Bioresource Technology 247 (2018) 357–362

Contents lists available at ScienceDirect

Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Resolving bacterial contamination of fuel ethanol fermentations with MARK

beneficial bacteria – An alternative to antibiotic treatment☆
⁎
Joseph O. Rich , Kenneth M. Bischoff, Timothy D. Leathers, Amber M. Anderson, Siqing Liu,
Christopher D. Skory
US Department of Agriculture, Agricultural Research Service, National Center for Agricultural Utilization Research, Renewable Product Technology, Peoria, IL, USA

A R T I C L E I N F O A B S T R A C T

Keywords: Fuel ethanol fermentations are not performed under aseptic conditions and microbial contamination reduces
Fuel ethanol contamination yields and can lead to costly “stuck fermentations”. Antibiotics are commonly used to combat contaminants, but
Lactic acid bacteria these may persist in the distillers grains co-product. Among contaminants, it is known that certain strains of
lactic acid bacteria are capable of causing stuck fermentations, while other strains appear to be harmless.
However, it was not previously known whether or how these strains interact one with another. In this study,
more than 500 harmless strains of lactic acid bacteria were tested in a model system in combination with strains
that cause stuck fermentations. Among these harmless strains, a group of beneficial strains was identified that
restored ethanol production to near normal levels. Such beneficial strains may serve as an alternative approach
to the use of antibiotics in fuel ethanol production.

1. Introduction infections occur unpredictably, and the accumulation of bacterial by-

Nearly 15 billion gallons of fuel ethanol are produced each year in
the US from the yeast fermentation of corn starch (ethanolrfa.org and
US Department of Energy, Energy Information Administration).
However, a variety of factors dictate that fuel ethanol fermentations are
not produced under aseptic conditions, and chronic and acute infections
are commonplace (Connolly, 1999; Beckner et al., 2011; Brexó and
Sant’Ana, 2017). A wide variety of bacterial and fungal contaminants
have been identified using both culture-dependent and independent
approaches (Skinner-Nemec et al., 2007; Beckner et al., 2011; Rich
et al., 2015; Li et al., 2016). Among these contaminating microorgan-
isms in the corn mash-to-ethanol process, the lactic acid bacteria (LAB)
are widely considered to be the most problematic (Skinner and
Leathers, 2004; Bischoff et al., 2009; Rich et al., 2015). Indeed, the
production of acetic acid by heterofermentative LAB is problematic for
yeast production of ethanol from corn mash (Rich et al., 2015). Ethanol
production from sugarcane is also plagued by chronic microbial con-
tamination (Brexó and Sant’Ana, 2017).
Chronic bacterial contamination reduces both the sugar available
for conversion to ethanol and the essential micronutrients required for
optimal yeast growth, resulting in reduced ethanol production. Acute
products, such as acetic and lactic acids, inhibits yeast growth and may
result in “stuck” fermentations that require costly shut-downs of facil-
ities for cleaning (Makanjuola et al., 1992; Narendranath et al., 1997).
Despite efforts to prevent contamination with extensive cleaning and
disinfecting procedures, numerous factors can impede these efforts,
such as contaminated saccharification tanks or continuous yeast pro-
pagation systems which can act as reservoirs of bacteria to continually
reintroduce contaminants (Skinner and Leathers, 2004; Skinner-Nemec
et al., 2007). Similarly, contaminants that form biofilms in fermentor
tanks and pipes are costly and difficult to remove (Skinner-Nemec et al.,
2007; Rich et al., 2015).
A variety of antimicrobial agents are used to treat chronic and acute
infections, including antibiotics used in clinical and veterinary medi-
cine (e.g., erythromycin, penicillin and tetracycline). The most com-
monly used antibiotics used in fuel ethanol production in the US are
virginiamycin, penicillin and erythromycin (Connolly, 1999; Lushia
and Heist, 2005). Regular dosing with antibiotics in industrial fer-
mentations is known to lead to bacterial antimicrobial resistance
(Bischoff et al., 2007). The problems with antimicrobial resistance are
well-known (Lutgring et al., 2017), and are further complicated by the
presence of biologically active virginiamycin in the distillers grains

Abbreviations: Lb., Lactobacillus; LAB, lactic acid bacteria

☆
Names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by USDA implies
no approval of the product to the exclusion of others that may also be suitable. USDA is an equal opportunity provider and employer.
⁎
Corresponding author.
E-mail address: joseph.rich@ars.usda.gov (J.O. Rich).

http://dx.doi.org/10.1016/j.biortech.2017.09.067
Received 18 July 2017; Received in revised form 7 September 2017; Accepted 8 September 2017
Available online 14 September 2017
0960-8524/ Published by Elsevier Ltd.
J.O. Rich et al. Bioresource Technology 247 (2018) 357–362

coproduct of fuel ethanol production (Benjamin, 2008; Luther, 2012; Table 1

Bischoff et al., 2016). Impact of beneficial bacterial treatment on ethanol production by yeast challenged with
Lactobacillus fermentum 0315-1.
Other commercially available chemical-based products include hop
acids and chlorine dioxide. Since treatment for contamination is often Species > 20 g/L 10–20 g/L 0–10 g/L < 0 g/L Total
prophylactic, necessitating the addition of antibiotics to each fermen-
tation batch, control and treatment of bacterial contamination is a Lactobacillus amylolyticus 2 2 3 3 10
Lb. amylovorus 1 1 1 2 5
critical control point for decreasing costs and improving efficiency of
Lb. brevis 0 1 0 0 1
ethanol production. We describe herein an alternative to antibiotics for Lb. casei 5 34 25 20 84
resolving the deleterious effects of bacterial contamination on yeast- Lb. diolivorans 0 0 2 0 2
production of fuel ethanol. Lb. fermentum 0 3 2 12 17
Lb. hamsteri 1 6 7 7 21
Lb. harbinensis 0 1 1 1 3
2. Materials and methods
Lb. helveticus 0 2 0 0 2
Lb. johnsonii 0 0 0 2 2
2.1. Materials Lb. mucosae 0 0 6 6 12
Lb. panis 0 0 2 2 4
Lb. paracasei 0 2 1 0 3
Corn mash was obtained from a commercial dry-grind ethanol fa-
Lb. paralimentaris 0 1 0 0 1
cility and stored at −20 °C and used as previously described (Bischoff Lb. plantarum 29 98 81 58 266
et al., 2009; Rich et al., 2015). Although the corn mash was not ster- Lb. pontis 1 16 12 5 34
ilized, plating of corn mash samples on MRS agar did not detect bacteria Lb. rossiae 0 0 0 2 2
in the mash (< 102 CFU/ml) (Bischoff et al., 2009). Microbial strains Lb. vaginalis 0 1 1 2 4
Lactococcus lactis 0 0 1 0 1
were isolated from a Midwestern dry-grind fuel ethanol plant and se-
Oenococcus oeni 0 0 1 1 2
lected from a previous screen (Rich et al., 2015). Specifically, 516 Staphylococcus epidermidis 0 0 2 11 13
strains that did not inhibit Saccharomyces cerevisiae production of Weissella confusa 0 1 1 0 2
ethanol from the previous screen and three additional strains were mixed 2 1 4 8 15
unknown 1 5 4 3 13
employed as potentially beneficial strains against a model inhibitory
Total 42 175 157 145 519
strain, Lactobacillus fermentum 0315-1 (Bischoff et al., 2009). Sacchar-
omyces cerevisiae (NRRL Y-2034), Lb. paracasei (B-50314) and Oeno-
coccus oeni (B-3472 and B-3473) strains were obtained from the ARS strains were used against an individual strain (either challenge or
(NRRL) Culture Collection (Peoria, IL). beneficial), the mixture and the individual strain were inoculated with
1 × 107 bacterial cells/mL. The mixture of beneficial strains (Mix B)
2.2. Stuck fermentation model was made by mixing equal amounts of Lb. plantarum (1101 5.22 and
1010 5.22), Lb. casei (1004 6.20), Lb. pontis (1004 5.35) and Lb. amy-
Saccharomyces cerevisiae NRRL Y-2034 pre-inocula were grown in lovorus (1101 7.24). The mixture of challenge strains (Mix C) was made
300 mL Erlenmeyer flasks with 50 mL yeast peptone (YP) media sup- by mixing equal amounts of Lb. fermentum (1001 5.32), Lb. plantarum
plemented with 5% (w/v) dextrose at 28 °C and 200 rpm for 24 h. (1101 7.25), Lb. brevis (1101 5.1), Lb. mucosae (1101 7.7) and Lb. casei
Bacterial isolates were grown in 15 mL conical tubes containing 10 mL (091009 7.3).
MRS liquid medium and incubated statically at 37 °C for 24 h. Cells
were harvested by centrifugation at 3,220g for 15 min at 4 °C and in-
2.4. Identification of contaminant species
ocula prepared by resuspending cells in phosphate buffered saline to a
density of OD600 equivalent to 400 and 80 for yeast and bacteria, re-
Bacteria were isolated from a variety of sampling points and iden-
spectively. One OD600 of S. cerevisiae corresponds to ca. 6 × 107 CFU/
tified by sequencing 16S rDNA as previously described (Rich et al.,
mL, and one OD600 of Lactobacillus sp. is equivalent to ca.
2015). Of the 768 strains previously identified, we selected 516 strains
1 × 108 CFU/mL.
(or mixtures of strains) for further examination (Table 1). Among these
Model fermentations were in duplicate 40 mL cultures in 50 mL
516 strains, 471 (91%) were identified as one of 18 different Lactoba-
flasks containing corn mash (33% solids) supplemented with 0.12% (w/
cillus species, with Lb. plantarum and Lb. casei being the most commonly
v) ammonium sulfate and 0.05% (v/v) glucoamylase (Optidex L-400,
tested species (51% and 16%, respectively). Fifteen (3%) mixed cul-
Genencor International Inc., Rochester, NY). Cultures were inoculated
tures and 13 (3%) unidentified strains were also included. Three other
with 50 µL of diluted potentially beneficial bacterial cells, equivalent to
strains from the ARS culture collection were also screened, Lb. paracasei
1 × 107 bacterial cells/mL. Sterile saline (50 µL) served as a negative
(B-50314) and Oenococcus oeni (B-3472 and B-3473).
control in the place of the potentially beneficial bacteria. Flasks were
capped with a vented rubber stopper and incubated at 32 °C and
100 rpm. After 3 h, 50 µL of deleterious bacterial challenge, Lb. fer- 2.5. HPLC analysis
mentum 0315-1 that causes a stuck fermentation (Bischoff et al., 2009),
was added to each flask resulting in a 1 × 107 cell/mL initial con- Samples (72 h) were harvested and clarified by centrifugation.
centration. Sterile saline (50 µL) served as a negative control in the Supernatants were diluted 10-fold into ddH2O. Ethanol, residual glu-
place of the bacterial challenge for unchallenged control flasks. All cose, lactic acid, and acetic acid concentrations were determined by
flasks were then inoculated with 100 µL of the diluted yeast, equivalent high performance liquid chromatography using a 300 mm Aminex HPX-
to final concentration of 6 × 107 yeast cells/mL. The flask cultures with 87H column (Bio-Rad) and a Shimadzu Prominence HPLC system
vented rubber stoppers were incubated at 32 °C and 100 rpm for 72 h. equipped with a refractive index detector and quantified using standard
curves of each compound. Samples (10 µL) were injected onto a column
2.3. Preparation of mixtures of challenge and beneficial strains at 65 °C and eluted with 5 mM H2SO4 mobile phase at 0.6 mL/min.

Model fermentations with mixtures of either beneficial or challenge 3. Results and discussion
strains were conducted as described above with inocula that contained
0.2 × 107 cells/mL of each strain to yield a total bacterial load LAB are Gram-positive, non-spore forming bacteria that are aero-
equivalent to 1 × 107 bacterial cells/mL. When a mixture of bacterial tolerant anaerobes (da Silva Sabo et al., 2014) and have a centuries-old

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J.O. Rich et al. Bioresource Technology 247 (2018) 357–362

Fig. 1. Treating Saccharomyces cerevisiae NRRL Y-2034 fermentation

infected with Lactobacillus fermentum 0315-1 with potentially bene-
ficial lactic acid bacteria impacts ethanol production. The baseline
ethanol levels of the no challenge and challenge controls are high-
lighted. The unchallenged and challenged control fermentations had
ethanol levels of 130 g/L and 105 g/L, respectively. All results are
normalized to the challenge control.

Table 2
Impact of selected beneficial bacteria on corn mash fermentation by yeast challenged with Lactobacillus fermentum 0315-1.

Beneficial Strain Strain ID Challenge Strain Ethanol, g/L Residual Glucose, g/L Acetic Acid, g/L Lactic Acid, g/L

none unchallenged none 130 ± 0.86 < 2.00 < 1.25 < 2.00
none challenged 0315-1 105 ± 1.27 39.8 ± 1.84 3.62 ± 0.75 3.59 ± 0.91
Lb. plantarum 1010 5.22 0315-1 133 ± 0.27 < 2.00 < 1.25 6.24 ± 0.04
Lb. plantarum 1004 5.37 0315-1 133 ± 1.92 < 2.00 < 1.25 5.74 ± 0.06
Lb. plantarum 1010 5.35 0315-1 132 ± 4.25 < 2.00 2.81 ± 2.11 9.43 ± 4.25
Lb. plantarum 1010 5.29 0315-1 132 ± 3.36 < 2.00 3.10 ± 1.77 8.06 ± 0.87
Lb. casei 1004 6.20 0315-1 131 ± 0.74 < 2.00 < 1.25 9.35 ± 0.54
Lb. casei 1004 6.28 0315-1 131 ± 3.56 < 2.00 < 1.25 7.84 ± 0.08
Lb. plantarum 091028 5.46 0315-1 131 ± 0.79 < 2.00 1.77 ± 0.00 4.70 ± 0.05
Lb. amylolyticus 1008 5.21 0315-1 131 ± 1.30 < 2.00 1.99 ± 0.12 2.41 ± 0.19
Mixed 1003 8.46 0315-1 131 ± 0.34 < 2.00 < 1.25 7.06 ± 0.31
Lb. plantarum 1004 5.16 0315-1 130 ± 1.06 < 2.00 1.63 ± 0.07 3.98 ± 0.86
Lb. plantarum 1010 5.8 0315-1 130 ± 0.36 < 2.00 < 1.25 7.11 ± 0.64
Lb. plantarum 1010 6.22 0315-1 130 ± 4.63 < 2.00 1.85 ± 0.28 7.27 ± 0.02
Lb. plantarum 1010 5.38 0315-1 130 ± 3.11 < 2.00 < 1.25 8.77 ± 0.41
Lb. amylolyticus 1004 5.21 0315-1 129 ± 0.20 4.10 ± 1.03 1.83 ± 0.04 2.17 ± 0.09
Lb. plantarum 1001 6.4 0315-1 129 ± 3.08 < 2.00 1.47 ± 0.76 4.43 ± 4.43
Lb. plantarum 1004 5.12 0315-1 129 ± 2.47 < 2.00 < 1.25 6.19 ± 0.63
Lb. plantarum 1003 6.37 0315-1 129 ± 1.83 < 2.00 1.59 ± 0.07 4.47 ± 0.10
Mixed 1010 5.34 0315-1 129 ± 0.96 < 2.00 < 1.25 6.36 ± 0.02
Lb. plantarum 1003 8.24 0315-1 129 ± 1.74 < 2.00 < 1.25 4.09 ± 0.01
Lb. plantarum 1010 6.1 0315-1 129 ± 4.22 < 2.00 1.29 ± 0.03 7.32 ± 0.82
Lb. plantarum 1107 5.2 0315-1 129 ± 4.77 < 2.00 < 1.25 5.97 ± 0.19
Lb. plantarum 1001 5.12 0315-1 129 ± 0.12 < 2.00 1.45 ± 0.03 4.76 ± 0.10
Lb. plantarum 1101 5.22 0315-1 128 ± 1.05 < 2.00 1.94 ± 0.06 4.09 ± 0.03
Lb. pontis 1004 5.35 0315-1 128 ± 2.18 4.70 ± 4.70 2.01 ± 0.10 2.13 ± 0.26
Lb. plantarum 1101 5.41 0315-1 128 ± 1.10 < 2.00 1.54 ± 0.06 5.98 ± 0.35
Lb. plantarum 090929 5.33 0315-1 128 ± 0.14 < 2.00 1.76 ± 0.01 7.90 ± 0.09

history of food preservation (Yang et al., 2012). LAB are well known to
employ antimicrobial peptides to outcompete related species of LAB
(Zacharof and Lovitt, 2012). The narrow antimicrobial specificities of
these bacteriocins have made them valuable food preservatives (da
Silva Sabo et al., 2014), and they have potential in controlling bacterial
contamination in fuel ethanol production (Liu and Summer, 2014).
The impact of several hundred bacterial contaminants isolated from
industrial fuel ethanol fermentations on yeast production of ethanol
from corn mash has recently been reported (Rich et al., 2015). This
screen revealed obligately heterofermentative lactic acid bacteria (LAB)
were the most inhibitory, and the inhibition was correlated with acetic
acid production. However, several hundred strains appeared to have
little effect on yeast ethanol production, and we selected 516 of these to
evaluate as possible beneficial bacteria that could outcompete deleter-
ious strains.
A stuck fermentation model with Lb. fermentum 0315-1 as the pro-
totypical challenge strain has been used to assess the impact of bacterial
contamination on yeast fermentation of corn mash (Bischoff et al., 2009;
Rich et al., 2015). Lb. fermentum 0315-1 has been a reliable challenge to
yeast production of ethanol from corn mash, showing decreased ethanol
production and glucose consumption. In this screen, over 500 strains that
were previously shown to have little or no impact on yeast were tested in
combination with the Lb. fermentum 0315-1 challenge. Interestingly, this
revealed these potentially beneficial microbes fell into three categories
(Fig. 1). 145 of the potentially beneficial microbes actually exacerbated
the Lb. fermentum 0315-1 challenge (Table 1), resulting in decreased

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ethanol production as compared to the Lb. fermentum 0315-1 challenge
alone (the 0315-1 challenge axis in Fig. 1).
Another group of these potentially beneficial microbes was over 300
strains that partly restored ethanol production in yeast challenged with
Lb. fermentum 0315-1 (Table 1). This category was certainly the most
prevalent, and suggests that many of LAB have benefit in the communal
health of the industrial fermentation.
The final group of 26 strains restored ethanol production to near the
unchallenged level (Fig. 1 and Table 2). Most of the beneficial strains
(73%) were identified as Lb. plantarum, although this species comprised
51% of the strains evaluated in this screen. Lb. casei and Lb. pontis were
also amongst the most beneficial, along with some mixtures and two
unidentified strains. It is interesting to note that all of the LAB species
that had more than 20 tested were divided amongst the three cate-
gories. There does not appear to be a universal beneficial or deleterious
species when used in mixed culture, rather the benefit or challenge
appears to be strain specific.
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Bioresource Technology 247 (2018) 357–362
Fig. 2. Changes in acetic acid (A) and lactic acid (B) levels do not
appear to directly relate to ethanol production in corn mash fermen-
tations infected with Lactobacillus fermentum 0315-1 treated with po-
tentially beneficial lactic acid bacteria. The baseline ethanol and
acetic acid levels of the no challenge (solid lines) and challenge (da-
shed lines) controls are highlighted. The no challenge control had
130 g/L, < 1.25 g/L and < 2.00 g/L of ethanol, acetic acid and lactic
acid, respectively. The challenge control had 105 g/L, 3.62 g/L and
3.59 g/L of ethanol, acetic acid and lactic acid, respectively. All results
are normalized to the challenge control.
Acetic acid production by obligately heterofermentative LAB is
correlated with stuck fermentations (Rich et al., 2015). In this screen
with yeast combined with pairs of bacterial contaminants, a challenge
strain and a potentially beneficial strain, we observed that there was no
relationship between acetic or lactic acid and ethanol production under
the employed conditions (Fig. 2). This begins to highlight the com-
plexity of mixed culture systems, and certainly could raise questions
about the utility of monitoring organic acid concentrations as an in-
dicator of problematic bacterial infections. It is clear from Fig. 2a that
the majority of infected reactions show increased acetic acid produc-
tion; although it also does not clearly show that increased acetic acid
concentration has a negative impact on the production of ethanol.
Furthermore, contamination with LAB does result in increased lactic
acid levels, although there is not a correlation between lactic acid and
ethanol production (Fig. 2b). Both acetic acid (Fig. 2a) and lactic acid
(Fig. 2b) levels were increased from the unchallenged fermentation in
nearly all of the challenge reactions, including in those reactions with
J.O. Rich et al. Bioresource Technology 247 (2018) 357–362

Fig. 3. Decreasing glucose utilization lowers ethanol production in

corn mash fermentations. The baseline ethanol levels of the no chal-
lenge (solid lines) and challenge (dashed lines) controls are high-
lighted. The no challenge control had 130 g/L and < 2.00 g/L of
ethanol and residual glucose, respectively. The challenge control had
105 g/L and 39.8 g/L of ethanol and residual glucose, respectively. All
results are normalized to the challenge control.

Table 3
Impact of treatment with beneficial bacteria on the production of ethanol by yeast challenged by various bacterial challenge strains.

Challenge Ethanol Production, g/L

None Lb. plantarum Lb. plantarum Lb. casei Lb. pontis Lb. amylovorus Mix Ba
Strain ID – 1101 5.22 1010 5.22 1004 6.20 1004 5.35 1101 7.24 –

None – 130 ± 0.86 129 ± 4.3 129 ± 0.06 125 ± 7.7 126 ± 2.1 129 ± 6.0 127 ± 1.2
Lb. fermentum 0315-1 105 ± 1.27 136 ± 2.6 133 ± 0.27 131 ± 0.74 128 ± 2.2 127 ± 0.63 128 ± 0.0
Lb. fermentum 1001 5.32 111 ± 6.1 124 ± 4.9 126 ± 1.2 123 ± 1.9 123 ± 5.0 122 ± 5.0 125 ± 1.3
Lb. plantarum 1101 7.25 114 ± 12 121 ± 7.8 120 ± 8.2 123 ± 4.6 122 ± 2.0 128 ± 3.4 127 ± 1.6
Lb. brevis 1101 5.1 114 ± 0.64 128 ± 0.93 128 ± 4.3 122 ± 1.2 125 ± 1.9 124 ± 1.7 123 ± 1.4
Lb. mucosae 1101 7.7 93.5 ± 0.91 124 ± 3.2 116 ± 3.5 120 ± 4.7 123 ± 3.1 103 ± 7.3 129 ± 3.3
Lb. casei 091009 7.3 117 ± 0.40 126 ± 0.19 125 ± 1.6 120 ± 5.3 123 ± 2.0 123 ± 1.8 129 ± 0.4
Mix Cb – 104 ± 0.8 126 ± 2.5 119 ± 2.1 123 ± 0.8 120 ± 8.2 119 ± 2.8 129 ± 1.3

a
Mix B is a mixture of selected beneficial strains Lb. plantarum (1101 5.22 and 1010 5.22), Lb. casei (1004 6.20), Lb. pontis (1004 5.35) and Lb. amylovorus (1101 7.24).
b
Mix C is a mixture of challenge strains Lb. fermentum (1001 5.32), Lb. plantarum (1101 7.25), Lb. brevis (1101 5.1), Lb. mucosae (1101 7.7) and Lb. casei (091009 7.3).

the most beneficial strains (Table 2).
Not surprisingly, decreasing glucose consumption results in de-
creasing ethanol production (Fig. 3). Treatment with several of the most
beneficial strains enabled the complete utilization of glucose after 72 h
(Table 2). However, when the culture uses less glucose (for whatever
reason), less ethanol is produced by the yeast. It may be that glucose
consumption is a more reliable monitor of fermentation health than
organic acid concentration.
Lactic acid bacteria are well known to produce peptides/proteins
that possess antimicrobial activity (Zacharof and Lovitt, 2012; da Silva
Sabo et al., 2014). These bacteriocins have biological activity targeted
at specific related microbes (Zacharof and Lovitt, 2012) and have great
potential in food preservation (Perez et al., 2014) and industrial fer-
mentations (Liu and Summer, 2014). Lb. plantarum is a known producer
of bacteriocins (da Silva Sabo et al., 2014), and could serve as a source
of novel antimicrobial compounds to combat bacterial infections of fuel
ethanol fermentations.
In order to further confirm the utility of this approach, five of the
top beneficial microbes from different species were tested against five
additional challenge strains identified from our previous study (Rich
et al., 2015). In all cases, the yeast in the presence of beneficial strains
had improved ethanol production as compared to the challenge
(Table 3). We also mixed all of the challenge strains and tested the best
Lb. plantarum strain (1101 5.22) and a mixture of the most beneficial
strains, and the beneficial strains were able to improve ethanol
production in the presence of a mixture of challengers.
4. Conclusions
Routine inoculation of beneficial bacteria may serve to control the
growth and minimize the impact of more deleterious bacterial con-
taminants and could provide an economically acceptable strategy for
industrial control of microbial contamination (Brexó and Sant’Ana,
2017). Furthermore, such treatment could lead to the reduction or
elimination of antibiotics currently used to control microbial con-
tamination in the industry, and also help address the growing concern
of antimicrobial resistance. Therefore, beneficial bacteria may offer
producers an alternative to antibiotics for control of unwanted bacterial
contamination.
Acknowledgements
The authors would like to acknowledge the excellent technical
support of Mr. Eric Hoecker. We would also like to honor the memory of
Dr. Ken Bischoff, our dear friend, colleague, and co-author, who re-
cently passed away after a lengthy illness. He will be greatly missed.
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