Renewable and Sustainable Energy Reviews 73 (2017) 423–434

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Renewable and Sustainable Energy Reviews

journal homepage: www.elsevier.com/locate/rser

Impact and significance of microbial contamination during fermentation for MARK

bioethanol production
⁎
Ramon Peres Brexó, Anderson S. Sant’Ana
Department of Food Science, Faculty of Food Engineering, University of Campinas, Campinas, SP, Brazil

A R T I C L E I N F O A BS T RAC T

Keywords: The bioethanol visibility in the global matrix of fuels, linked to ecological appeal and the possibility of using new
Ethanol raw materials justifies the increasing investments for the development of new processes. This fuel results from
Bioenergy fermentation of sugars by yeasts, however the growth of exogenous microorganisms is sometimes unavoidable.
Fuel The lactic acid bacteria and wild yeasts are the main contaminants, affecting yeasts’ performance and leading to
Feedstock
negative impacts such as the formation of organic acids, polysaccharides and gummy biofilms. Further
Wild yeasts
Saccharomyces
undesirable effects of the microbial contamination during bioethanol processing comprise flocculation,
Lactic acid bacteria reduction of yeast cells viability and decrease in process yield. In extreme cases, the reduction in process yield
Bioprocesses, microbial contamination can reach 20–30%, making the process impracticable. In this article, the impact and significance of microbial
Sugarcane contaminants (lactic acid bacteria and wild yeasts) during fermentation of must for bioethanol production are
reviewed.

1. Introduction conditions to prevent contaminant levels above a certain level.

Nonetheless, bioethanol processing plants may be not capable to
Ethanol, ethyl alcohol or bioethanol is chemically composed by promote sterilization of raw materials and coadjutants, making the
molecules of carbon, hydrogen and oxygen, with a hydroxyl group process susceptible to bacterial and wild yeast contamination [18,19].
attached to an alkyl radical as its main chemical characteristic [1,2]. It It is know that microbial contamination has been a limiting factor for
is a colorless liquid, clear, with characteristic and pleasing aroma, better performance of the process, despite all the techno-scientific
strongly penetrating with caustic and burning taste. This compound is structure developed by the bioethanol industry [16,17]. The aggravat-
highly flammable and a viable fuel with lower vapor pressure than ing contamination factor in fermentation of must for bioethanol
gasoline, resulting in reduced evaporative emissions. Ethanol high production is the shift in the metabolism of sugars [19]. Bacterial
octane rating serves as an important regulator of detonation potential contamination in concentration above 107 colony forming units (CFU)
when used as gasoline additive, thus replacing toxic additives such as per mL may result in reduction from 1% to 5% of the process yield [20–
tetraethyl and tetra methyl lead [3]. 22]. For instance, in Brazilian distilleries loss of approximately
The ecological appeal of biofuels in reducing pollutant gas emis- 20,000 l/day of bioethanol are predicted when contamination reaches
sions, the possibility of using regional raw materials in the process, and 107–108 CFU per mL [23]. These losses are caused by the stress and
the carbon recovery through photosynthesis stand out as advantages of consequent decrease in yeast cells viability [23–25]. Stressed yeast cells
bioethanol in the matrix of fuels in the world [4]. The increase in with reduced viability, will be weak competitors for substrate and
demand of this fuel over the past few years can also be attributed to the micro-nutrients [24]. In addition, the losses are related to the synthesis
introduction of hybrid engines in the market (flex fuel, which uses of bacterial co-products such as organic acids [19,23,26], proteinac-
blends from 0% to 100% of ethanol and gasoline) [5,6]. Currently, the eous compounds and cyclic peptides [24], hydroxylated fatty acids [24].
projection on increasing global demand for bioethanol is mainly Another important reason for the decrease in bioethanol production
supported by government policies for blending ethanol in gasoline yield is the competition between yeasts and microbial contaminants for
used in passenger light vehicles, and through incentives for controlling space in the fermentation environment [19]. In extreme cases, the
prices in order to keep bioethanol competitive with fossil fuels [7–14]. reduction in yield of bioethanol production can reach 30% resulting in
Bioethanol process consists in the biochemical transformation of huge economic losses [27–29]. Microbiological contamination also
fermentable sugars by yeasts [15–17]. This process requires controlled poses operational difficulties due to the production of gums [30,31],

⁎
Corresponding author.
E-mail address: [email protected] (A.S. Sant’Ana).

http://dx.doi.org/10.1016/j.rser.2017.01.151
Received 27 February 2016; Received in revised form 5 November 2016; Accepted 25 January 2017
Available online 31 January 2017
1364-0321/ © 2017 Elsevier Ltd. All rights reserved.
R.P. Brexó, A.S. Sant’Ana Renewable and Sustainable Energy Reviews 73 (2017) 423–434

and necessity to use acids, antibiotics and antifoam agents [32,33]. the process follows the same steps of saccharides-rich raw materials
This in turn culminate with the increase in production costs and [47,48] (Fig. 1).
changes in quality of the final product [24]. Thus, given its importance, The cellulosic materials are the third class of raw materials used in
in this article the impact and significance of microbial contaminants fermentation processes [8,44]. Eucalyptus, quince, sawdust, bagasse,
during fermentation of must for bioethanol production are reviewed. rice hulls, and many other agro industrial residues [49,50] are
examples of these raw materials which present high amounts of
2. An overview of bioethanol processing lignocellulosic compounds. The technology for bioethanol production
using cellulosic materials comprises the physicochemical treatment,
2.1. Raw materials delignification procedures to separate cellulose and hemicellulose
fractions, and the acid and/or enzymatic treatment for cellulose
Chemically, the energy formed in oxidation reactions of fermen- saccharification [35,47,49] (Fig. 1). In addition to common crops such
table carbohydrates can be used for both yeast growth (and increase of as corn and sugarcane, algae and microorganisms are gaining relevance
biomass) and partial anaerobic oxidation, resulting in the production of as adjuncts for bioethanol production [51–53]. Because of the wide
ethanol and CO2 [34]. Biochemically, fermentation is an anaerobic range of raw materials with potential use for bioethanol production, the
catabolic process that does not involve the respiratory chain or technologies have been classified by generation (1st to 4th) [54–59].
cytochromes [15,16]. During fermentation, sugar molecules are broken
within the cell by enzymatic action and converted in energy with 2.2. Bioethanol processing technologies from sugarcane must
production of ethanol and carbon dioxide [35]. Bioethanol (i.e., ethanol
derived from fermentation of plant materials) is produced by the Corn (Zea mays) and sugarcane (Saccharum spp. L) are the most
abundant availability of fermentable sugars present in must obtained used raw materials for bioethanol production worldwide [15,60]. In
from specific raw materials [15,36]. Although any fermentable organic this article, a focus will be given on the bioethanol production from
matter can be used to produce bioethanol, several factors may restrict sugarcane considering the importance of this raw material in Brazil,
the application of some raw materials in the process, such as the which is the world largest sugarcane ethanol producer.
suitability of the crop for the local farming practices, environmental Fig. 2 illustrates the production process of bioethanol from
and food security aspects [8]. In addition, restrictions for the applica- sugarcane juice/molasses. The first stages of processing are cleaning
tion of some raw materials for bioethanol production may be related to and preparation of the raw material for obtaining the juice rich in
the need to carry out pretreatment of lignocellulosic feedstock before sucrose. This step can be conducted through dry cleaning or cleaning
fermentation [8,16]. The main concerns with the need to apply these with water [48]. Although cleaning with recycled water may be applied,
treatments rely on their high cost considering that is necessary to it should be noticed that it results in losses of fermentable sugars from
develop technologies that avoid the synthesis of cytotoxic compounds the sugarcane to the water and that this process also results in an
for fermenting yeasts, such as furfural, hydroxymethylfurfural, phe- increased microbial contamination of the obtained sugarcane juice for
nols, formic acid, acetic acid, levulinic acid [37–40]. fermentation [48,61]. Because of these limitations, cleaning of sugar-
The amount of fermentable sugars in the must for bioethanol cane with water has been replaced by dry cleaning using mechanical
production is intrinsically related to the process productivity, as the removal of vegetable particles through the application of fans, and
fermentable sugars will be metabolized by the yeast cells [41]. In separation of mineral contaminants in special tables with roles [48,61].
addition to the sugars, yeasts cells demand other chemical elements for After the cleaning process, the sugarcane cells are disrupted by
the maintenance of their physiological processes [15]. Carbon, hydro- leveling (rotary knives), chippers (oscillating knives) and shredders
gen, oxygen, nitrogen, phosphorus, potassium, sulfur, magnesium, (rotary hammer) to reduce the particle size and improve sucrose
iron, zinc, as well as vitamins and other growth factors in small extraction [48,61]. Sucrose extraction can be achieved by pressing or
concentrations are essential components in raw materials needed for diffusion. Milling is performed by one or more mills that break the cells
the fermentation process [15,42]. through compression [48]. Sucrose diffusion takes place in the counter-
In order to be economically viable, the raw material used for flow of the processed sugarcane with water at 70–80 °C pumped by
bioethanol production should result in high rates of fermentable centrifugal pumps [61].
sugars, high yield and low production cost per hectare [43]. In Once sugarcane juice is obtained, it is sieved in rotating and static
addition, it should not favor the synthesis of inhibitory compounds sieves and further added of sulfur dioxide (SO2) and limewater
during the process [39], providing an adequate environment for growth [calcium hydroxide, Ca(OH2)]. The mixture is heated to catalyze the
of fermenting yeast [33]. Furthermore, the obtainment of fermentable flocculation and sedimentation of impurities [63]. In addition, this is
substrates from raw materials should involve simple and low cost done to degrade starch and proteins, and to facilitate gas removal. The
operations, such as milling, pre-treatments, among others [44–46]. sugarcane juice is then decanted to remove colloids, organic and
Several sources may serve as raw materials for bioethanol produc- inorganic non-fermentable molecules, and emulsified lipids [63]. The
tion [43]. These sources are classified into three categories according to fermentable sugar content is adjusted by the addition of sugarcane
availability of fermentable sugars, and different technologies may be molasses and sugar wastes (sugar not intended for human consump-
needed to be applied to obtain fermentable substrates [43,47]. tion) [15]. Thus, the sugarcane must is obtained, which is a substrate
Saccharides or sugar-rich raw materials have abundant amount of rich in sucrose and reducing sugars of plant origin, suitably diluted to
available sucrose, such as beets, sweet sorghum stalk, fruit juices, and undergo alcoholic fermentation [59,63].
sugarcane [35]. For these raw materials, bioethanol production in- The industrial fermentation occurs in three distinct stages: pre-
cludes selection of raw materials, extraction of sugar by milling or paration (pre-fermentation or preliminary phase), fermentation per se
diffusion, and treatments for cleaning and reduction of microbial load (tumultuous phase) and post-fermentation (complementary phase)
[48]. Subsequently, the fermentation, rectification and dehydration [17,36,64]. The pre-fermentation phase is characterized by the lag
steps take place, resulting in the anhydrous alcohol [47,48] (Fig. 1). phase of yeasts cells after their inoculation in a ratio of 20% v/v with
Another class of raw materials that can be used for bioethanol sugarcane must. This phase is characterized by a slow and gradual
production and that present large amounts of starch, include cassava, increase in temperature with tiny carbon dioxide release. The aim of
sorghum grain, potatoes, babassu mesocarp, and cereals such as corn, this phase of fermentation is to obtain large amount of cells with
rice and barley, among others [43]. In this case, for the fermentation maximum fermentation power within about five hours [36,64].
process, starch should be subjected to saccharification by thermal and Further, the tumultuous phase, which lasts about ten hours takes
enzymatic treatment. Once starch is converted into fermentable sugars, place. This phase is characterized by a rapid increase in temperature,

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R.P. Brexó, A.S. Sant’Ana Renewable and Sustainable Energy Reviews 73 (2017) 423–434

Fig. 1. Overview of the processes for bioethanol production from: (1) lignocellulosic raw materials, (2) starchy raw materials and (3) sugar-rich raw materials.

release of high amounts of carbon dioxide, increase of acidity, decrease [65].

in must density due to the formation of alcohol and equivalent In Brazil, which is a major global producer of bioethanol from
consumption of sugars [36,64]. The complementary phase, that lasts sugarcane, fed-batch and continuous fermentation are the most
about seven hours, is characterized by a continued carbon dioxide commonly used processes [6,66]. The first is still dominant in the
release and slow and steady decrease in temperature [36,64]. The majority (83%) of bioethanol plants [5]. In contrast, the continuous
fermented must called “wine” obtained a complex composition of fermentation process represents about 17% of the production of
liquid, solid and gaseous components and should contain small bioethanol [5,65,67–69]. The fed-batch fermentation consists of feed-
amounts of sugars [36]. ing the bioreactor with fermentable substrate under controlled condi-
For the optimization of bioethanol production by fermentation, tions. In this process, the fermentation broth is supplemented with one
bioreactors that favor the maintenance of physicochemical conditions or more substrates for maintenance of the yeast strains and of the
and yeast cells integrity are needed [65]. From the 50 s, both the product up to the end of the fermentation [15,36,63]. The supplemen-
reactors (also called bioreactors and biological or biochemical reactors) tation of the fermentation broth with nutrients can occur intermittently
and the fermentation processes have been modified to reach this or continuously [65,69]. Thus, fermentation process becomes more
purpose [65]. The reactors for ethanol fermentation can work in three flexible, making it possible to apply different feeding rates and to
operational modes: semi-continuous, fed batch or continuous phases control nutrients added, favoring the metabolic pathway of interest

Fig. 2. Flowchart of bioethanol processing from sugarcane. After harvesting, sugarcane buds are transported to the sugar and bioethanol plant. Sugarcane buds are cleaned, cut and
mixed with water. Then, the fragments are crushed between rollers in the milling tandem and the juice is subjected to chemical (SO2 e CaO) and thermal (105 °C) treatments. This
treatment results in flocculation and decantation of non-sugar residues. Molasses are obtained if the clarified juice is heated. The must for bioethanol production can be prepared with
the clarified juice, molasses or a mixture of both to reach approximately 18°Brix. In the fermentation vats, yeasts are added and the fermentation takes place at 30 °C for 12–18 h. After
this period, the fermented must (wine) is centrifuged for separation of wine and yeast cells. The wine is destined to distillation while the yeast cells can be recycled through the
application of acid washing. The sugarcane bagasse can be burnt for steam or electricity production. In addition, sugarcane bagasse can be used for second-generation ethanol
production.

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Fig. 3. Yeast cells recycling (acid washing process). The process consists of applying an acid washing treatment (sulfuric acid solution pH=2.0–3.0) in the concentrated yeast cells,
following agitation for 1.0−1.5 h. After acid washing, the yeast cells are centrifuged and hydrated. These washed cells are ready for use in a new fermentation process.

[65,69,70]. When the concentration of essential nutrients is suitable This operation is only feasible due to the different volatility of the
and viable yeast cells are present, a great productivity is obtained as components in the solution [36]. When the fermented must is
ethanol production is favored by minimizing the substrate inhibiting subjected to such process, two fractions are obtained, the phlegm
effects [71]. Furthermore, in the fed-batch process, successive batches (main distillate) and vinasse. The vinasse is the result of distillation of
can be performed in the same container due to strict control of yeast fermented must and its alcohol level must be zero [3,36,74]. However,
viability and traceability of all components involved in the batch all fermented must substances accumulate in the vinasse, including
process [70,72]. volatile compounds [3]. For the bioethanol industry, continuous
A great advance in ethanol production occurred with the use of cell distillation in columns is applied [36,74]. The distillation column is
recycle in the process [5,25]. The "Melle-Boinot" process was created in comprised of trays or plates that act as a simple distillation apparatus
the 30 s by Firmino Boinot (Les Usine de Melle), France [5,15,16]. The [36]. The fermented must presenting higher alcohol levels is brought to
process consists of yeast cell reuse multiple times (recycling) preceded boil in the upper plate, generating concentrated vapor [36]. Thus, the
by their separation from wine to be distilled using continuous column temperature decreases from the bottom to the top, while the
centrifuges [15,16]. Before their reuse, the yeast cream (concentrate alcohol level increases proportionally [36].
of yeast cells) is subjected to a decontamination process often Hydrous alcohol is a binary mixture with 96° GL at the end of the
performed with organic acids (acid washing) [15,33,65,68]. At the distillation process [36]. Alcohol is formed of an azeotropic mixture
end of harvest season, the yeast may be recycled many times 400–600 incapable of being separated by distillation, which can be subjected to
times) (Fig. 3), depending on yeast cell viability and bacterial contam- other processes to achieve the anhydrous level [3]. Currently, two
ination [16]. methods can be used to obtain anhydrous ethanol: Azeotropic
In Brazil, the implementation of the continuous process was Distillation and Extractive Distillation. Azeotropic distillation, also
delayed due to low productivity problems found in the first plant called Melle-Guinot, consists of adding a third component to the
established in the country [63]. In addition, the high cost of imple- phlegm to form an azeotrope product with a lower boiling point [3].
mentation and greater susceptibility to bacterial contamination, op- Benzene has been replaced by cyclohexane due to its environmental
poses the efficiency of the well-established Melle-Boinot process and occupational health issues [3]. The principle of this technique is to
[5,63,69]. Even with lower acceptance, the continuous process is obtain a mixture of water with the third component with the lowest
referred to as a natural evolution of the fed-batch process, even though boiling point used in distillation, which is removed at the top of the
this technology has not been modern since some industrial facilities column, while the product containing approximately 99.3% alcohol is
have processed beet molasses in France in the 30 s [69]. The recovered at the bottom portion of the column [3]. Despite the
continuous process consists of constantly feeding a culture medium possibility to recover and reuse the component added, the process is
under a constant flow rate with the maintenance of the reaction volume considered too energetic [3]. The Extractive Distillation, also called
through removal of the fermented juice [65,68,73]. The continuous Mariller technique, is not widely applied by bioethanol processing
process presents greater fermentation capacity, leads to uniform plants due to its high cost of implementation. Nonetheless, the long-
fermentation broth, lower labor costs, and allow the implementation term cost-benefit and the best quality of the ethanol produced have
of advanced automation systems. Despite that, the microbial contam- motivated investments in the application of extractive distillation [3].
ination and spontaneous genetic mutations in yeast can compromise The simplest extractive distillation method uses monoethylene glycol
entire batches, affecting yield [68,69,73]. The use of continuous process (MEG) or glycerin as a phase separator, which carry water to the
involves the reduction with fixed capital in 57–71% in comparison to bottom of the column, allowing the recovery of anhydrous ethanol
the batch system when cell recycle and operation under vacuum are vapors at its top [3].
applied [69].
The bioethanol production is finished with the distillation process,
which enables the separation of liquid mixtures to obtain higher 2.3. Yeasts in sugarcane must for bioethanol production
bioethanol concentrations [74]. The distillation is performed with the
vaporization and subsequent condensation of the fermented must, Yeasts are the most important microorganisms for production of
which originally presents 7–12% by volume of ethanol, and 88–93% by bioethanol through fermentation [35]. Yeasts are unicellular fungi,
volume of water [3,74]. A purification process is of fundamental with ovoid, elliptical or rounded shaped cells and rigid cell wall, with
importance as the fermented must can also contain glycerol, higher the same eukaryotic organelles found in other higher cells [35,75].
alcohols, furfural, acetic aldehyde, acids, and solids such as sugarcane Most yeasts in nature belong to the subdivision of Ascomycetes and
fragments, non-fermentable sugars, and gases such as CO2 and SO2 [3]. Deuteromycetes (Fungi imperfect) and a limited number belong to
Basidiomycetes [75,76]. Yeasts have asexual reproduction via budding,

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binary fission and sexual reproduction by forming spores [35,75,77]. In biomass production, and lower or similar glycerol synthesis rate when
nature, yeasts have wide distribution, being found in flowers, fruits, compared to non-selected yeast strains [33,85,87,88]. Despite this, the
soil, rivers, seas, in insects and mammals [75]. Most yeasts present search for better strains or engineering of strains currently in use has
optimum growth temperature from 20 to 30 °C, pH 3.2–6.0 and high been a noticeable challenge [18,81]. In this scenario, the use of
osmotic tolerance [15,35,36,64,77]. molecular biology comprises an important tool aiming to select better
The facultative respiration allows the use of two distinct pathways strains or to engineer (improve) strains in use [82,89].
for sugar assimilation [15,19,78]. The yeasts able to perform both For decades, yeast identification was based on morphological and
respiration and fermentation are called facultative aerobic [19]. For physiological approaches [15]. Although very useful for the develop-
both processes, sucrose is cleaved by glycolysis to pyruvic acid by an ment of yeasts taxonomy, these techniques are time consuming and not
ordered sequence of catalyzed enzymatic reactions [78]. The difference always conclusive [25]. In this scenario, uncertainty played a major role
of aerobic and anaerobic processes lies on the fate of pyruvic acid in the making it difficult to monitor and type species and strains present in
metabolic pathway and the energy result of this route [78]. In the the fermentation processes [25,33,90,91].
presence of oxygen, pyruvic acid is destined to the Krebs cycle and Molecular techniques have revolutionized the identification and
converted into biomass, carbon dioxide and water, while each glucose typing of microorganisms. The application of nucleic acid fragments
molecule generates 38 ATP molecules [78]. Under anaerobic condi- techniques allowed distinction of different strains present in the
tions, the enzymes pyruvate-decarboxylase and alcohol-dehydrogenase fermentation vats, and favored the development of studies on new
use pyruvic acid as substrate for the synthesis of ethanol and water, genetic resources present during sugarcane must fermentation
yielding only 2 ATP molecules [15,78]. In total, 12 ordered sequential [15,25,33,86]. Karyotyping electrophoresis, analysis of microsatellites,
reactions occur, from sucrose breakdown to ethanol synthesis [15,18]. mitochondrial DNA (mtDNA) polymorphism, DNA restriction frag-
These reactions may be affected by several factors such as pH, ment length polymorphism (RFLP), random amplified polymorphic
temperature, inhibitors, and substrate concentration [15,18,19,78]. DNA (RAPD), and low weight RNA analysis molecular (LMW-RNA) are
In addition to the bioethanol and biomass, yeasts present alter- techniques to be considered in technology of microbial fermentation
native metabolic pathways for synthesis of secondary products. These [15,90,92]. Some of these can be simple, quick, and effective techni-
products can be directly and indirectly related to adaptation and ques for the quality control of fermentation processes [25,89,92].
survival as well as can be produced aerobically and anaerobically Genetic studies on fermentative yeasts are also extremely important
[24,79]. The yield of these pathways includes cell biomass production because they can serve as valuable tools aiming to breed specific yeast
(nucleic acids, lipids, and proteins), glycerol, succinate, higher alcohols, strains. The understanding of the mechanism leading to increased
organic acids and others [24,33,63]. According to the equation resistance of S. cerevisiae towards UBP15, THI12, CLA4, DIT1 and
described by Gay Lussac, the theoretical yield is 0.511 g of ethanol MTC7 gene shutdown was possible by through the discovery of heat-
per gram of glucose used as substrate [78]. However it is known that and ethanol-resistant strains [18,42]. Despite this, the effects of
under industrial conditions, yield reaches a maximum value of about random mutagenesis techniques are also considered for the exploration
90% when production conditions are exceptional [15,25]. of different phenotypes [93,94]. Among the techniques, the multiple
There are many yeasts genera able to synthesize ethanol, particu- exposure to ultra violet (UV), and the protoplasmic fusion called
larly Saccharomyces, Schizosaccharamyes, and Pichia [35]. Genome Shuffling have stood out. Conversely, the strains obtained by
Saccharomyces cerevisiae is the most widely used for fermentation mutagenesis often present difficulties to compete with wild strains
processes [18], which presents the following important characteristics: when applied industrial scale [15,93–95].
i) high capacity to hydrolyze sucrose, the most widely used substrate for
industrial production of bioethanol [17,33]; ii) selective permeability of 2.4. Microbial contaminants during sugarcane must fermentation for
the cytoplasmic membrane [17–19]; iii) high resistance to stress bioethanol production
conditions (pH, osmolality, temperature), and survival in the presence
of up to 18% ethanol [17–19,33,43]; iv) rapid growth in the presence of Several factors such as environmental conditions, quality and
O2 and substrate, and production of ethanol under anaerobiosis practices applied to the raw material, and the reuse of yeast cells
[17,33,43]. innumerous times during season can influence on the level of con-
Bioethanol is produced through sugarcane must fermentation by tamination of sugarcane must by wild yeasts and bacteria [5,25,66].
natural selection (native dominant yeast strain) or intentionally added This in turns will affect the performance and preservation of yeast's
specific yeast strain [5,17,25,33,74]. In the first case, yeasts have the fermentation parameters, which may influence process parameters and
ability of withstand the physicochemical conditions and microbial yield [24,25,33,79,86].
communities present in the fermentation process and dominate the
ecological interactions [74,80]. Thus, by natural selection, the most 2.5. Wild yeasts
adapted strains survive and predominate in the process [33,81–83].
These yeasts are able to dominate the fermentation process because Classic and molecular studies on sugarcane juice, dilution water,
they tend to organize chromosomes and change their genetic features, yeast cream, recycled and acid washed yeast, and fermented must have
providing survival advantages against other yeasts [84]. Nonetheless, revealed the presence of Candida, Pichia, Hansenula,
the predominance of these yeasts can be disturbed any time yeasts Schizosaccharomyces, Kloeckera, Dekkera, Cryptococcus,
displaying flocculation capacity [18], higher glucose and ethanol Rhodotorula, Torulopsis, Trichosporon, and Yarrowia
resistance [5,33], and ability to express the Killer factor are present [24,45,85,89,96–99].
during sugarcane must fermentation [85]. On the other hand, strains The development and predominance of Saccharomyces cerevisiae-
that do not exhibit the advantageous combinations of variables for the and non-Saccharomyces-wild strains with lower productivity, floccula-
fermentation process are rapidly eliminated or replaced dynamically tion, foaming, pseudo hyphae formation, biofilm and invasive growth,
and continuously by the most resistant and adapted strains is undesirable for the sugar-bioethanol industries [17,24,62,89,100].
[5,16,17,25,33]. Dekkera bruxellensis is an example of detrimental species. In fed-batch
The use of intentionally added yeasts to the fermentation process fermentation, D. bruxellensis together with S. cerevisiae causes reduc-
can be advantageous because these strains may have superior resis- tion in pH of the broth, adversely affecting the efficiency of ethanol
tance to stress factors such as acidity, temperature, osmolality, and production by S. cerevisiae [5,24]. An increase in investment in
ethanol concentration when compared to non-selected yeast strains materials such as anti-foaming agents, steam, dispersants, and anti-
[5,23,33,86]. Moreover, these strains commonly present high, better microbials is required for processes facing contamination problems

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[21,97,101,102]. Despite the problems caused by contamination of industrial plants, the stored sugarcane presents accentuated spoilage,
fermentation process with wild yeasts, it should be highlighted that with losses of 6–11% sugars due to the increased contaminant levels
these yeasts may present relevant characteristics for industrial uses [109]. These contaminants are mainly Leuconostoc mesenteroides and
[25,33]. For instance, mutants and wild yeasts isolated from fermenta- L. dextranicum in the first 92 h of storage, with consequent production
tion processes have been applied with excellent results in many of gum, and later with predominance of Lactobacillus [112,117]. In
countries, not only in the bioethanol industry, but also in beverages addition, further spoilage can occur in the summer due to the
and bakery industries [5,15,25,33,103]. Isolation and breeding of temperature rise inside the stem and higher vulnerability to microbial
yeasts is gaining more space in the bioethanol sector [5,33,44,86]. establishment [109,117,118]. Some authors have shown the predomi-
Such approach is an important strategy to ensure complete substrate nance of the genera Flavobacterium, Xanthomonas, Pseudomonas,
fermentation, higher yields, and adaptation to stressful and more Enterobacter, Erwinia, Leuconostoc, Bacillus and Corynebacterium,
profitable processes [42,85,86,104]. Characteristics such as high Klebsiella, Eschrerichia and Aerobacter in sugarcane stems
efficiency in ethanol synthesis, linked to the high tolerance to osmotic [112,119,120]. In the non-thermally processed sugarcane juice, the
factors, temperature, acidity and other stressing factors arising from microorganisms found were Lactobacillus, Staphylococcus,
must processing are of great interest to producers [17,33,42,86]. In Leuconostoc, Bacillus, Klebsiella, Achromobacter, Flavobacterium
contrast, these strains must present low metabolic shift for production and Micrococos [121,122]. In clarified, pasteurized and pre-cooled
of biomass, glycerol, and other metabolites, otherwise such yeasts are sugarcane juice, Lactobacillus, Leuconostoc and Bacillus have predo-
considered contaminants [3,62,85]. Although the wild yeasts are seen minated [121].
as contaminants and dangerous to the bioethanol production, when Sugarcane, as well as transient microbiota, has several endophytic
selected they can present interesting characteristics [5,17,33,86,92]. microorganisms that can be carried by sugarcane fragments not filtered
Thus, wild yeasts can also serve for understanding the genetic in the process, or dragged along with the juice extracted in milling
machinery that enables survival under the environmental and ecologi- operations. Representative endophytes in sugarcane are Burkholderia,
cal stress of the sugarcane must fermentation for bioethanol produc- Pantoea, Klebsiella, Pseudomonas, Enterobacter, Herbaspirillum,
tion [17,18,86,92]. Erwinia, Bacillus and Gluconacetobacter [111,117,123,124].
The microbiota found in the unprocessed sugarcane can be
2.6. Bacteria transferred to the crude juice and further persist throughout the
process [102]. The concentration of these contaminants can increase
The physicochemical characteristics of the raw material and in prolonged periods between cutting and milling operations, or due to
manufacturing process (78–86% water, 10–20% sucrose 0.1–2% poor cleaning of the mill, filters, pumps and pipes [113]. The washing
reducing sugars 0.3–0.5% ash, and 0.5–1.0% nitrogenous com- of sugarcane prior to milling, to partially remove external contami-
pounds/mineral micronutrients, pH in the range of 4.5–5.5, and nants, may result in an increase of about 10–20 times the microbial
temperature from 26 to 35 °C) favor the establishment of various community when performed using adequate procedures and treated
bacteria [15,24,30,36,97,105]. The presence of different bacterial water [125–127]. Thus, the quality of both the washing water and
contaminants in the fermentation process has been reported by dilution water is very important for a proper microbiological fermenta-
Desonghe [106] and Guichard [107]. Even with so many advances in tion [109].
the bioethanol industry, it is not feasible to work with sterile sugarcane Studies on fermented sugarcane must have shown a predominance
must and under aseptic conditions [13,23,60,101]. Contaminants can of Gram-positive microorganisms, which can represent up to 98%
originate from either farm or the pipes of processing plants [24,36]. contaminants [30,102,128]. Lactobacillus, Bacillus, Staphylococcus,
Both sugarcane as raw material as its products (mixed or clarified Micrococcus, Pediococcus, Streptococcus, Leuconostoc, Acetobacter,
sugarcane juice, syrup, filter juice, and final molasses), and other Sporolactobacillus, Pseudomonas, Citrobacter have been found as the
sources such as washing and dilution water may represent important main genera [105,112,121,125,129,130]. In sugarcane syrup, the
contamination sources of the fermentation process [108]. The wastes presence of Bacillus and Clostridium was observed, emphasizing the
and the plant environment can constitute micro-niche for the devel- osmotic resistance of these genera [131], while Lactobacillus strains
opment of a wide variety of microorganisms [108]. Sugarcane trans- have been found when yeast recycle is performed by centrifugation
ported from the field to the processing plant can carry microbiota [112,132].
attached externally to the stem, roots, and rhizoids, or endophytically Advances in molecular biology and microbial identification techni-
to the stem [109–111]. The mineral levels of the impurities in ques have also favored the assessment of contaminants in the
sugarcane vary from 0.3% to 1.6% by mass, and considering the variety fermentation process [102,128]. Through the use of PCR based on
and abundance of microorganisms in the soil, it can be assumed that repetitive sequences (repPCR), amplified ribosomal DNA restriction
this is a relevant source of microbial introduction to the process analysis (ARDRA), and 16S and PHES (ribosomal RNA) sequences, it
[30,61]. has been reported the predominance of Lactobacillus, Oenococcus and
Healthy sugarcane stems contain about 101–108 bacteria per gram Weissella, Leuconostoc, Bacillus, Acetobacter, Staphylococcus,
[112]. The massive presence of contaminants in the field can be Lactococcus and Streptococcus in sugarcane juice and molasses
correlated with pest infestations during harvest and transportation [116,125]. Analysis of the terminal restriction fragment length poly-
that can facilitate contamination [113]. Manual cutting results in stems morphism (T-RFLP) and quantitative PCR (qPCR) of samples collected
with a smaller exposure area for the penetration of microorganisms from the centrifuge plate, wall fermenter, heat exchanger, water pipes,
when compared to the mechanical technique [109]. Thus, from the molasses, treated yeast, and yeast starters revealed the presence of the
microbiological point of view, the former should be preferred instead of genera Alicyclobacter, Bacillus, Lactococcus, Pseudomonas,
the latter [109]. Nevertheless, it is known that the mechanical harvest- Halomonas, Lactobacillus and Streptococcus, but the predominance
ing of raw sugarcane for certain purposes was prioritized to produce of Lactobacillus [67]. The microbiological contamination patterns in
second-generation bioethanol [59]. It avoids the burning of straw sugar and bioethanol industry can vary, although the genera
sugarcane used to facilitate manual harvesting, resulting in higher Lactobacillus, Bacillus, Lactococcus, Leuconostoc, Pseudomonas,
recovery of lignocellulosic material, reducing the demand for human Staphylococcus and Streptococcus have stood out (Table 1).
resources and also results in the standardization of harvest [61,114– In general and according to the data, the main contaminants of
116]. bioethanol production from sugarcane must fermentation are the lactic
The storage of the harvested stem also represents process losses acid bacteria (LAB) [21,67,97,102,109,112,117,130,133–137]. Such
[113]. When the raw material is not immediately processed in contaminants reduce yeast cell viability by competition for nutrients

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Table 1
Bacterial contaminants found in different sampling points throughout the bioethanol processing.

Microorganisms Sugarcane buds Sugarcane (endophyitic Sugarcane Molasse4 Recycled Wine6 Vat and Heat exchanger
(surface)1 contaminants)2 (must)3 yeast5 centrifuge7 and pipes8

Acetobacter ✓ ✓ ✓
Acromobacter ✓
Aerobacter ✓
Alycyclobacillus ✓ ✓ ✓
Azospirillum ✓
Bacillus ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓
Burkholderia ✓ ✓
Clostridium ✓
Corynebacterium ✓
Enterobacter ✓ ✓
Enterococcus ✓
Eschrerichia ✓
Erwinia ✓ ✓
Flavobacterium ✓ ✓
Gluconacetobacter ✓
Halomonas ✓ ✓ ✓ ✓ ✓
Herbaspirillum ✓
Klebisiella ✓ ✓ ✓
Lactobacillus ✓ ✓ ✓ ✓ ✓ ✓
Lactococcus ✓ ✓ ✓ ✓ ✓
Leuconostoc ✓ ✓ ✓
Micrococcus ✓ ✓
Oenococcus ✓ ✓
Paenibacillus ✓
Pantoea ✓
Pediococcus ✓ ✓ ✓ ✓
Pseudomonas ✓ ✓ ✓ ✓
Sporolactobacillus ✓
Staphylococcus ✓ ✓ ✓ ✓
Streptococcus ✓ ✓ ✓
Tatumella ✓
Xanthomonas ✓
Weissella ✓ ✓

References: 1 [30,112,119,120], 2 [85,110,111,123,124], 3 [30,112,121,122,130], 4 [67,131,140], 5 [67,112,130,132], 6 [2,105,109,112,121,129,130,137], 7–8 [67]

and by the deleterious effects of acids and other active compounds may present in capsules in log and stationary phases [75,139,143].
derived from the bacterial metabolism [19,138]. In bioethanol corn The genera Bacillus and Staphylococcus are also Gram-positive and
processing plants, Lactobacillus are also the most representative catalase positive bacteria [75]. Bacillus genera present rod-shaped cells
bacterial contaminant. Some authors also suggest Acetobacter and and aerobic or facultative aerobic metabolism [75,139]. These are
Enterobacter as important contaminants of ethanol fermentation endospore-producing microorganisms, and present relevant produc-
[24,102,112,128]. tion of secondary metabolites such as enzymes and antibiotics [75].
The LAB are generally non-motile and non-sporeforming, catalase The great microbial diversity in sugarcane juice and during
negative, and stand out by producing lactic acid as the major product of fermentation process indicate that different intrinsic and extrinsic
fermentative metabolism [75]. LAB exhibit anaerobic profile, ability to factors govern the fermentation process as a whole. Among factors
rapidly grow, and can survive in ethanol production plants, being leading to the great microbial diversity in sugarcane juice and during
tolerant to high temperatures (30−45 °C) and low pH [75,102,138]. fermentation process, the following could be highlighted: i) microbial
LAB account for a large natural group of non-sporeforming bacteria composition of soil, ii) differences in sugarcane varieties, iii) harvest
consisting of rod- and cocci-shaped cells [22,75]. Due to lack of technologies, iv) occurrence and prevalence of pests and diseases, and
porphyrins, cytochromes, and absence of oxidative phosphorylation, v) transportation and storage conditions [5,25]. All these factors will
the lactic acid bacteria can obtain energy only by substrate-level influence the microbiological state of the raw material at the time of
phosphorylation [75,78]. These microorganisms are aerotolerant bac- processing, such as clean or dirty, wet or dry, burnt, spoiled, with or
teria, and are classified into homofermentative when the single without roots, or perforated by insects [63,102,113,144]. Other factors
fermentation product is lactic acid or heterofermentative when other including humidity and room temperature, handling, cleaning meth-
metabolic products are formed [21,22,75,139,140]. ods, and sampling techniques can explain the large microbial diversity
Lactobacillus are rod-shaped LAB that present cells with variable during bioethanol processing [67,145,146]. Thus, the magnitude of
length, thickness and curvature. Lactobacillus are predominantly bioethanol processing imparts unique characteristics for each produc-
homofermentative strains, and in general resistant to low pH, low tion unit [63,102,144]. Consequently, each processing plant should
oxygen concentrations, and high ethanol concentrations, justifying improve and standardize the most susceptible steps to microbial
their prevalence in ethanol production [24,75]. The genus contamination [102,128]. They should also regularly monitor the
Leuconostoc present as cocci when occurring alone, and a little more process to take preventive and corrective decisions in order to obtain
elongated when in pairs or short chains, are heterofermentative better results in terms of yield and productivity [15,25].
[75,139]. Some species can produce polysaccharides like dextran and
levan in the presence of sucrose and fructose favoring the formation of 2.7. Negative aspects of microbial contamination during sugarcane
bacterial biofilms and yeast flocculation [141,142]. Bacteria of the must fermentation for bioethanol production
genus Lactococcus have spherical or ovoid cells, while Streptococcus
are round cells, forming long and short chains, or grouped in pairs, and Microbial contamination in bioethanol processing plants account

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Fig. 4. Negative aspects of microbial contamination during fermentation process for bioethanol production.

for numerous losses due to the presence of bacterial contaminants in Lactobacillus bacteria are important in inducing yeast flocculation,
their equipament [102]. The cost of aseptic processing is high and the especially Lactobacillus fermentum, often isolated from flocculated
routes of entrance of microbial contaminants are diverse [15,22]. Thus, vats [18,118,138]. From the ecological point of view, the ability of some
bacterial contamination is a cause of great concern to the sugar- bacteria to stimulate flocculation hinders yeast cell growth and
bioethanol sector [24,30,97]. nutrients capture from the environment, favoring the establishment
Recent data have shown that bacteria can lead to 1.5–5% losses in of bacteria [118,148]. The bacteria and yeast metabolism can also
yield, depending on the fermentation conditions due to metabolic shift promote flocculation [118,149,152]. Some alcohols such as, including
in sucrose [20–22]. In some cases, the reduction of yield can reach n-propyl, n-butyl, iso-butyl, 1,3-butylene glycol and ethanol, have been
30%, leading to a loss of commercial viability of the processing plant shown to stimulate yeast flocculation at concentrations above 0.5%
[27–29,32,129]. Other aspects of extreme concern arising from [154].
bacterial contamination in the fermentation vat are the formation of Apart from their influence in yeast flocculation, several authors
gums, toxins, and other secondary compounds, as well as yeast have stated that the acids produced by bacteria are responsible for the
flocculation and foaming [23,24,147] (Fig. 4). lower yeast cell viability and reduction in bioethanol production yield
The main sucrose deviation routes in the bioethanol industry occurs [112,155]. The non-dissociated forms of acids are capable of diffusing
due to the formation of lactic and butyric acids, produced mainly by the through the cell membrane, thus affecting fermentation [24]. Within
species Lactobacillus acidophilus, L. bulgaricus, L. casei and L. the cell, they dissociate releasing H+ ions; acidifying the cytoplasm,
leischmanii, Streptococcus lactis, and Clostridium pasteurianum, C. altering cellular homeostasis, decreasing specific growth rate and
saccharobutyricum [112]. In production steps such as milling and increasing the lag phase [24]. This phenomenon can be illustrated by
sieves, in which there is contact with oxygen, the acetic fermentation by the activity of acetic acid, which is a metabolic product of yeasts and
Acetobacter aceti, A. pasteurianum, A. acetosum, A. kuntzegianum bacteria and that can present toxicity up to thirty times greater than
and A. suboxydans can take place [109,112]. ethanol [23,138,140]. In synergy with ethanol, acetic acid may confer
The formation of gum increases sugarcane juice viscosity and can non-selective antibacterial activity, and significantly affect yeast per-
cause clogging in pipes, centrifuges, and heat exchangers. Bacillus, formance [23,97,138,140]. However, the effect of acids on the yeast
Aerobacter and Streptococcus produce levan gum whereas viability is controversial [138,150,156,157].
Leuconostoc mesenteroides produces dextran gum [109,112,148].
Yeast flocculation can decrease fermentation rate and hinder
centrifugation operations, since the yeast sediments and remains in 2.8. Interaction between yeasts and lactic acid bacteria during
the bottom of the fermentation vats [24,97,109,142,149]. In addition, sugarcane must fermentation for bioethanol production
yeast flocculation favor losses due to metabolism deviation resulting in
the formation of gum, acids, and toxins, which culminated with There are many queries about the interactions between yeasts and
reduction of yeast cells viability that negatively affect productivity lactic acid bacteria during sugarcane must fermentation for bioethanol
[24,97,109,118,142,149]. In spite of the fact that some S. cerevisiae production [19,138,157]. The conclusions about the drastic reduction
species naturally express flocculin, cell-cell interactions and the activity of the yeast viability passes from competition for trace nutrients as
of bacterial contaminants have also found to stimulate floculation thiamine, vitamins, and amino acids such as glutamic acid, or the
[63,118,149,150]. FLo1, Flo5, Flo9, and Flo10 proteins promote cell action of compounds not yet identified [19,23,157]. However, from the
adhesion in response to the sugar concentration in the substrate for microbial ecology point of view, what are these compounds and what is
fermentation [149]. The phenomenon of flocculation can also occur in their relevance on the evolutionary history of these two microbial
the absence of cell division and under defined environmental condi- groups? The knowledge of the ecological interactions within the
tions involving the presence of divalent cations, usually calcium, which fermentation vat can offer great returns for the bioethanol industry
binds to anionic groups of the yeast cells [18,151]. Flocculation by the [18,19,24,138]. This type of approach may indicate the need of new
interaction between yeast cells is due to the activation of surface technologies for control and maintenance of the fermentative commu-
lectins, responsible for the strong interaction to the N-glycosylated nity, resulting in increased yield of the final product [18,19,24,138].
mannans of the adjacent cell [18,152]. The interaction with starchy Both lactic acid bacteria and yeasts have developed physiological
polysaccharides and the direct contact with bacteria can also be mechanisms for survival [19,24,138,157]. Because of the close rela-
flocculation factors [31,118,149,152,153]. Sporolactobacillus and tionship between lactic acid bacteria and yeasts, antagonistic mechan-
isms are evident [19,24]. In this context, literature highlights the

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R.P. Brexó, A.S. Sant’Ana Renewable and Sustainable Energy Reviews 73 (2017) 423–434

production of lactic acid, butyric acid, and acetic acid, diacetyl (2,3- Compliance with ethical standards
butanedione), hydroxylated fatty acids and reuterin (3-hydroxypropio-
naldehyde) by lactic acid bacteria so as to affect yeast growth [24]. Funding
Diacetyl, reuterin, and hydroxylated fatty acids act mainly on the
inhibition of ethanol production [24,79]. Diacetyl is produced by many This study was funded by Conselho Nacional de Desenvolvimento
acid lactic bacteria species, while Gram-negative bacteria and yeasts Cientifico e Tecnológico (CNPq) (Grant CNPq 302763/2014-7) and
are more sensitive to this metabolite. Reuterin is a broad-spectrum Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
antibiotic affecting Gram-positive and Gram-negative bacteria, fungi, (CAPES).
and yeasts, especially the genera Aspergillus, Fusarium, Torulopsis,
Candida and Saccharomyces [24,79]. On the other hand, some Conflict of interest
Lactobacillus species can produce broad-spectrum fatty acids and
peptides for yeast metabolism [24,79]. Despite the intense competitive R.P. Brexó and A.S. Sant’Ana declare that they have no conflict of
interaction, it is believed that there is a considerable dependence of the interest.
lactic acid bacteria for nutrients derived from yeast [24,26,79]. These
nutrients would include amino acids, small peptides, vitamins, or Ethical approval
certain types of sugars that are deemed to favor their co-existence
[24,26,79]. This article does not contain any studies with human participants or
The stress caused by contamination favors the production of animals performed by any of the authors.
secondary metabolites by yeasts [18,19]. In this way, glycerol and
succinic acid are the most important compounds produced in great Acknowledgements
quantities [19,79,158]. Glycerol is closely correlated with the redox and
osmotic balance within the cell [159], whereas the succinic acid acts The authors would like to thank the support of Conselho Nacional
mainly on the suppression of lactic acid bacteria growth [5,158]. In this de Desenvolvimento Cientifico e Tecnológico (CNPq) (Grant CNPq
competitive environment, the synergy between ethanol and succinic 302763/2014-7) and Coordenação de Aperfeiçoamento de Pessoal de
acid enhances the antibacterial activity, favoring yeast viability and Nível Superior (CAPES) for the financial support.
permanence in the process [22,118,158]. As seen, numerous survival
strategies employed by lactic acid bacteria and yeasts in the fermenta- References
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