewable biofuels that are currently available in the market are biodiesel (green diesel) and bioethanol (with both types of these fuels already being used for various types of diesel engines and heating systems) .

Biodiesel is prepared through trans-esterification of vegetable oils, animal fats, waste/used fats/oils and microbial oils with short chain alcohols (principally methanol and to lesser extent ethanol or butanol) . Despite the continuous need for biodiesel production, the last year's lack of oil feedstocks has created problems related with the production of conventional or so-called first generation biodiesel, since between 2007 and 2008 there has been an almost two-fold increase of the price of conventional plant commodity oils . Potential responses to the above-mentioned situation was the production of “non-conventional oils” with the aid of microorganisms capable of producing intracellular lipids, the so-called single cell oils (SCOs), that would further be amenable to be converted into biodiesel (the so-called second-generation biodiesel or third generation biodiesel, derived through atmospheric CO₂ sequestration ). However, up-to-date, the production cost of SCOs remains always much higher than that of plant oils .

While in the case of biodiesel the microbial technology and the applied microbiology are implicated only concerning the production of second- or third-generation biodiesel (production that is restricted in industrial level), in all stages and all cases of bioethanol production there is an enormous implication of the above-mentioned scientific domains. Most probably, the case of bioethanol production (together with the production of edible fungi growing on several types of solid agro-industrial resources; for a review see ) represents the most industrialized processes of the applied microbiology worldwide. The production of bioethanol is accompanied by serious economic and environmental benefits, since ethanol as a fuel presents a high octane number , while even small amounts of ethanol added into the gasoline can significantly increase the octane number of the blend . Moreover, the higher oxygen content improves the efficiency of the combustion . Also greenhouse gas emissions are generally considered to be reduced and ethanol burn has as result lower emission in carbon monoxide (CO), volatile organic compounds, sulfur oxides, etc. in comparison with the burn of the typical fossil fuels. However, recent findings have indicated that the employment of bioethanol as fuel increased nitrogen oxides, acetaldehyde and formaldehyde emissions compared with the typical fossil fuels . Moreover, blends of ethanol up to 10% w/w in gasoline results in increase of the reid vapor pressure, which has as result the increment of the volatility of the blend compared with the gasoline without added ethanol .

Globally, bioethanol is the largest volume biofuel used in the transportation sector, with corn-derived (starch-based) ethanol production occurring mostly in the US and sugarcane-derived (saccharose-based) ethanol production occurring mostly in Brazil . First generation (derived from fermentation of hydrolyzed edible starchy materials or edible simple sugars) and especially second-generation bioethanol (derived through valorization–conversion of several waste streams) are the most promising renewable energy sources applied worldwide . On the other hand, despite its recent tremendous expansion, use of ethanol as a vehicle fuel or energy source is known already from the early 1800s; in 1826, Samuel Morey used alcohol in the first American internal combustion engine prototype, while in the 1860s in the US, thousands of distilleries produced at least 90 million US gallons (340 000 m³) of ethanol per year, that were mainly used for the purpose of lighting. In 1860, the German inventor Nikolaus Otto used ethanol for the first time as a fuel in an early internal combustion engine . The first cars produced by the “Henry Ford” companies, (e.g. the “Model T”) were capable of running by using exclusively ethanol. However, the decreasing cost of gasoline and the later introduction of the “Prohibition in the US” rendered ethanol as an impractical fuel for most users, resulting in the utilization of other types of fuels, viz. gasoline and kerosene. Currently, about 820 million cars and light trucks are working with ethanol . Ethanol can be used in blends with gasoline in which the percentage of ethanol into the mixture varies from 5% (E5) to 100% (E100). E10, a fuel mixture of 10% anhydrous ethanol and 90% gasoline, sometimes called “gasohol”, is used in the internal combustion engines of modern automobiles and light-duty vehicles without modifications on the engine or fuel system. E10 blends are typically rated as being 2 to 3 octane numbers higher than regular gasoline and are approved for use in all new US automobiles, and mandated in some areas for emissions and other reasons . However, as the ethanol percentage in the blend increases some modifications are necessary (e.g. in the fuel injection system, in the evaporation system, etc.). Finally, for ethanol addition higher than 25% into the gasoline, engine modifications should also be realized .

Research on bioethanol production is mainly performed along two axes. The first axis refers to the discovery of new natural microorganisms (or the “construction” of genetically engineered ones) that produce ethanol at significant final product concentrations and high volumetric productivities and/or in small quantities antagonistic to ethanol metabolites (i.e. glycerol). Under this optic, genetic engineering works have been developed in order to construct strains (mainly of the microorganisms S. cerevisiae and Zymomonas mobilis) capable of consuming pentoses (e.g. xylose, arabinose), sugars that are found in significant quantities into the lignocellulosic biomass . The second axis is focused on both process optimization, modeling and development of new fermentation configurations (e.g. simultaneous saccharification and fermentation, consolidated bioprocess, etc.) as also in the potential of the efficient utilization of various by-products and waste or raw renewable materials used as substrates in this type of conversion . Finally, with the rationale of achieving significant reduction of the operating costs of the process an important innovation recently developed in biotechnological processes refers to the accomplishment of the bioprocess under completely nonaseptic conditions .

2 Global ethanol production

Ethanol is used as a raw material for a wide range of applications, including chemicals, fuel (bioethanol), beverages, pharmaceuticals and cosmetics . The majority (90-95%) of the percentage of ethanol that is produced globally derives from biological fermentation technology (bioethanol), whereas the rest is produced using ethene (coming from cracking of crude oil and/or natural gas) using steam and phosphoric acid as catalyst (synthetic ethanol). The reaction of synthetic ethanol production also produces toxic by-products, thus is not used for human consumption. Bioethanol as fermentation product stream is processed with subsequent enrichment by distillation/rectification and dehydration. During World War II, when wartime conditions changed economy and priorities, several ethanol-from-cellulose (EFC) plants were built in Germany, Russia, China, Korea, Switzerland and the US, among other countries, to provide an alternative fuel source. Since the end of the war, competition from synthetically produced ethanol has forced many of these plants to close . Since April 2004, the first demonstration plant using lignocellulosic feedstocks in Canada has been in operation . In 2006, for the Global ethanol market Brazil disposed more than 300 bioethanol-producing plants, producing 15 billion liters per year and supplying 3 million cars with pure ethanol. In the US, there were more than 80 plants producing 10 billion liters per year . However, the last year's USA took the lead presenting an impressive increase in the annual ethanol production. On the other hand, ethanol production in Europe represented 5% of the global production in 2008, with Germany and France being the main producers . Despite the fact that there is an increasing interest in utilizing alternative sources for ethanol fermentation (viz. waste materials), the main sources of ethanol production in Europe are cereals and sugar beet . On the other hand, the US has produced about 50 billion liters in 2012 alone. As previously indicated, the majority of the world's ethanol is produced by the US and Brazil together reaching values from 62 to 87% of the global ethanol production . The vast majority of US ethanol is produced from hydrolyzed starch deriving from corn, while bioethanol in Brazil primarily derives from sugar originated from sugar-cane. China, India, Eastern Europe, Western Europe and finally Canada are following.

3 Ethanol as fuel

With its clean burning characteristics ethanol as a fuel can play a significant role in the reduction of the greenhouse gas emissions given that it reduces polluting gases, mainly carbon dioxide. Moreover, by using crude agro-industrial residues as raw alcoholic fermentation starting materials, not only alternative substrates are provided but also their disposal problem can be solved. Moreover, ethanol as a biodegradable and relatively highly soluble in water material, has low toxicity, therefore the consequences of large fuel spilling threaten far less the environment than those associated with crude oil or gasoline . Finally, the potential of bioethanol production under completely nonaseptic conditions renders this bioprocess as a very important one with imminent applications in the industrial sector.

Ethanol fuel can be used as blend with gasoline in percentages from 5 to 85% . The most popular blends are known as E85 (85% bioethanol, 15% gasoline), E20 (20% bioethanol, 80% gasoline) and E10 (10% bioethanol, 90% gasoline; called also gasohol in USA) . There is also a mixture used in Brazil, called gasohol and contains 24% bioethanol blended with 76% gasoline . Blends having higher concentrations of bioethanol can be used in flexible-fuel vehicles (FFVs) that can operate on mixtures up to 85% bioethanol (E85) . Bioethanol can be also converted into etherized bioethanol (ethyl tertiary butyr ether; ETBE), which can be used as a 15% blend with gasoline . Finally, ethanol may be used in the trans-esterification of vegetable oils for the production of fatty acid ethyl esters (one of the commonly used forms of biodiesel) . As far as biodiesel blends with petroleum are concerned, their nomenclature is similar with the one of bioethanol/gasoline blends, where the letter “E” is changed to the letter “B”; blends up to B20 can be directly used in common engines whereas higher blends might require some few engine modifications .

4 Biochemistry of alcoholic fermentation 4.1 General concepts

Irrespective of the (simple or complex) substrates utilized as microbial carbon sources amenable to be converted into ethanol, finally, the (enzymatic, physical, chemical, mechanical, etc.) pretreatment of the several types of substrates results in the generation of hexoses, pentoses or glycerol that will be fermented by the relevant microorganisms in order to be converted into bioethanol. The biochemical processes which produce “the universal money of free energy”, namely the ATP are substrate-level phosphorylation and oxidative phosphorylation. Substrate-level phosphorylation takes place during glycolysis and can be either aerobic or anaerobic. On the other hand, oxidative phosphorylation is an aerobic process and occurs in the mitochondria. The production of ATP is linked to the transport of electrons to an oxygen molecule by the cytochromic respiratory chain. This oxygen molecule is the final acceptor of the electrons .

The major simple carbon sources amenable to be converted into ethanol are glucose (and other hexoses), disaccharides (mainly saccharose), xylose and glycerol. Glucose can be converted into ethanol with the aid of the most important ethanol producers, mainly the yeast S. cerevisiae and the bacterium Z. mobilis . Xylose can be directly fermented with yeasts, such as Pachysolen tannophilus, Candida shehatae and Pichia stipis, or by isomerization of xylose to xylulose with the enzyme glucose (xylose) isomerase (XI; EC 5.3.1.5), and subsequent fermentation with bakers' yeast, S. cerevisiae . Glucose transporters mediate xylose uptake, but no transporter specific for xylose has yet been identified for the wild-type strains of S. cerevisiae. Overexpressing genes for aldose (xylose) reductase, xylitol dehydrogenase, xylose isomerase and moderate levels of xylulokinase could potentially enable xylose assimilation and fermentation by genetically engineered S. cerevisiae strains . Finally, in a not very high (but increasing) number of reports, glycerol has been converted into ethanol with the aid of bacterial or yeast strains .

Alcoholic fermentation biochemistry includes substrate degradation pathways (glycolysis, alcoholic fermentation, glyceropyruvic fermentation and respiration for the case of the utilization of hexoses, xylose catabolic pathways for the case of utilization of pentoses and glycerol assimilation and glycolysis for the case of glycerol-converting microorganisms) and regulation between fermentation and respiration (Pasteur effect, Crabtree effect, Kluyver effect and Custers effect) . The glycolysis will be of the Embdem-Meyenhorf-Parnas (EMP) pathway (for S. cerevisiae and glycerol-consuming microorganisms), of the pentose-phosphate (PP) pathway (for the xylose-consuming microorganisms) and of the Entner-Dudoroff pathway (for Z. mobilis bacteria) .

4.2 Embdem-Meyenhorf-Parnas (EMP) pathway

Embdem-Meyenhorf-Parnas (EMP) pathway, a series of reactions that take place completely in the cytosol, is the process of intracellular transformation of glucose (and fructose) into pyruvate together with the formation of ATP and NADH . Initially, hexoses are transported inside the cell, principally by facilitated diffusion without the need of energy consumption . The yeast contains several glucose transporters namely Hxt1, Hxt2, Hxt3, Hxt4, Hxt6, Hxt7 and Gal2 .

The first stage of glycolysis is the conversion of glucose into fructose 1,6-biphosphate, requiring 2 ATP molecules, which comprises three steps: an initial phosphorylation of glucose toward the formation of glucose 6-phosphate, catalyzed by a family of enzymes called hexokinases (PI hexokinase is not active until stationary phase, as it is repressed partially by glucose; PII hexokinase is essential and predominantly active during the log phase in a high sugar concentration medium). This reaction consumes ATP, but it keeps the intracellular hexose concentration low and thus favors the continuous transport of sugars into the cell through the plasma membrane transporters. Afterward, the isomerization of glucose 6-phosphate into fructose-6-phosphate by phosphoglucose isomerase takes place, followed by the phosphorylation of fructose-6-phosphate by the action of phosphofructokinase (PFK), forming fructose-1,6-biphosphate and consuming one ATP molecule . The enzyme PFK is one of the limiting steps of the EMP glycolytic pathway, since it is allosterically activated by the presence of AMP into the microbial cell (in AMP-rich cells there is acceleration of glucose uptake and glucose catabolism since microbial cell needs ATP), while it is allosterically deactivated by the presence of ATP (the microbial cell does not need more glucose since it is “energy rich”) . On the other hand, in the case of imbalanced growth conditions (e.g. depletion of nitrogen into the culture medium), and due to the rapid disappearance of the cellular AMP (that is degraded by AMP desaminase in order for the cell to be provided by nitrogen), the absence of AMP blocks in this level the glycolytic pathway, resulting in the accumulation of storage polysaccharides .

At the second stage of glycolysis (C3 pathway), two triose phosphate isomers, namely glyceraldehyde-3-phosphate (G3P) and dihydroxyacetone phosphate are formed when fructose 1,6-biphosphate is cleaved by the catalytic action of aldolase. This reaction produces a much greater proportion of dihydroxyacetone phosphate (DHAP) (96%), which is rapidly transformed into G3P by triose phosphate isomerase . Then, G3P is converted to 1,3-biphosphoglycerate (1,3-BGP), reaction catalyzed by glyceraldehyde 3-phosphate dehydrogenase. This reaction involves the synthesis of one mole of NADH. Thereafter, 1,3-BGP is converted into 3-phosphoglycerate, reaction catalyzed by phosphoglycerate kinase, with simultaneous release of one mole of ATP. The last step of glycolysis transforms 3-phosphoglycerate into pyruvate which is the final product of glucolysis. Phosphoglycero-mutase catalyzes the conversion of 3-phospho-glycerate into 2-phosphoglycerate which is dehydrated by the enzyme enolase, forming phosphoenolpyruvate (PEP). Finally, PEP is converted into pyruvic acid, reaction catalyzed by pyruvate kinase, with simultaneous formation of another mole of ATP . In this manner, from one mole of glucose, glycolysis creates two moles of pyruvic acid, four moles of ATP and two moles of NADH. Since two molecules of ATP had been immediately used to activate a new hexose molecule, the net energy gain of glycolysis for the cell is therefore two ATP molecules per molecule of hexose metabolized. Pyruvate produced by glycolysis can be used by yeasts for several metabolic pathways. Evidently, the microorganism must regenerate NAD⁺ from the NADH to reestablish the oxido-reduction potential of the cell. This can be done by fermentation or respiration. This stage marks the end of the common trunk of glycolysis. The reactions of alcoholic fermentation, glyceropyruvic fermentation or respiration follow, depending on various conditions . On the other hand, during glycerol assimilation by fungi, yeasts, (heterotrophic) algae or bacteria, by one mole of glycerol there will be the formation of 1 mole of DHAP that will be subsequently converted into pyruvate with the reactions mentioned above.

It should be stressed out that the microorganism Z. mobilis catabolizes the sugar substrate through the Entner-Dudoroff glycolysis pathway whereas, yeast strains in general break down sugar substrates through the EMP glycolysis pathway (for more information concerning the Entner-Dudoroff see next paragraphs).

4.3 Alcoholic fermentation

The reducing power of NADH produced through glycolysis must be transferred to an electron acceptor to regenerate NAD⁺ consumed by glycolysis. In the case of S. cerevisiae and other yeast species, this process is called alcoholic fermentation and takes place within the cytoplasm, where acetaldehyde (ethanal; the decarboxylation product of pyruvate acid) serves as the terminal electron acceptor . With respect to glycolysis, alcoholic fermentation contains two additional enzymatic reactions. Pyruvate is initially decarboxylated into acetaldehyde by pyruvate decarboxylase. The cofactors are thiamine pyrophosphate (TPP) and magnesium. Then, acetaldehyde is reduced into ethanol recycling NADH to NAD⁺. This reaction is catalyzed by the alcohol dehydrogenase using zinc as cofactor. Both final products of alcoholic fermentation, ethanol and carbon dioxide, are transported outside the cell by simple diffusion .

4.4 Glyceropyruvic fermentation

Besides production of ethanol as the most important pathway to regenerate NAD+, glyceropyruvic fermentation is the alternative pathway for this purpose (generating glycerol as its final product . Dihydroxyacetone phosphate (DHAP; the main product of aldolase reaction) is oxidized to glycerol-3-phosphate (by the enzyme glycerol-3-phosphate dehydrogenase) while a molecule of NADH is simultaneously oxidized to NAD⁺. Then, the enzyme glycerol-3-phosphate phosphatase catalyzes the production of glycerol by dephosphorylating glycerol-3-phosphate. The production of glycerol consumes ATP but it is necessary to compensating for the redox imbalance in the cell. In general, at the first stages of fermentation, when yeasts perform a very active growth rate accompanied by the biosynthesis of proteins, lipids and nucleotides, glyceropyruvic fermentation occurs preferentially. Most of the aforementioned biomolecules are synthesized using pyruvate as substrate. Each time a molecule of pyruvate is used anabolically, an NAD⁺ deficit is created which must be recovered through the glyceropyruvic pathway. For this reason, glycerol is mainly produced during the first steps of alcoholic fermentation, when yeasts are growing and they need a large proportion of pyruvate to increase their biomass. Furthermore, yeasts produce glycerol as a protector against high osmotic pressures .

Glyceropyruvic fermentation can a completely desirable process (in the case in which it is desirable to convert glucose into glycerol; this was the case of several types of works that predominated 10–15 years ago and before the large expansion of biodiesel production and the subsequent tremendous increase of glycerol into the market volume) or an undesirable metabolic pathway (in the case in which the main metabolic pathway that is desired to be produced is that of ethanol, and, thus, the carbon flow of glucose toward ethanol should be maximized) . Two different strategies for modulating (maximizing or minimizing) the production of glycerol by S. cerevisiae have been reported in the literature; one principal strategy is related with the change the operating conditions of the fermentation process (i.e. alteration of aeration levels, addition of vitamins, stress conditions like osmotic stress, etc) that can maximize or minimize the produced glycerol through glucose break-down . The other approach is related with the use of genetically modified strains. Engineered strains were constructed by overexpressing and repressing components of the glycerol synthesis pathway in order to maximize or minimize the production of glycerol by the tested strains (e.g. construction of mutants presenting over-expression or disruption of GPD1 gene encoding glycerol 3-phosphate dehydrogenase , construction of mutants in which GLN1 gene, encoding glutamine synthetase, and GLT1 gene, encoding glutamate synthase, were over-expressed, and GDH1 gene, encoding the NADPH-dependent glutamate dehydrogenase, was deleted ).

4.5 Respiration

Yeasts are facultative anaerobic microorganisms because they possess the genetic equipment for metabolizing sugars aerobically or anaerobically. Therefore, sugars can be consumed also by respiration, in which when the implicated sugars (or glycerol) are used by the respiratory pathway, pyruvic acid undergoes an oxidative decarboxylation, catalyzed by pyruvate dehydrogenase in the interior of mitochondria. TPP, lipoamide and flavin-adenine dinucleotide (FAD) serve as catalytic cofactors . This reaction reduces NAD⁺ to NADH and must incorporate acetyl coenzyme A (acetyl-CoA) (the acetyl unit issued from pyruvate is activated in the form of acetyl-CoA). Acetyl-CoA can then be incorporated to the citric acid cycle (tricarboxylic acids cycle or Krebs cycle), so as to be completely oxidized into carbon dioxide and to produce reducing power (NADH and FADH). These reactions also occur in the mitochondria . The respiration of 1 mole of glucose yields an overall energy gain of 36–38 moles of ATP . Thus, respiration of the same amount of sugar produces 18 to 19 times more biologically usable energy available to yeasts than fermentation indicating that this is a much more beneficial in terms of energy process than the one of fermentation. In any case, the transformation of pyruvate into acetaldehyde or acetyl-CoA is therefore a key point for regulating yeast metabolism . All processes previously indicated (EMP glycolysis, respiration, glyceropyruvic fermentation, alcoholic fermentation) are described in Fig. .

Biochemical pathways of glycolysis, alcoholic fermentation, respiration and glyceropyruvic fermentation (adapted by Zamora ).

4.6 Regulation between respiration and fermentation

For low concentrations of glucose on culture media (where glucose does not suppress oxidative phosphorylation), S. cerevisiae yeast (or other glucose-fermenting yeasts) utilizes glucose through either respiration or fermentation. The presence of oxygen induces the respiratory chain and the production of biomass but simultaneously decreases the kinetics of sugar fermentation and simultaneous ethanol production. In fact, these conditions are used for the industrial production of selected dry yeast . On the other hand, low concentrations of oxygen result in the catabolite repression of the first enzymes of the Krebs cycle with simultaneous increase of enzymes-keys of the EMP glycolysis (e.g. phospho-fructokinase), resulting in the direction of the cellular metabolism toward ethanol production. This is the so-called “Pasteur effect”, while yeast species are generally categorized as either Pasteur positive or negative . Therefore, according to Pasteur effect in several yeast strains, there is suppression of alcoholic fermentation in the presence of oxygen into the culture medium . Once the microorganism starts to consume sugars, large quantities of carbon dioxide are produced, the oxygen is displaced and semi anaerobic conditions that favor fermentation are created. However, even in the presence of oxygen, if sugar concentration is higher than a critical value (e.g. ∼9 g/L) respiration is impossible and the microorganism only metabolizes sugars by the fermentative pathway. This phenomenon is known as the “Crabtree effect” (named after the English biochemist Herbert Grace Crabtree), Pasteur contrary effect and as catabolic repression by glucose . In a high sugar concentration into the culture medium, the mitochondria degenerate, the proportion of cellular sterols and fatty acids decreases and both the enzymes (e.g. iso-citrate dehydrogenases) involved in the oxidative part of the metabolism (namely the Krebs cycle and the oxidative phosphorylation chain) and the constituents of the respiratory chain undergo catabolite repression . The metabolic network is directed toward the synthesis of ethanol via fermentative conversion, despite the presence of oxygen. On the other hand, S. cerevisiae cells need oxygen (at least during the first hours after inoculation). Oxygen-dependent reactions result in the biosynthesis of sterols, unsaturated fatty acids and phospholipids needed in the formation of cell membranes and, thus, the continuation of growth. S. cerevisiae can only, therefore, use respiration when both the sugar concentration is very low and oxygen is present in the medium .

The last year's several investigations in the tumor cell metabolism have demonstrated that tumors display enhanced glycolytic activity and impaired oxidative phosphorylation indicating that from a metabolic point of view, the yeast S. cerevisiae and tumor cells present several common features (in the cancer cells the above-mentioned phenomenon is called “Warburg effect”) . Therefore, it seems potential that disruption of glycolysis in the cancer cells could have been a promising tool for the development of specific anticancer therapies. Regarding energy metabolism, the glucose-induced repression of oxidative metabolism of yeast and the Warburg effect of tumor cells are very similar, since in both cell types, the oxidative metabolism is downregulated and fermentation enhanced despite the presence of oxygen . As indicated in the previous paragraphs, S. cerevisiae is a Crabtree-positive microorganism , and based on the above-mentioned effect, S. cerevisiae could be considered as an amenable metabolic model microorganism for the screening of metabolism-targeted drugs employed for antitumor therapy .

It should be also mentioned that the yeast strains capable of producing ethanol by fermentation, present a so-called “diauxic shift” (or “biphasic growth”), if oxygen is found into the growth medium. Given that they metabolize available sugar by fermentation, biomass is created and after depletion of sugar from the growth medium ethanol is oxidized (re-consumed) and new cell material is created . Specifically, in this shift two phases are observed together with two different maximum specific growth rates (μ_max), during the growth of the microorganism . At the first growth phase ethanol and carbon dioxide are the major products from assimilation of glucose via aerobic fermentation . When ethanol is available but no sugars are found in the medium and the dissolved oxygen concentration is above a critical level, then, previously produced ethanol is the only carbon source and serves as a substrate for further yeast growth . At this point, a lag in the yeast growth is observed due to occurrence of an onset of biosynthesis of the enzymes responsible for the gluconeogenesis . According to Piškur et al. the metabolism in Crabtree-positive yeast strains changes after exhaustion of glucose and accumulation of ethanol, with the requirement of certain transcription factors and enzymes. The (ethanol) “make-accumulate-consume” strategy (Fig. ) relies on the evolution of Saccharomyces against its competitors as ethanol is toxic to most other microbes. Therefore it is considered that in a (nonaseptic) sugar-rich environment, Saccharomyces kills its competitors by producing ethanol, but can also consume the generated ethanol later. Alcohol dehydrogenase (Adh) catalyzes the acetaldehyde-to-ethanol conversion (during aerobic or anaerobic fermentation) in both directions. Genes ADH1 (expressed constitutively) and ADH2 (expressed only when the internal sugar concentration drops) encode cytoplasmic Adh activity.

Glucose and ethanol assimilation by Saccharomyces cerevisiae under aerobic conditions. The conversion between acetaldehyde and ethanol is catalyzed by alcohol dehydrogenase (Adh). Gene ADH1 is expressed constitutively, whereas gene ADH2 is expressed only when the intracellular sugar concentration drops (adapted from Sarris et al. ).

Besides Pasteur and Crabtree effects, there exist two other types of regulation of fermentative metabolism in the alcohol-producing yeast strains, namely Kluyver and Custers effects. The former (Kluyver effect) indicates the absence of alcoholic fermentation during oxygen-limited growth on a sugar (often a disaccharide), even though glucose is readily fermented . It is known that yeast species can grow on various sugars. However, sometimes growth on certain sugars (especially oligosaccharides) occurs only under aerobic conditions, and not in anaerobiosis or in the absence of respiration. Fermentation is blocked under these conditions. This dependence of sugar utilization on respiration is called the Kluyver effect, and respiration-dependent species are called Kluyver-positive . Finally, the Custers effect refers to the inhibition of fermentation under anaerobic conditions, and has been considered as a common biochemical characteristic of the yeasts belonging mainly to the genera Brettanomyces and Dekkera . These yeasts ferment glucose into ethanol and acetic acid under aerobic conditions . However, upon a shift to anaerobic conditions, fermentation is strongly inhibited. This phenotype can be fully rescued by the reintroduction of oxygen into the medium .

4.7 Entner-Dudoroff pathway: Ethanol production by the bacterial strain Zymomonas mobilis

Besides eukaryotic microorganisms such as S. cerevisiae, Saccharomyces uvarum (carlsbergensis), Saccharomyces rouxii, Kluyveromyces fragilis, Kluyveromyces lactis, Candida tropicalis, Candida oleophila, Hanseniaspora spp. and Mucor rouxianus that have been reported capable of producing alcohol in various culture conditions and configurations, ethanol can be produced by various prokaryotic microorganisms belonging to the species Z. mobilis, Clostridium acetobutylicum, Clostridium thermosaccharolyticum, Clostridium sporogenes, Thermoanaerobacter ethanolicus and Bacillus stearothermophilus . Given that in an industrial point of view, bioethanol has been in general produced by strains belonging to the species Z. mobilis and Saccharomyces spp. (usual species that are mentioned in the literature to produce large amounts of ethanol are S. cerevisiae and Saccharomyces carlsbergensis - uvarum), the present part will focus upon the biosynthesis of ethanol by Z. mobilis. Like the yeast Saccharomyces spp., the bacterium Z. mobilis is incapable of metabolizing pentoses (e.g. xylose or urban, industrial or other agro-industrial wastes containing xylose), hence, genetically engineered microorganisms of the above species capable of breaking down xylose are needed in order to proceed with this fermentation . Zymomonas mobilis produces ethanol under anaerobic conditions whereas Saccharomyces spp. produces ethanol under both aerobic and anaerobic conditions (see previous Crabtree effect). The reaction efficiency in both instances is the same, giving generation of 2 moles of ethanol and 2 moles of carbon dioxide from 1 mole of catabolized sugar . Fundamental biochemical differences exist between the fermentation carried out by Z. mobilis compared with the one conducted by the yeast strains. The microorganism Z. mobilis catabolizes the sugar substrate through the Entner-Dudoroff glycolysis pathway (Fig. ) whereas yeast strains break down sugar substrates via the EMP glycolysis pathway (Fig. ) .

Glucose metabolic pathways in Zymomonas mobilis (GK: glucokinase, GPDH: glucose-6-phosphate dehydrogenase, PGL: phosphogluconolactonase, EDD: 6-phosphogluconate dehydratase, KDPG: 2-keto-3-deoxy-6-phosphogluconate, GAPDH: glyceraldehydes-3-phosphate dehydrogenase, PGK: phosphoglycerate kinase, PGM: phosphoglyceromutase, ENO: enolase, PYK: pyruvate kinase, PDC: pyruvate decarboxylase) (adapted from Koutinas et al. ).

From this analysis it can be deduced that in the fermentation carried out by Z. mobilis strains, for 1 mol of catabolized hexose there is a generation of 1 mole of ATP produced, while in the conversion carried out by yeast strains, 2 moles of ATP are produced per mole of hexose catabolized. The above fact has as a consequence, hence, production of less biomass when Z. mobilis strains are used compared with the fermentation carried out by yeasts. In contrast, as it has been already reported, no alterations in the final spectrum of end-fermentation products is observed, given that in both instances the stoichiometric reaction is the same, giving generation of 2 moles of ethanol and 2 moles of carbon dioxide from 1 mole of catabolized sugar, the maximum theoretical yield, hence, in both cases (utilization of both Z. mobilis or yeast strains) is ∼0.51 g of ethanol produced per 1 g of sugar consumed .

Various other biochemical differences are observed between the fermentation carried out by Z. mobilis or yeast strains, given that in the former case the metabolism of Z. mobilis can also lead to the production of fermentation by-products such as mannitol, sorvitol, dihydroxy-acetone and principally, the exo-polysaccharide levane, while Saccharomyces spp. strains produce (besides ethanol) mainly glycerol . It is also noted that pyruvic acid decarboxylase of Z. mobilis strains is not common (it does not require as activation co-factor pyrophosphate thiamine – TPP) while in bacterial membranes the presence of various opanoids (specific triterpenoids), the biosynthesis of which is not related at all to oxygenation of the culture medium, is common. Additionally, uncommon cellular fatty acids (e.g. ^∆11C18:1) are detected in the membranes of Z. mobilis strains . In contrast, the necessity of slight oxygenation even at the early growth stage is obligatory for the case of Saccharomyces spp. strains, in order for the yeasts to synthesize their cellular unsaturated fatty acids (e.g. ^∆9C16:1, ^∆9C18:1, ^∆9,12C18:2 – it is known that reactions of dehydrogenation occur only under aerobic conditions ), while unusual cellular fatty acids are not common . Yeast strains also present in non-negligible quantities ergosterol in their total cellular lipids, given that this component provides cellular stability and tolerance against ethanol . Both Z. mobilis and Saccharomyces spp. strains can tolerate up to 120–140 g/L of ethanol .

As presented above, biomass production by Z. mobilis strains is almost two-fold less, compared with the alcoholic fermentation performed by S. cerevisiae. The ethanol yield on sugar assimilated for both microbial sources used is comparable, with maximum theoretical ethanol (EtOH) yield per unit of total sugars (TS) consumed (Y_EtOH/TS) being 0.51 g/g for both microorganisms used. Therefore, although the conversion carried out by Z. mobilis strains can, in some cases, have as result slightly higher volumetric productivities achieved compared with the yeast fermentation , cultures led by S. cerevisiae attract interest due to the (higher) concentration of the process by-product (biomass) which can be utilized as animal feed. Finally, other “nonclassical” ethanol producers (e.g. bacteria of the genus Thermoanaerobacter) have been successfully performed in several types of sugar substrates .

4.8 Xylose catabolism and ethanol production

By taking into consideration that the pentose sugar D-xylose comprises about 1/3rd of the total carbohydrate sugars in the lignocellulosic biomass, significant scientific work has been dedicated to the understanding of the microbial metabolism of xylose and the subsequent production of ethanol by this pentose. As it has been already demonstrated in several review-articles related with the xylose fermentation, numerous microorganisms are capable of carrying out direct fermentation of xylose to ethanol . Yeasts with the capacity for direct fermentation of xylose include but are not limited to the genera Brettanomyces, Candida, Kluyveromyces, Clavispora, Pachysolen, Pichia and Schizosaccharomyces. The most potential wild-type ethanol-producing yeast species that have been reported in the literature belong to the species C. shehatae, P. tannophilus, Kluyveromyces marxianus and Pichia stipitis (for reviews see: ). On the other hand, a variety of wild-type fungal and bacterial genera can carry out direct fermentation of xylose; fungal genera, which in many instances exhibit high yields but suffer from low rates, include but are not limited to the genera Fusarium, Monilia, Mucor, Neurospora, Paecilomyces, Polyporus and Rhizopus . It must be pointed out here, that in several instances there has been employed the so-called consolidated bioprocess (CBP), in which lignocellulosic materials (rich in xylose and glucose) were employed as substrates and secretion of hydrolytic enzymes and production of ethanol by one and the same microorganism had been performed, by species of the above-mentioned fungal species, due to the ability of these microorganisms to convert xylose (besides glucose) into ethanol . (In this process configuration, ethanol productivities are generally lower than the typical conversions of simple sugars into ethanol by S. cerevisiae; see: Koutinas et al. ). Bacterial species found in both mesophilic and thermophilic genera can convert xylose into ethanol; mesophilic genera include Aerobacter, Aeromonas, Bacillus, Bacteroides, Erwinia and Klebsiella, while thermophilic bacteria include the genera Clostridium and Thermoanaerobacter .

The major metabolic pathways for xylose fermentation are similar as regards bacterial, yeast, and fungal species; after transport into the cell and conversion to xylulose-5-phosphate, ethanol production from xylose is performed by way of the PP and EMP pathways (Fig. ) . Xylose must be converted to xylulose and then should be phosphorylated to xylulose-5-phosphate before entering into the PP cycle. Finally, within the PP cycle, xylulose-5-phosphate is metabolized to glyceraldehyde-3-phosphate and fructose-6-phosphate, and then these compounds are converted to pyruvate in the EMP pathway. The pyruvate is finally converted to ethanol. Besides PP pathway, xylose can also be metabolized through the phosphoketolase reaction, which converts xylulose-5-phosphate to glyceraldehyde-3-phosphate and acetyl-phosphate . Glyceraldehyde-3-phosphate enters glycolysis, whereas acetyl-phosphate is converted to acetate . It is noted that the phosphoketolase reaction is slightly more efficient pathway yielding ∼1.2 moles of acetyl-CoA per 100 g of catabolized xylose (∼0.66 moles) compared with the PP pathway, wherein ∼1.0 mole of acetyl-CoA is generated per 100 g of xylose utilized . The maximum theoretical yield of ethanol produced per unit of xylose consumed is 0.51 g/g . Finally, intense research in the past 15 years has been elaborated focusing on metabolic engineering of pentose utilization in S. cerevisiae or Z. mobilis (equally incapable to assimilate pentoses) .

Ethanol production by xylose-consuming microorganisms (scheme by McMillan ).

4.9 Glycerol catabolism and ethanol production

The last years, due to the tremendous expansion of biodiesel production units, there has been an excessively abundant supply of glycerol into the market volume , that necessitated the potential of valorization of this low- (or almost zero-) cost material . Conversion, therefore, of this material to higher added-value compounds via fermentation currently attracts high and continuously increasing interest and various microbial added-value metabolites (e.g. 1,3-propanediol, citric acid, 2,3-butanediol, single cell oil, etc.) have been produced through utilization of this waste stream as microbial substrate in conversions performed under aerobic or anaerobic conditions . The last years, there have been a continuously increasing number of reports dealing with the conversion of glycerol into ethanol with the aid of either bacterial or yeast strains (see i.e. ).

The most important yeast candidate capable to convert glycerol is the species P. tannophilus . In the yeast cells, two major pathways of glycerol assimilation are implicated; the phosphorylation pathway, that is the most common one and the oxidation pathway. Phosphorylation pathway involves the phosphorylation of glycerol by a glycerol kinase (GK) that yields in the generation of 3-P-glycerol (G-3-P), which is subsequently oxidized by an NAD-dependent dehydrogenase to yield in the synthesis of 3-P-dihydroxyacetone (DHA-P) . Alternatively, in the oxidative pathway, glycerol is first oxidized into DHA (reaction catalyzed by an NAD- or FAD-dependent dehydrogenase), and thereafter, the yielded DHA is phosphorylated into DHA-P, by a specific DHA kinase . The main pathways of glycerol assimilation performed by eukaryotic microorganisms are illustrated in Fig. (adapted from ).

Pathways involved in the glycerol catabolism by eukaryotic microorganisms (adapted from Fakas et al. ).

Concerning prokaryotic microbial candidates capable to convert glycerol into ethanol, these are more numerous as compared with the yeasts, belonging but not limited to the species Klebsiella pneumoniae, Klebsiella oxytoca, Citrobacter freundii, Kluyvera cryocrescens, etc. . In prokaryotic microorganisms capable of growing on glycerol utilized as the sole carbon source, the events related with the uptake of glycerol are different compared with the eukaryotes and are diversified with the presence or absence of oxygen in the fermentation medium; glycerol assimilation under aerobic and/or anaerobic fermentation conditions can be effectuated by facultative anaerobic microorganisms (principally) belonging to the family Enterobacteriaceae . In contrast, microorganisms belonging to the species Clostridium sp. can breakdown glycerol utilized as substrate only under strictly anaerobic conditions . In the presence of oxygen, as in the previous section, glycerol is first converted into G-3-P, reaction catalyzed by GK, and then G-3-P is oxidized to DHA-P, reaction catalyzed by an aerobic dehydrogenase . The genes of those enzymes constitute a regulon, the so-called GLP-regulon . On the other hand, various strains of the enteric group of bacteria can also carry out glycerol breakdown under strictly anaerobic fermentation conditions. The above-mentioned biochemical pathway (namely the GLP-regulon) can be implicated under anaerobic conditions , but in most cases, this event is effectuated only in the presence of an exogenous electron acceptor (e.g. nitrate, fumarate, etc.) and, as previously, glycerol is primarily phosphorylated to G-3-P by an ATP-dependent GK. On the other hand, under anaerobic conditions and in the case that there is not any available external electron acceptor, in order for glycerol assimilation to be effectuated an internal (intracellular) acceptor of electrons is needed. In this case, glycerol assimilation occurs via a completely different biochemical mechanism, and glycerol itself (by virtue of its dehydrated derivative 3-hydroxypropionaldehyde - 3-HPA) is the final electron acceptor; in general, anaerobic assimilation of glycerol in the absence of external electron acceptor is accompanied by biosynthesis of 1,3-propanediol (1,3-PD), which is the product formed in order for NAD⁺ compounds to be re-generated. In this case, glycerol assimilation occurs via an “oxidative” and a “reductive” branch. A portion of glycerol is transformed into DHA (reaction catalyzed by a NAD⁺-dependent glycerol dehydrogenase - GDH). DHA is then phosphorylated to DHA-P by DHA-kinase (DHA-K). GLP- and DHA-regulons are shown in Fig. (adapted from ).

Pathways involved in the glycerol assimilation by prokaryotic microorganisms (adapted from Papanikolaou ).

According to the above-mentioned analysis, theoretically when glycerol assimilation is performed under anaerobic conditions, 1,3-PD should absolutely be created and detected into the culture medium. Thus, bacteria capable to synthesize ethanol during anaerobic glycerol conversion, should absolutely produce in blends ethanol and 1,3-PD[107]. Nevertheless, recent developments have indicated that a newly isolated C. freundii strain has performed glycerol assimilation under anaerobic conditions and no 1,3-PDat all was detected into the culture medium (almost the sole product synthesized was ethanol) suggesting the utilization of the GLP-regulon under anaerobic conditions, without implication of external electron acceptors .

Whatever happens and irrespective of the conversion of glycerol (aerobic or anaerobic, conversion performed by prokaryotic or eukaryotic microbial cells), this compound enters into the C3 pathway of EMP glycolysis, resulting finally in the generation of pyruvic acid, through the following generalized stoichiometric reaction : [Image Omitted. See PDF]Then, in order for ethanol to be created, pyruvic acid is subjected to oxidative decarboxylation and the maximum theoretical of ethanol produced per glycerol consumed is 1.0 mole/mole (= 0.50 g/g).

5 Technology of alcoholic fermentation 5.1 General concepts; raw materials used as substrates and fermentation configurations

Fermentation processes of any material that contains carbohydrates can produce ethanol. These can be categorized into four main types of raw materials: simple hexoses or disaccharides (deriving from sugar-beets, molasses, fruits, sweet sorghum), starch (deriving from potato, wheat, oat, rice), lignocellulosic materials (agricultural residues, grasses, forestry wastes, sawdust) and urban – industrial wastes . Glycerol represents another individual category of substrates amenable to be converted into ethanol. Lignocellulosic biomass and starch may represent cheaper material than simple sugar, however, the energy (therefore the funds) needed for converting the starch or the lignocellulosic biomass into simple fermentable sugars is the major disadvantage concerning the utilization of these substrates . On the other hand, the principal microbial ethanol producers (S. cerevisiae and Z. mobilis) cannot break down xylose, thus, this point needs to be solved by discovery of xylose-consuming ethanol producers or by the means of genetic engineering (creation of mutant strains expressing the enzymes involved in the catabolism of xylose). Moreover, as far as complex substrates are concerned, in most of the cases a sugar recovery is needed through enzymatic or acid hydrolysis , while, as previously indicated, crude - waste agro-industrial residues could be envisaged as raw alcoholic fermentation materials. Thus, not only alternative substrates are provided but also their disposal problem can be solved. The substrates already used in the literature will be presented in the following paragraphs.

5.2 Directly fermentable feedstock

Simple sugar-based materials [raw glucose or raw saccharose that are the low-added value products used in confectionary industries, sugar beets, molasses, fruits, fruit juices potentially not possible to be consumed by humans (i.e. contaminated with fungicides grape juices), sweet sorghum juice, cane juice, whey permeate, waste-waters containing significant quantities of simple sugars or waste-waters enriched with sugars, etc.] can readily be utilized as the sole substrate by microbial strains capable to produce ethanol. In this case the process cost is decreased given that the pretreatment of substrate in many cases is not needed at all . The raw material that is used widely for ethanol fermentation is that of molasses, derived from the sugar industry . Various types of molasses exist, and these low-added value materials contain about 45–50 % (w/w) of fermentable sugars and 50–55% organic and inorganic compounds . In order to carry out the fermentation process, the medium is first diluted with water (for reducing the sugar concentration so as to avoid toxicity and, therefore, the abrupt end of the bioprocess due to inhibition exerted by the substrate). After dilution with water, pH adjustment, sterilization and inoculation (with yeasts or bacteria) are needed. As stated, literature reports the use of various microorganisms grown on molasses for the production of added value products such as gluconic acid, citric acid, fructo-oligosaccharides, pullulan, succinic acid, microbial oil and erythromycin .

Concerning ethanol production from molasses fermentation, Baptista et al. have performed immobilization in polyurethane foam cubes in a fluidized-bed fermentor of two S. cerevisiae strains for continuous ethanol production using cane black-strap molasses as growth medium (maximum ethanol [EtOH_max], 40 g/L; initial total sugars [TS], 100 g/L). Kopsahelis et al. performed repeated batch bioreactor trials with S. cerevisiae strain AXAZ-1 immobilized in various supports (e.g. delignified Brewer's spent grains) with waste molasses utilized as carbon source, and ethanol up to 9.03% v/v (yield of ethanol per total sugars – Y_EtOH/TS∼0.45 g/g) was reported. Nahvi et al. have used a flocculating S. cerevisiae strain for the production of ethanol when growing on beet molasses (EtOH_max = 5.6% v/v). Cáceres-Farfán et al. used an inoculum that was composed of the yeasts K. marxianus and S. cerevisiae grown on henequen (Agave fourcroydes) juice and molasses medium resulting in the production of EtOHmax∼5.22% v/v. Ergun and Mutlu cultivated a S. cerevisiae strain on molasses resulting in the production of EtOH_max equal to 18.4 g/L. S. cerevisiae strain C10 was cultivated on sugar beet low purity syrup, different fermentation strategies were applied and it was established that the fed-batch process was the most efficient process reaching ethanol concentration of 15.2% (v/v) in 53 h without residual sucrose and with productivity of 2.3 g/L/h .

Another important category of sugar-based (or sugar-enriched) waste wasters is that of the olive mill waste-waters (OMWs), that are the principal waste stream deriving from olive oil production process . These residues are considered as one of the most difficult to treat effluents, since they contain in non-negligible quantities phenolic compounds (up to 10 g/L), while is some cases (viz. from the press-extraction systems) they can contain sugars (mainly glucose) to quantities higher than 70 g/L . Concerning OMWs, there are only a few reports in the literature suggesting the use of such effluents as fermentation process water and substrate directly for the production of ethanol, while either the dilution of this residue or the necessity for removing the phenolic (toxic) fraction by an efficient pretreatment of the waste before alcoholic fermentation, was considered of primordial importance. Various Saccharomyces strains that were grown on olive oil extraction effluents which contained around 8.0 g/L of phenolic substances were used by Bambalov et al. . Three strains grew only in the three-fold diluted medium, producing ethanol (EtOH_max = 10.8-11.7 g/L; Y_EtOH/TS = 0.38-0.41 g/g). Zanichelli et al. , before inoculating a S. cerevisiae strain, removed the phenolic fraction and pretreated the waste enzymatically. The ethanol production reached was 63–95 g/L while the initial phenolic compounds concentration (after dilution) was 2.1 g/L; the glucose supplementation of the medium was up to 200 g/L and the total fermentation time was 65 days. OMWs were also considered as valuable effluents for the production of ethanol by Massadeh and Modallal , who used Pleurotus sajor-caju for the pretreatment of the waste so as to degrade phenols. Following pretreatment, 50% diluted and thermally processed OMW medium was inoculated with a S. cerevisiae strain leading to maximal ethanol production (14.2 g/L) after 48 h of fermentation.

In last developments, OMWs were proposed either as a diluted and enriched with low-cost glucose substrate or as a promising tap water substitute and substrate when blended with molasses. At the first case (initial reducing sugars concentration at ∼115 g/L), when S. cerevisiae strain MAK-1 grown on OMWs-based media enriched with commercial glucose, EtOH_max that produced was 52.0 g/L with Y_EtOH/TS equal to 0.46 g/g . At the latter case, the same strain MAK-1 grown on blends of OMWs with molasses (initial total sugars concentration at ∼135 g/L), was reported capable to produce 52.4 g/Lof EtOH_max with Y_EtOH/TS equal to 0.48 g/g . What was interesting in both of the above-mentioned cases, was that the strain was capable to resist at significant initial concentrations of phenolic compounds (up to ∼ 6.5 g/L) without significant viability loss, whereas very interestingly, up to a given initial quantity of phenolic compounds added into the medium, the quantity of maximum DCW and ethanol produced was stimulated by the addition of the residue . Moreover, another important issue of both the above-mentioned bioprocesses was related with the fact that the utilized strain, together with the significant bioethanol and biomass production reported during growth on these residues (blends of OMWs and industrial glucose or blends of OMWs and raw molasses) was capable of simultaneously performing significant removal of the color and interesting removal of phenolic compounds from the medium , increasing the interest of the performed bioprocess. Under the same optics, this strain was cultivated on pasteurized grape must in which the fungicide quinoxyfen had been added in various concentrations (this fungicide can be found in significant concentrations into the grape must, rendering the process of vinification very problematic). In the above-mentioned trials, significant quantities of biomass were produced (∼10.0 g/L) regardless of the addition of fungicide into the medium (in several cases quinoxyfen was added in high quantities viz. 2.4 mg/L). Ethanol was synthesized in very high quantities (∼106.0-119.0 g/L) irrespective of the addition of the fungicide, whereas the fungicide concentration itself was reduced to ∼79–82% w/w . Literature also reports the use of S. cerevisiae strains grown on various media for the production of ethanol. Ethanol production of around 50 g/L was achieved during growth of two S. cerevisiae strains on blends of molasses with commercial sucrose . Çaylak and Sukan performed bioreactor trials using free or immobilized cells of S. cerevisiae strains on sucrose-based media and achieved final ethanol concentrations ranging between 88 and 110 g/L.

5.3 Starch

Starch, an abundant and important substrate, is the polymer of glucose and it first needs to be converted into glucose (saccharification), which will be used by Saccharomyces spp. and Z. mobilis to produce ethanol. It is composed by the polymers amylose and amylopectin in different proportions based on its source. Glucose linked mainly in linear chains by α-1,4 bonds gives the amylose polymer whereas amylopectin is a highly branch polymer of glucose also including at the branch point α-1,6 bonds. Starch has to be broken down by the combination of two enzymes. It is first hydrolyzed by the enzyme α-amylase, an endo-amylase that attacks α-1,4 bonds having as a result the reduction of starch molecular size. The starch is then cooked at high temperature and in its liquefied form, the exo-amylase enzyme glucoamylase hydrolyzes amylose and amylopectin chains to produce glucose . Microorganisms in general prefer saccharified starch but this needs a high amount of energy to be consumed, thus, research is focused upon the production and study of enzymes (therefore microorganisms, mainly fungi) that are able of degrading raw (nonsaccharified) starch . It should be also noted that various approaches in genetic engineering level include insertion of genes encoding for the synthesis of enzymes capable of breaking down starch (e.g. α-amylase, isoamylase, amyloglucosidase) in the genome of microorganisms carrying out alcoholic fermentation process (e.g. Z. mobilis, Saccharomyces spp.) so that to obtain simultaneously and by one and the same microorganism the secretion of hydrolytic enzymes and the production of ethanol (it is the consolidated bioprocess by using starchy materials). Industrial ethanol production could take place using starchy materials as corn, wheat, potatoes, cassava root, etc.

In most of the bioethanol-producing plants in the US, first-generation bioethanol is produced through corn starch conversion. Ethanol production from corn should no longer be considered as practical because of the competition with the agricultural land needed for food production. Finally, instead of utilizing edible starch, the utilization of waste starchy materials (e.g. waste breads, waste potato chips, flour hydrolysates, cassava hydrolysates, etc.) represents an interesting option . In some of the above-mentioned cases, interesting concentrations of ethanol and comparable with the ones reported during first-generation bioprocess have been reported (e.g. utilization of Saccharomyces cerevisiae ATCC 26602 that produced >75 g/L of ethanol in shake flasks when cultivated with flour hydrolysates ).

5.4 Lignocellulosic biomass

Lignocellulosic biomass includes wood (wood chips, forestry wastes), agricultural crop residues, bagasse, grass, straw, ground nut shell, sawdust, cotton, rapeseed, mustard, mulberry, sunflower stalks, etc. It is an alternative energy source as it is renewable and available throughout the world . Therefore it represents an enormous and zero (or even negative) cost raw material compared with directly fermentable feedstock (sugars) and a competitive biomass resource as compared with the starchy materials (mainly corn). Therefore, lignocellulosic biomass is a promising resource for various fermentation technologies including bioethanol production. Lignocellulose is a more complex substrate than starch. It is a mixture of lignin (nonfermentable phenylopropene) and the carbohydrate polymers cellulose (glucose polymer) and hemicellulose (sugar heteropolymer). The carbohydrate polymers have tight hydrogen bonds with lignin, which represents a physical barrier to be removed from them so as to be available for further transformations. The three steps of the biological process for converting the lignocellulosic biomass to ethanol are as follows: delignification which sets free the cellulose and hemicellulose from lignin, depolymerization which produces free sugars and fermentation of the hexoses and pentoses blends with the most difficult and, therefore, limiting step the delignification process . Lignocellulosic biomass needs to be pretreated so as to increase the surface area and the porosity of the material, to increase the bulk density and decrease the crystallinity of the cellulose in order finally to make it accessible for hydrolysis . Pretreatment is the most important processing challenge in the production of ethanol, being one of the most costly steps and is of crucial importance because it influences the whole ethanol production process. There are various techniques that can be used for this scope, some of which are going to be briefly mentioned in the following paragraphs. The lignocellulosic biomass pretreatment can be divided into four main categories: physical, physicochemical, chemical and biological .

Pyrolysis is a physical endothermic process which includes the material treatment at temperatures higher than 300°C leading to the rapid decomposition of cellulose toward gaseous products and residual char . Following, a mild acid hydrolysis has as a result the conversion of the pyrolysis residues (cellulose) into reducing sugars. Mechanical size reduction (usually wet or dry milling, vibratory ball milling, compression milling) is another physical pretreatment. It is a process that reduces cellulose crystallinity and improves the efficiency of downstream process . Steam explosion (or autohydrolysis) is a physicochemical pretreatment that is commonly used over lignocellulosic biomass and makes it more accessible to cellulase attack . This process includes the treatment of chipped biomass with high- pressure saturated steam and the following pressure reduction, which leads to the explosive decompression of the materials . As a result, lignin is transformed and hemicellulose is lead to degradation. This is a fact that increases the potential cellulose hydrolysis. By adding H₂SO₄, SO₂ or CO₂ the production of inhibitory compounds decreases and improvement of enzymatic hydrolysis occurs. Steam explosion requires lower energy compared to conventional mechanical methods but it also includes the incomplete disruption of the lignin-carbohydrate matrix and might produce inhibitory of the process compounds to the microorganisms, therefore, treated biomass might need to be washed with water . Ammonia fiber explosion (AFEX) is also a physicochemical (alkaline thermal) pretreatment of lignocellulosic biomass; lignocellulose is exposed to liquid ammonia under pressure at high temperature for certain period of time and rapid pressure release follows. The above process improves the saccharification rates of various crops. An advantage for this method is that no inhibitors are produced for the rest of the process, and, hence, washing with water is not necessary . For reducing the cost and for protecting the environment, ammonia should be recycled at the end of the pretreatment. Carbon dioxide (CO₂) explosion is the third physicochemical pretreatment similar to the other two (however more cost effective than AFEX, not causing formation of inhibitors and with higher conversion yields compared to steam explosion) which is based on the formation of carbonic acid leading to the increase of the hydrolysis rate.

Ozonolysis, acid hydrolysis, alkaline hydrolysis, oxidative delignification and organosolv process can be categorized as chemical pretreatment processes of lignocellulosic biomass. Such methods are easy in operation and demonstrate good conversion yields in short period of time . Ozonolysis is mainly focused upon degradation of the lignin and hemicellulose whereas it can hardly affect cellulose. The application of mild operating conditions constitutes great advantage of this method. Moreover, ozonolysis removes effectively the lignin without producing toxic inhibitors for the rest of the process. In acid hydrolysis, concentrated and hazardous acids such as H₂SO₄ and HCl are used. They demand corrosion resistant reactors as also their recovery for financial reasons. A variation of the method is dilute acid hydrolysis (like cellulose hydrolysis) that has less severe process conditions and improves the reaction rates . It has been demonstrated that this method achieves high conversion yields of xylan into xylose. This fact affects the process financially, since xylan constitutes almost the one third of the total carbohydrate in several types of lignocellulosic biomass . On the other hand, this pretreatment method results in the production of several growth inhibitors of microorganisms such as acetic acid, furfural and 5-hydroxymethylfurfural . The above-mentioned compounds can present, in several cases, significant problems in the carbon metabolism (e.g. sugar uptake, impact in the activity of several enzymes like hexokinase, glucose-P isomerase and glyceraldehyde-3P-dehydrogenase, glucose-6P-dehydrogenase, etc.) .

Alkaline hydrolysis digests the lignin matrix, producing cellulose and hemicellulose amenable to be subjected to enzymatic degradation . This mechanism is based on the use of bases for the saponification of intermolecular ester bonds cross-linking xylan, hemicellulose and various components such as lignin and hemicelluloses . An increase of the internal surface area and, at the same time, a decrease in the crystallinity and the degree of polymerization as also the separation disruption of the lignin structure and separation of structural linkages between carbohydrates and lignin can be caused by treatment with diluted NaOH solutions . The use of alkaline chemicals for improving the cellulose digestibility is supposed to be effective but not of financial interest (recovery and recycling of bases) for the production of fuel. In the oxidative delignification the pretreatment is done with the aid of hydrogen peroxide (H₂O₂), which catalyzes the lignin biodegradation. Finally, the ogranosolv (or organic solvent) chemical process breaks down the internal lignin and hemicellulose bonds with the aid of a mixture consisted of an organic or aqueous solvent with inorganic acid catalysts . For reducing the process cost, the solvents used should be removed (for avoiding also inhibitory actions) and recycled.

Ending, the fourth main pretreatment category is biological pretreatment, where fungi are used to degrade hemicellulose and lignin that are contained in several waste and residue materials. Certain genera of fungi have been reported capable of breaking down cellulose (brown rot fungi). Others attack both cellulose and lignin (white and soft rot fungi) . The essential enzymes secreted and involved in the biodegradation lignin compounds during cultivation of various molds lignocellulosic materials are laccase, lignin-peroxidase, manganese-independent peroxidase and manganese-dependent peroxidase, the secretion of which is strain-dependent and is influenced by various culture conditions . The mild environmental conditions as also the low energy requirement are the advantages of this process. On the other hand, serious disadvantages of the biological process, constituting in fact major limiting steps, refer to the very low rate of hydrolysis and the low yields as well as the fact that a limited number of naturally occurring microorganisms is capable of breaking down these compounds .

Nasidi et al. proposed an improved ethanol production from whole sorghum residues (bagasse). Hydrolysate samples (pretreated with dilute sulfuric acid, followed by enzymatic hydrolysis and sequential detoxification by Ca(OH)₂ over-liming and charcoal filtration) were fermented with the yeasts S. cerevisiae DCLM and P. tannophilus NCYC614. Detoxification of pretreated sorghum (bagasse to remove potential yeast inhibitors) following fermentation with P. tannophilus resulted in 23 g/L of ethanol produced (representing 72% of theoretical yield). Matsakas and Christakopoulos , used sweet sorghum stalks, as substrate for ethanol production with the use of S. cerevisiae strain MAK-2. After being dried, it was liquefied with the use of a commercial solution so as fermentation to be permitted under high-solid concentrations. When the optimum conditions were tested, high productivity (3.0 g/L/h) and final ethanol production (62.5 g/L) were achieved. In another case, sweet sorghum bagasse was utilized as feedstock for ethanol production with the use of compressed baker's yeast. Prior to liquefaction and saccharification, a hydrothermal pretreatment was applied, in order to achieve high enzymatic hydrolysis yield. Ethanol production of 41.4 g/L and a volumetric productivity of 1.88 g/L/h were achieved .

5.5 Glycerol

Depending on the type and the technology imposed by various biodiesel plants, as well as the purity of the utilized fatty biomass as a starting material for biodiesel generation, glycerol wastes may contain numerous kinds of impurities such as methanol, salts, soaps, heavy metals and residual fatty acids (for review see: Chatzifragkou and Papanikolaou ). This often renders biodiesel-derived glycerol unprofitable for further purification, especially with reference to small- and medium scale biodiesel plants, in order to be used as a component in food, pharmaceutical or cosmetics industries . Significant quantities of glycerol-containing water can also be generated through bioethanol and/or alcoholic beverages production units (see previously sub-chapter “Glyceropyruvic fermentation”). Also, liquid waste streams containing high levels of glycerol (glycerol quantities of 55–90% w/v) are generated in the various oleochemical facilities employing transformations of vegetable or animal fats, and this additional surplus will inevitably negatively affect the price of glycerol . Finally, even larger quantities of glycerol feedstocks may be produced due to the very high intra-cellular accumulation of glycerol (in quantities up to c. 85% w/w) in several algal species like Dunaliella sp. .

While many studies dealing with the fermentative conversions of glycerol have appeared that are related with conversion of this residue into microbial lipid, 1,3-propanediol, citric acid, etc. (for reviews see: Xiu and Zeng ; Papanikolaou ; Papanikolaou and Aggelis ; Zeng and Sabra ; Clomburg and Gonzalez ), recently interesting results related with the conversion of glycerol into ethanol have appeared. The maximum EtOH concentrations achieved through utilization of glycerol are certainly lower compared with the typical fermentations of hexoses performed by Saccharomyces spp. and Z. mobilis, but due to the abundance of glycerol as feedstock, this fermentation attracts interest . Significant EtOH concentrations from glycerol conversion have been reported by a newly isolated K. cryocrescens strain, with EtOH_max quantity achieved being at ∼ 27 g/L. The respective values of conversion yield of ethanol produced per glycerol consumed (Y_EtOH/Gly) and volumetric productivity were ∼0.40 g/g and 0.61 g/L/h . An EtOH_max value of 28.1 g/L but with lower volumetric productivity (∼0.06 g/L/h) has been achieved by P. tannophilus CBS4044 . Metsoviti et al. reported EtOH_max production in 25.2 g/L in fed-batch fermentations of the microorganism K. oxytoca FMCC-197 cultivated under anaerobic conditions on waste glycerol employed as substrate. The respective Y_EtOH/Gly and volumetric productivity values were ∼0.20 g/g and ∼0.28 g/L/h. (In the above-mentioned work, ethanol was typically produced in blend with 1,3-propanediol, that presented a non-negligible final value of 50.1 g/L). Furthermore when raw glycerol was utilized as substrate, an EtOH_max quantity of 10.0 g/L (volumetric productivity 0.83 g/L/h) has been reported by Enterobacter aerogenes Hu-101 in batch bioreactor cultures , while Mu et al. have reported production of ethanol from crude glycerol to a maximum level of 11.9 g/L (volumetric productivity 0.5 g/L/h). Rossi et al. isolated a strain of K. pneumoniae able to convert raw glycerol to 6.1 g/L of EtOH, under anaerobic conditions, while Metsoviti et al. have revealed a newly isolated C. freundii strain that produced ∼15 g/L of EtOH from waste glycerol under strictly anaerobic conditions. Interestingly, in the above-mentioned case, no 1,3-propanediol at all was detected into the medium, suggesting employment of the GLP-regulon on glycerol catabolism, despite the absence of extracellular electron acceptor into the medium . Furthermore, reports indicate usage of genetically modified bacterial strains in the conversion of glycerol into ethanol, with EtOH_max concentrations being in the previously indicated order of magnitude; for instance, Oh et al. reported the production of 21.5 g/L of EtOH in fed-batch bioreactor experiments, by the mutant strain K. pneumoniae GEM167. This concentration was somehow improved to the value of 25.0 g/L by overexpressing the pdc and adhII genes encoding pyruvate decarboxylase (Pdc) and aldehyde dehydrogenase (Adh) of the ethanol-producer Z. mobilis, in the genetically engineered strain GEM 167 . Finally, Yazdani and Gonzalez demonstrated anaerobic fermentation of glycerol by a genetically engineered Escherichia coli, a species that had long been considered to be incapable of glycerol utilization, and production of ethanol from this substrate.

5.6 Fermentation configurations

In the case in which complex carbon sources are employed in order to be converted into bioethanol, several types of fermentation configurations can be applied. In these configurations, pretreatment should take place prior to enzymatic saccharification. In the first type of configuration, namely separate hydrolysis and fermentation (SHF), the implicated enzymes amenable to hydrolyze the complex material are acting for a specific amount of time in a first stage. Thereafter, the second stage of the bioprocess is applied, in which the microbial conversion takes place . The main benefit of this configuration is the application of optimal temperature conditions of both the enzymatic saccharification and the microbial fermentation, resulting in more efficient conversions at a relatively shorter duration. On the other hand, an important drawback of SHF is related with the possible inhibition exerted by the liberated glucose on the enzymes during the first step. In the simultaneous saccharification and fermentation (SSF) process, enzymatic hydrolysis and alcoholic fermentation are combined. Glucose is fermented to ethanol as soon as it appears in the solution, thus, the concentration of the substrate (glucose) is kept low. Hydrolysis products (glucose and short cellulose chains) inhibit strongly cellulase, the responsible enzyme for the enzymatic hydrolysis of pretreated cellulose. SSF achieves the removal of the end-product inhibition, eliminating the need for separate reactors (one for saccharification and another for fermentation) and reduces the fermentation time . It also reduces the contamination risk of external microflora because of high process temperature, anaerobic conditions and ethanol presence in the reaction medium, while it requires lower amounts of enzyme. On the other hand, the different optimum temperature needed for the process of hydrolysis and that of fermentation as also the ethanol itself (as an inhibitor in the fermenting action of microorganisms and cellulose activity) introduce the method's disadvantages. More method disadvantages are the low rates of cellulose hydrolysis and the fact that most of the microorganisms used in the procedure cannot utilize xylose, the hemicellulose hydrolysis product . Hence this procedure requires research to increase the rate of hydrolysis, fact that will reduce the ethanol production cost. On the other hand, further consolidation may include co-fermentation of the hemicellulose hydrolysate, either by a separate pentose-utilizing microorganism or by an engineered strain capable of efficient co-utilization of hexoses and pentoses. This configuration is termed as simultaneous saccharification and co-fermentation (SSCF). Finally, the most desirable set-up that minimizes utility costs is referred to the direct fermentation of biomass to ethanol with the aid of a cellulase expressing and hexose/pentose co-fermenting microorganism. This approach, (secretion of hydrolytic enzymes and production of ethanol by one and the same microorganism; see ) is termed as consolidated bioprocess (CBP) , but in general, is accompanied by ethanol productivities that are lower than the typical conversions of simple sugars into ethanol by S. cerevisiae . The four types of bioprocesses are schematically illustrated in Fig. .

Configurations of lignocellulosic materials utilization as fermentation feedstocks for the production of bioethanol (scheme from Philbrook et al. ).

Lezinou et al. studied the SSF of sweet sorghum carbohydrates to ethanol by Fusarium oxysporum strain F3 alone or in blended cultures with S. cerevisiae strain 2541 or Z. mobilis strain CP4 in a fed-batch fermentation process. The maximum value of the process was achieved by the mixed culture of the fungus and yeast [ethanol yield 29.7% (w/w) and ethanol concentration 7.5 % (w/v)]. Tropea et al. investigated the potential to transform pineapple residues into ethanol after enzymatic saccharification of plant cell walls, and fermentation of the resulting simple sugars using S. cerevisiae strain NCYC 2826, comparing direct fermentation, separate hydrolysis and fermentation and finally SSF. The highest ethanol yield was achieved SSF reaching 3.9% (v/v), corresponding to the 96% of the theoretical yield.

5.7 Nonaseptic ethanol production processes

Generally, a viable perspective of industrial-scale bioethanol is both the utilization of the appropriate microbial strains and the potential of carrying out the bioprocess under nonaseptic conditions. Significantly lower energy consumption due to the absence of sterilization attributes a notable advantage of this production approach. Roukas has proposed ethanol production from nonsterilized beet molasses by free and immobilized S. cerevisiae cells using fed-batch culture (EtOH_max = 53 g/L; Y_EtOH/TS = 0.31 g/g; TS₀ = 250 g/L). Kopsahelis et al. have proposed a continuous ethanol production process performed in a multistage fixed bed tower bioreactor, in which trials with immobilized S. cerevisiae strain AXAZ-1 were performed with waste molasses (at TS₀ = 115 g/L) utilized as substrate. Trials were performed under both aseptic and completely nonaseptic conditions and ethanol production was almost completely unaffected by the nonaseptic conditions employed (ethanol up to 51.4 g/L with conversion yield Y_EtOH/TS∼0.47 g/g was reported for the nonsterile trial). As mentioned above, nonsterile batch bioreactor trials were performed with the use of S. cerevisiae strain MAK-1 grown on OMWs-based media enriched with commercial glucose (EtOH_max = 52.0 g/L, Y_EtOH/TS = 0.46 g/g; ) and blends of OMWs with molasses (EtOH_max = 52.4 g/L, Y_EtOH/TS = 0.48 g/g; ) for the biotechnological production of ethanol. Additionally, the mutant strain of Z. mobilis NS-7 has been cultivated on nonsterile glucose-based media. An ethanol concentration of 73 g/L was achieved with the curves of glucose consumption and ethanol biosynthesis being almost equivalent compared with the sterile media . Finally Weuster-Botz et al. have developed a continuous fluidized bed reactor operation system for ethanol production by Z. mobilis strain ATCC 31821 using hydrolyzed starch without sterilization. The unsterile, 99% hydrolyzed, starch conversion resulted in 50 g/L ethanol production. A summary of the aforementioned findings as also more reports for the conversion of various carbon sources to ethanol in various fermentation configurations is given in Table .

Production of bioethanol by several strains cultivated on various fermentation configurations

	Strain	Carbon source	Substrate concentration	EtOH concentration	Reference
	S. cerevisiae 2541 & F. oxysporum F3 blends	Sweet sorghum	29.7 % (w/w)	7.5 % (w/w)	Lezinou et al.
		Carob pod	200–350 g/L	∼62.0 g/L	Roukas
	Baker's yeast
		Molasses	150–300 g/L	53.0 g/L	Roukas
	Bakers' yeast	Sucrose	220 g/L	96.7 g/L	Caylak and Sukan
	27817	Glucose	50–200 g/L	5.1–91.8 g/L	Vallet et al.
	L-041	Sucrose	100 g/L	25.0–50.0 g/L	Pinal et al.
	ATCC 24860	Molasses	1.6–50 g/L	5.0–18.4 g/L	Ergun and Multu
	CMI237	Sugar	160 g/L	70.0 g/L	Navarro et al.
			100 g/L (fed-batch	126–147 g/L	Alfenore et al.
	CBS8066	Glucose	strategy)	147 g/L	Alfenore et al.
	S. cerevisiae sp.	Molasses	–	44.3 g/L	Nahvi et al.
	132	OMW (pretreated)	200 g/L	63–95 g/L	Zanichelli et al.
	Fiso			4.8–40.0 g/L
	A3	Galactose	20–150 g/L	4.8–36.8 g/L	Cruz et al.
	L52			2.4–32.0 g/L
	GCB-K5			27.0 g/L
		Sucrose	30 g/L		Kiran et al.
	GCA-II			42.0 g/L
	KR18			22.5 g/L
		Glucose	31.6 g/L		Yu and Zhang
	2.399			13.7 g/L
	24860	Glucose	150 g/L	48.0 g/L	Najafpour et al.
	NCYC 1119	Molasses	100 g/L	40.0 g/L	Baptista et al.
	AXAZ-1	Molasses	∼216 g/L	71.3 g/L	Kopsahelis et al.
	ATCC 26602	Flour hydrolysates	150 g/L	76 g/L	Wang et al.
	S. cerevisiae sp. & K. marxianus blends	Henequen juice & molasses blends	∼215 g/L	41.2 g/L	Cáceres-Farfán et al.
	L-6	OMW (pretreated)	–	14.2 g/L	Massadeh and Modallal
	MAK-1	Grape must	250 g/L	106.4–119.2 g/L	Sarris et al.
	ATCC 24858	Sweet and forage sorghum	76–113 g/L	39.0–56.0 g/L
	Baker's yeast	Sweet sorghum bagasse	–	41.43 g/L	Matsakas and Christakopoulos
	MAK-2	Sweet sorghum	100–500 kg m−3	20.2–62.5 kg m−3	Matsakas and Christakopoulos
		OMW/glucose	115 g/L	52.0 g/L	Sarris et al.
	MAK-1
		OMW/molasses	135 g/L	52.4 g/L	Sarris et al.
	S. cerevisiae sp.	Cassava starch hydrolysate	100–200 g/L	38.0–48.0 g/L	Betiku and Alade
	C10	Sugarbeet low purity syrup 2	–	13.4–15.2 % (v/v)	Joannis-Cassan et al.
	S. cerevisiae DCLM & P. tannophilus NCYC614	Sorghum bagasse	26–69% (w/w)	23.0 g/L	Nasidi et al.
	NCYC 2826	Pineapple wastes	–	3.9% (v/v)	Tropea et al.
	ZM4 & ZMI2	Glucose	100–200 g/L	47.6–78.0 g/L	Sreekumar and Basappa
	ATCC 29191	Hydrolyzed starch	120 g/L	50.0 g/L	Weuster-Botz et al.
	NS-7	Glucose	150 g/L	73.2 g/L	Tao et al.
	ZM4	Hydrolyzed starch stream	80–110 g/L	39–54 g/L	Davis et al.
	NRRL-B-14023	Glucose	100 g/L	40–55 g/L	Ruanglek et al.
	ATCC 29191	Sucrose	200 g/L	55.8 g/L	Cazetta et al.
	ZM4	Rice bran hydrolysate	–	13.4 g/L	Todhanakasem et al.
		Alkali–treated cellulose 123	5 g/L	9.6 g/L	Christakopoulos et al.
	Fusarium oxysporum F3			0.5–9.4 g/L	Christakopoulos et al.
		Brewer's spent grain (BG) (alkali-pretreated)	75 g/L	109 g ethanol/kg of dry BG	Xiros et al.
	Klebsiella pneumoniae GEM167	Crude glycerol	20 g/L (fed-batch strategy)	21.5–25 g/L	Oh et al.
	Pachysolen tannophilus CBS4044	Crude glycerol	5% (v/v)	17.5 g/L	Liu et al.
	Enterobacter aerogenes ATCC 13048 mutant strains	Glycerol	20–50 g/L	12–20 g/L	Nwachukwu et al.

6 Concluding remarks

The majority of the environmental problems are created due to the use of conventional energy sources. The constant enlargement of such problems as also the reduction of fossil energy resources has led to the worldwide rise of the need for alternative renewable energy sources. Therefore, utilization of various renewable biofuels as energy sources in engines and heating systems is of remarkable and continuously growing importance. The potential of producing bioethanol through conversion of waste and residual biomass can be a viable and important perspective as regards both the synthesis of a renewable fuel as well as by solving the important and pressing problem of safe waste and residues disposal.

The authors have declared no conflict of interest.