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Animal Feed Science and Technology 208 (2015) 7985

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Animal Feed Science and Technology

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

Effects of potassium sorbate and Lactobacillus plantarum

MTD1 on production of ethanol and other volatile organic
compounds in corn silage
Sasha D. Hafner a , Michelle Windle b,1 , Caitlyn Merrill b,2 , Megan L. Smith b ,
Roberta B. Franco c , Limin Kung Jr. b,
Institute of Chemical Engineering, Biotechnology and Environmental Technology, University of Southern Denmark, 5230 Odense M,
Department of Animal and Food Sciences, University of Delaware, Newark, DE 19716, USA
Department of Land, Water and Air Resources, University of California, Davis, One Shields Avenue, Davis, CA 95616, USA

a r t i c l e i n f o a b s t r a c t

Article history: Ethanol and other volatile organic compounds (VOC) are formed during ensiling, and may
Received 5 February 2015 contribute to poor air quality and reduce feed intake by animals. Chemical or biological
Received in revised form 10 July 2015 additives may help address these problems by reducing production of VOC during ensiling.
Accepted 11 July 2015
The purpose of this study was to evaluate the effect of additives on production of nine silage
VOC in corn silage, including compounds thought to contribute to poor air quality or affect
Keywords: feed intake (alcohols: methanol, ethanol, 1-propanol; esters: methyl acetate, ethyl acetate,
ethyl lactate; and aldehydes: acetaldehyde, valeraldehyde, hexanal). Potassium sorbate,
Volatile organic compounds
an anti-fungal additive that has shown promising results in reducing ethanol and ester
Potassium sorbate concentrations (1 g/kg on a fresh mass basis); a facultative heterofermentative lactic acid
bacterium, Lactobacillus plantarum MTD1 (105 cfu/g on a fresh mass basis); and a combina-
tion of both additives were compared to a control treatment, which received only water.
Silage was made in bucket silos which were opened after 119 days of ensiling. Potassium
sorbate reduced ethanol production by >70% and ethyl lactate and ethyl acetate by >65%
whether or not L. plantarum was included. Other compounds were not clearly affected by
either additive when used individually, but the combined treatment reduced 1-propanol
and valeraldehyde. The concentration of valeraldehyde was increased by L. plantarum. Nei-
ther potassium sorbate treatment increased concentrations of any measured VOC. These
results provide additional evidence that potassium sorbate is an effective additive for reduc-
ing production of ethanol and ethyl esters in corn silage. Combining potassium sorbate
with L. plantarum may provide additional benets, although the persistence of this effect
for silages with higher VOC concentrations needs to be evaluated.
2015 Elsevier B.V. All rights reserved.

Abbreviations: ADF, acid detergent ber; aNDF, amylase-treated neutral detergent ber; cfu, colony-forming unit; CP, crude protein; DM, dry matter; LAB,
lactic acid bacteria; LP, L. plantarum MTD1 treatment; LP + PS, combined potassium sorbate and L. plantarum treatment; PS, potassium sorbate treatment;
SP, soluble protein; VOC, volatile organic compounds; WSC, water-soluble carbohydrate.
Corresponding author.
E-mail address: lksilage@udel.edu (L. Kung Jr.).
Present address: Vita Plus, 2514 Fish Hatchery Road, Madison, WI 53725-9126, USA.
Present address: Pzer, 401 North Middletown Rd, Pearl River, NY 10965-1215, USA.

0377-8401/ 2015 Elsevier B.V. All rights reserved.
80 S.D. Hafner et al. / Animal Feed Science and Technology 208 (2015) 7985

1. Introduction

The production of alcohols, esters, and other volatile organic compounds (VOC) during ensiling has recently received
attention because of possible effects on air quality (Howard et al., 2010; Hu et al., 2012; Hafner et al., 2013) and feed intake
(Raun and Kristensen, 2010; Daniel et al., 2013; Gerlach et al., 2013). Reducing VOC production through the use of silage
additives could address both problems. Ethanol is the single most important VOC in corn silage from the perspective of
air quality, primarily due to high concentrations (Hafner et al., 2013), and it may also affect feed intake by contributing to
the production of ethyl esters (Wei and Auerbach, 2012; Gerlach et al., 2013). Several studies have reported reductions in
ethanol concentration in silage by the addition of potassium sorbate, which is commonly applied to limit growth of spoilage
yeasts. Application of potassium sorbate at 0.5 g/kg or higher (fresh mass basis), alone or in combination with other chemical
inhibitors, has been shown to reduce ethanol production by 58%85% (Kleinschmit et al., 2005; Teller et al., 2012; Wei and
Auerbach, 2012; Bernardes et al., 2014; Hafner et al., 2014).
Two studies have also quantied the effect of yeast inhibitors on a small number of esters in corn silage. Wei and
Auerbach (2012) reported 45% reductions in ethyl lactate and ethyl acetate, along with a 70% reduction in ethanol, through
the use of a commercial mixture of sodium benzoate and potassium sorbate (21.9% sodium benzoate, 13.2% potassium
sorbate, applied at 2 g/kg (Kirsten Wei, Humboldt Universitt Berlin, Germany, personal communication)). Hafner et al.
(2014) found that a 1 g/kg dose of potassium sorbate reduced production of methyl acetate (by 24%) and ethyl acetate (by
Other compounds may affect air quality and feed intake, and so it is useful to determine the effects of additives on VOC
other than ethanol. For example, the alcohol 1-propanol is generally present at lower concentrations than ethanol, and seems
to make a much smaller contribution to air quality problems (Hafner et al., 2013), despite a higher reactivity than ethanol
(Carter, 2009). However, its concentration varies widely in corn silage, and it may make more of a contribution to poor air
quality than ethanol in some cases (Raun and Kristensen, 2010; Hafner et al., 2013, 2014).
These results described above are encouraging, but it is important to know whether reductions are consistent for silage
made in different locations and under different conditions. The only study to measure the effect of pure potassium sorbate
on ester production was done in California, USA, and used forage with a relatively low DM content of 250 g/kg (Hafner
et al., 2014). Furthermore, the response of aldehydes to potassium sorbate addition has not been evaluated. The primary
objectives of this work were to further test the effectiveness of this additive on alcohol production, and determine its effect on
production of esters and aldehydes. The effect of potassium sorbate addition (1 g/kg, fresh mass basis) on production of three
alcohols, three esters, and three aldehydes within corn silage, along with effects on the microbial community and aerobic
stability, were quantied and compared to a facultative heterofermentative biological additive (Lactobacillus plantarum) and
a combination of the two additives. The combination of a biological and chemical additive could provide reductions beyond
those provided by potassium sorbate alone.

2. Materials and methods

2.1. Silage production and treatments

Corn plants (Augusta Seed A6867GTCBLLC, Verona, VA) grown at the University of Delaware (Newark, DE) were harvested
by hand from 4 random locations in a single eld. Plants were chopped to a theoretical length of 19 mm using a pull-type
chopper equipped with a kernel-processor (John Deere 3975, Moline, IL). To prepare replicated treatments for ensiling, four
replicated piles of 6.5 kg of forage were prepared from each of the four locations (to yield 16 total piles). Treatments were
mixed in 300 mL of distilled water and manually sprayed to fresh forage while the pile was being mixed. Treatments were
potassium sorbate (99%, Acros Organics, New Jersey, USA) applied at 1 g/kg on a fresh weight basis (PS); L. plantarum MTD1
(Ecosyl, Ltd., Stokesly, UK), applied at a rate of 105 cfu/g fresh weight (LP); a combination of these two additives (LP + PS);
and a control (CON), which received only distilled water. Initial forage samples were taken after addition of additives, and
were immediately stored on ice and later frozen until analysis. Treated forage was packed into 7.5 L buckets at a DM density
of about 230 kg/m3 using a hydraulic press. Buckets were sealed with lids with O-rings and stored at 22 2 C for 119 days
before opening.

2.2. Forage and silage analysis

The DM content of samples was determined in a 60 C forced-air oven for 48 h. Dried samples were analyzed by a
commercial laboratory (Cumberland Valley Analytical Services, Inc., Hagerstown, MD) for aNDF, ADF, CP, and soluble protein
(SP, fraction of CP). A portion of each dried sample was ground using an Udy Cyclone Sample Mill (Udy Corp., Fort Collins,
CO) to pass through a 1-mm screen and analyzed for aNDF via the procedures of Van Soest et al. (1991) using heat-stable
alpha-amylase (Termamyl 120L, Novo Nordisk Biochem, Franklinton, NC, USA) with sodium sulte. Acid detergent ber was
quantied on dried ground samples according to procedures described in AOAC (1995; method 973.18) with the modication
that the ber residue from the ADF was recovered on a 1.5 m particle retention 7 cm Whatman lter in a California Buchner
Funnel (934-AH Whatman Inc., Clifton, NJ) instead of a Gooch crucible, to allow for better ltration. The concentrations of
ADF and aNDF are reported inclusive of ash. Total N was determined by combustion of the sample (LECO CNS 2000 Analyzer,
S.D. Hafner et al. / Animal Feed Science and Technology 208 (2015) 7985 81

Table 1
Initial composition of fresh forage after addition of additives but before ensiling. All values are in g/kg on an oven dry matter basis unless indicated.

Variable CON LP PS LP + PS PSE P

DM (g/kg fresh) 390 380 374 395 5.3 0.064

pH 5.43 5.50 5.52+ 5.54* 0.025 0.046
WSC 60.4 56.4 57.4 51.3 5.24 0.68
CP 78.8 77.8 76.8 80.2 0.90 0.089
Soluble protein (g/kg CP) 160 172 184 208* 110 0.054
Ammonia 0.278 0.235 0.262 0.248 0.0196 0.48
ADF 186 186 184 168 5.9 0.16
aNDF 340 354 344 340 6.2 0.32
LAB (log10 cfu/g fresh weight) 7.04 6.98 6.86 6.95 0.096 0.64
Mold (log10 cfu/g fresh weight) 5.52 5.02 3.70 3.74 0.929 0.44
Yeast (log10 cfu/g fresh weight) 5.77 5.74 5.40 5.62 0.224 0.64

Notes: CON = control treatment (water only), LP = Lactobacillus plantarum MTD1 (105 cfu/g), PS = potassium sorbate (1 g/kg on a fresh mass basis),
LP + PS = combination of LP and PS. For each mean, n = 4 bucket silos. Symbols show difference from CON based on Dunnets test: P < 0.10 (+ ) or P < 0.05 (*).
Column P gives the P value for an overall treatment effect based on an ANOVA.

LECO Corporation, St. Joseph, MI) and CP was calculated by multiplying the resulting total N by 6.25. Soluble protein was
determined by the method of Krishnamoorthy et al. (1982).
Water extracts were made by homogenizing 25 g of forage or silage with 225 mL of one-fourth strength Ringer solution
(Oxoid BR52; Oxoid, Unipath Ltd., Basingstoke, UK) for 1 min in a Proctor-Silex 57171 blender (Hamilton Beach/Proctor-
Silex Inc., Washington, NC, USA). The pH of the extract was measured directly after blending. A subsample of the extract
was ltered through four layers of cheesecloth and used for enumeration of yeasts and molds by pour-plating 10-fold
serial dilutions in malt extract agar (Oxoid CM59; Oxoid, Unipath Ltd.). Colonies were counted after incubation for 23 d
at 32 C. The numbers of LAB was determined by pour-plating 10-fold serial dilutions in de Man, Rogosa, and Sharpe agar
(CM3651, Oxoid, Unipath, Basingstoke, UK). Plates were incubated aerobically at 32 C for 23 d. The remainder of the water
extract was ltered through Whatman 54 lter paper (Whatman Inc., Clifton, NJ), acidied with 50% H2 SO4 , and analyzed for
ammonia, water soluble carbohydrates (WSC), organic acids, 1,2-propanediol, and ethanol. Ammonia was measured using
the colorimetric phenol-hypochlorite method (Weatherburn, 1967). The WSC were quantied by a colorimetric procedure
(Nelson, 1944). The remaining compounds were quantied using HPLC following Muck and Dickerson (1988).
Concentrations of methanol, 1-propanol, methyl acetate, ethyl acetate, acetaldehyde, valeraldehyde, and hexanal were
measured in silage samples using a headspace gas chromatography (GC) method, following Hafner et al. (2014). After opening
the silos, silage samples were sealed in vacuum bags and frozen until analysis. Prior to analysis, vacuum bags containing
frozen silage were injected with 100300 mL of air and allowed to warm to room temperature over 2.55.5 h. Gas samples
(1.0 mL) were then removed from individual bags and VOC in the silage solution in equilibrium with the gas samples were
quantied by GC as described in Hafner et al. (2014). Dry matter-based VOC concentrations were calculated based on the
oven DM.
After silo opening, aerobic stability was also determined using 2 kg (1 g) of material from each silo. Representative silage
samples were placed in clean buckets. A thermocouple wire was placed in the center of each silage mass and temperatures
were recorded every 30 min using a data logger (AM416 Relay Multiplexer, Campbell Scientic, Inc., Logan, UT). Buckets
were covered with 2 layers of cheesecloth and exposed to air in the laboratory (22 2 C). Aerobic stability was calculated
as the number of hr before the temperature of the forage mass rose 2 C above baseline temperature.

2.3. Statistical analysis

Linear regression with dummy variables to represent the treatments was used in R (v. 3.1.1; R Core Team, 2015) for data
analysis. Treatment coding was used with the control group as the baseline. Where an overall effect of treatment was
found ( = 0.05) based on an F test, each additive was compared to the control using Dunnets test (using the glht function
from the multcomp package (Hothorn et al., 2008)). Measurements made at the start and end of the ensiling period were
analyzed using separate analyses. Concentrations of VOC were log10 -transformed to account for error distributions closer
to log-normal than normal and to eliminate heteroskedasticity. Standard errors for VOC data were back-transformed and
expressed as a relative value, using the formula 10SEl 1.0 where SEl is the standard error of log10 -transformed data. Aerobic
stability data were square root-transformed prior to analysis (Zar, 1999). Linear regression was used to quantify correlation
between esters and ethanol and acids.

3. Results

No overall treatment effects were detected in the initial chemical and microbiological results from fresh forage, with
the exception of pH, which was slightly higher (0.1 unit, P = 0.023) in the LP + PS forage (Table 1). Most properties of the
nished silage (Table 2) were within the range typically observed for corn silage, with the exception of pH, which ranged
from 3.4 to 3.6. The combined treatment LP + PS increased lactic acid (P = 0.004), slightly reduced pH (P = 0.033), and slightly
82 S.D. Hafner et al. / Animal Feed Science and Technology 208 (2015) 7985

Table 2
Final composition of corn silage, after 119 days of ensiling. All values are in g/kg on an oven dry matter basis unless indicated.

Variable CON LP PS LP + PS PSE P

DM (g/kg fresh) 374 367 378 378 3.7 0.15

pH 3.53 3.58 3.61+ 3.43* 0.024 0.001
WSC 8.33 6.05 16.5** 8.60 1.32 <0.001
CP 79.2 82.0* 77.2 81.0 0.70 0.002
Soluble protein (g/kg CP) 472 500* 437** 483 596 <0.001
Ammonia 0.755 0.822 0.643 0.590 0.0987 0.37
ADF 175 192 180 191 7.6 0.37
aNDF 305 333 311 323 9.0 0.18
Lactic acid 44.3 44.5 38.9 59.3** 2.55 <0.001
Acetic acid 10.1 11.9 11.6 10.7 1.04 0.60
LAB (log10 cfu/g fresh weight) 8.70 7.41 7.89 6.61 0.688 0.24
Aerobic stability (h) 47.8 49 169 218+ 49.2 0.068

Notes: for each mean, n = 4 bucket silos. Column P gives the P value for an overall treatment effect based on an ANOVA. Symbols show difference from CON
based on Dunnets test: P < 0.10 (+ ), P < 0.05 (*), P < 0.01 (**), or P < 0.001 (***).

Table 3
Concentrations of volatile organic compounds in corn silage after ensiling for 119 days. All values are in mg/kg on an oven dry matter basis.

Variable CON LP PS LP + PS PSE P

Methanol 200 169 201 182 0.066 0.26

Ethanol 8770 9840 1800*** 2210*** 0.146 <0.001
1-Propanol 12.0 24.6+ 10.9 5.13* 0.177 <0.001
Methyl acetate 5.00 5.09 4.55 2.94 0.160 0.23
Ethyl acetate 98.5 117 23.9*** 20.8*** 0.216 <0.001
Ethyl lactate 341 350 119*** 105*** 0.164 <0.001
Acetaldehyde 6.87 10.4 4.73 1.01 0.332 0.087
Valeraldehyde 0.754 1.28* 0.841 0.335* 0.066 <0.001
Hexanal 0.331 0.669 1.61* 0.523 0.167 0.089

Notes: data were log10 -transformed for analysis, and PSE values shown here are back-transformed and expressed as a relative value by 10SEl 1.0 where
SEl is the standard error of log10 -transformed data. For each mean, n = 4 bucket silos, except for acetaldehyde, which is based on 4 samples for CON and PS,
2 for LP + PS, and 4 for LP, due to interference from an unidentied peak. Column P gives the P value for an overall treatment effect based on an ANOVA.
Symbols show difference from CON based on Dunnets test: P < 0.10 (+ ), P < 0.05 (*), P < 0.01 (**), or P < 0.001 (***).

increased soluble CP (P = 0.047). Lactic acid was the highest in this treatment, while in the other treatments it did not differ
from the control. No effect of additives on the concentration of acetic acid was detected (P = 0.60). The concentration of WSC
was almost doubled by PS (P = 0.002), while effects were not detected for other treatments. Soluble protein concentration
(as a fraction of crude protein) was slightly reduced by PS (P = 0.001) and slightly increased by LP (P = 0.0004). Propionic
acid, butyric acid, succinic acid, and 1,2-propanediol concentrations were below the quantication limit of 1 g/kg for most
samples and values are not reported here. Fiber fractions were not affected by any of the additives (P > 0.18). Aerobic stability
ranged from 10 h for one of the LP replicates to >340 h for two LP + PS replicates and one PS replicate. Mean values were
numerically higher for both PS and LP + PS. However, variability was high (the lowest value for a PS replicate was 58 h), and
no overall difference was detected. Numbers of yeasts and molds in the nal silages could not be determined because of an
unexpected contamination issue during sample preparation, and these data are not shown.
Ethanol approached 10 g/kg in the control treatment, while methanol (about 200 mg/kg), and 1-propanol (<25 mg/kg)
were much lower (Table 3). Both the PS and LP + PS treatments reduced ethanol production by more than 70% (P < 3 105 ).
LP provided no detectable reductions in VOC production. Although neither additive alone affected 1-propanol, the combined
(LP + PS) treatment reduced it by more than 50% (P = 0.022). Ethyl acetate and ethyl lactate were reduced by >65% by both
PS and LP + PS (P < 7 104 ). Neither methanol nor methyl acetate was affected by any of the treatments (P > 0.23).
Aldehydes were present at much lower concentrations than were alcohols. The highest mean acetaldehyde concentration
was 10 mg/kg (LP). Variability in the concentration of this compound was high, and quantication was impossible for ve
samples (one replicate each for CON and PS, and three replicates for LP + PS) due to interference from an unidentied com-
pound. No treatment effects were detected. The LP treatment elevated valeraldehyde by 70% (P = 0.031). Conversely, LP + PS
reduced concentrations of valeraldehyde by >50% compared to the control treatment (P = 0.028). However, concentrations
were very low even without additives (0.75 mg/kg for CON). Hexanal was also present at very low concentrations (<2 mg/kg).
There was some evidence of an increase in hexanal in PS, but the overall F test did not show any differences. There was no
evidence of an increase in any measured VOC by either PS or LP + PS treatments.
Concentrations of ethyl esters were strongly correlated with ethanol concentrations (based on all replicates)
(Figs. 1 and 2). Ethyl acetate concentration could be related to ethanol (e) and acetic acid (a) concentrations (all mg/kg)
by: 128 + 0.0115e + 0.0115a (P < 0.0001 for e and 0.0046 for a, adjusted R2 = 0.77). The largest residuals were for two sam-
ples in the LP and CON treatments. Ethyl lactate concentration could be related to ethanol by: 48.7 + 0.0318e (P < 106 ,
S.D. Hafner et al. / Animal Feed Science and Technology 208 (2015) 7985 83

0.20 CON

Ethyl acetate (g/kg)




2 4 6 8 10
Ethanol (g/kg)

Fig. 1. Ethyl acetate concentration versus ethanol concentration (oven dry matter basis) in corn silages made in laboratory bucket silos with or with-
out additives. CON = control treatment (water only), LP = Lactobacillus plantarum MTD1 (105 cfu/g), PS = potassium sorbate (1 g/kg on a fresh mass basis),
LP + PS = combination of LP and PS.

adjusted R2 = 0.91). Partial correlation to lactic acid was not statistically signicant (P = 0.96). Methyl acetate was only weakly
correlated with methanol (P = 0.014, adjusted R2 = 0.33) and not acetic acid (P = 0.84).

4. Discussion

Concentrations of VOC t a typical pattern observed for corn silage, with ethanol being the most concentrated VOC,
followed by methanol and ethyl lactate, other esters, and nally, aldehydes (Hafner et al., 2013). Concentrations of 1-
propanol were within the wide range of earlier measurements reported in Hafner et al. (2013), but near the bottom. Ethanol
concentrations in the CON silage were typical, and values for other treatments were within the range of values reported in
earlier studies (Hafner et al., 2013).
The relative effect of potassium sorbate on ethanol measured here was similar to reductions reported previously (Teller
et al., 2012; Bernardes et al., 2014; Hafner et al., 2014). Together, these results provide strong evidence that this additive is
effective at reducing ethanol production under a range of conditions. Ethanol in silage can be produced by multiple groups
of microorganisms, but yeasts and heterolactic acid bacteria are generally thought to be the most important in corn silage.
Conrmation of the primary sources of the ethanol in corn silage with and without additives would be useful.
Reductions in ethyl esters are expected as well, based on esterication as the likely route of ester formation and the
observed correlations between ethanol and ethyl ester concentrations (Wei et al., 2009; Raun and Kristensen, 2010; Wei
and Auerbach, 2012, 2013; Hafner et al., 2014). Esterication of carboxylic acids by alcohols is a reversible reaction with an
equilibrium constant that favors the ester. The reaction can proceed with only the acid as a catalyst, although very slowly at
room temperature (Simons, 1993; Calvar et al., 2007). Correlations observed in the current experiment support this pathway
of production. However, the relationship among ethyl acetate and ethanol and acetic acid differ from results from an earlier
experiment (Hafner et al., 2014). The coefcient for ethanol is about 2.5-fold and the coefcient for acetic acid about 4-fold

Fig. 2. Ethyl lactate concentration versus ethanol concentration (oven dry matter basis) in corn silages made in laboratory bucket silos with or with-
out additives. CON = control treatment (water only), LP = Lactobacillus plantarum MTD1 (105 cfu/g), PS = potassium sorbate (1 g/kg on a fresh mass basis),
LP + PS = combination of LP and PS.
84 S.D. Hafner et al. / Animal Feed Science and Technology 208 (2015) 7985

earlier results. While these correlations are useful for understanding how esters are formed and how their concentrations
could be reduced, it is not possible to use them for predicting ester concentrations.
It is unclear why the combined treatment LP + PS affected 1-propanol concentrations. L. plantarum may have reduced
the activity of endogenous lactic acid bacteria through competition, including those indirectly contributing to 1-propanol
production. However, alone, L. plantarum increased 1-propanol concentrations. Additionally, at least one study has shown
that yeasts can produce 1-propanol (Kibe et al., 1977), and yeasts may have been responsible for some of the production
in these silages. From the perspective of VOC emission, 1-propanol concentrations were low in all samples in this study,
and so even the observed >50% reduction would have a negligible effect on air quality for these silages. However, if the
relative effect of the combined treatment holds for ensiling conditions that generally have high 1-propanol concentrations,
this response could have a major effect on VOC emission.
Because of their very low concentrations, all the measured aldehydes would also make a negligible contribution to air
quality compared to ethanol for these silages. Few measurements of aldehydes in corn silage are available, but studies have
reported concentrations of acetaldehyde as high as 380 mg/kg (Langin et al., 1989), and valeraldehyde as high as 200 mg/kg
(Chmelova et al., 2009). Additive effects on aldehydes could be important for both air quality and feed intake for some silages.
Since there was evidence here that additives may increase or decrease aldehyde concentrations, additional measurements
of their effects on this group of compounds would be useful.
Because many compounds may contribute to VOC emission from silage and affect air quality, effects of additives on
production of individual compounds will not translate into proportional reductions in air quality effects. This is most obvious
for cases where an additive has different effects of different compounds, such as the effect of Lactobacillus buchneri reported
by Hafner et al. (2014), where the relative increase in 1-propanol was much greater than the effect on ethanol. Accurately
estimating the effect of an additive on VOC emission will either require accurate emission measurements or another approach
for estimating emission, such as emission models. Estimating effects on air quality will require application of an air quality
model (e.g., Hu et al., 2012). Despite the difculty in accurately estimating effects of additives on emission and air quality,
a 70% reduction in ethanol is expected to result in a similar reduction in ethanol emission (Hafner et al., 2012). Given the
high concentration of ethanol relative to other measured VOC in this work (Table 3) and in general (Hafner et al., 2013), this
reduction in ethanol production alone would provide a substantial reduction in VOC emission and air quality effects. It is
important that neither potassium sorbate treatment increased concentrations of any measured VOC. There is no evidence
that these treatments would exacerbate VOC problems for silage made under the conditions of this study. However, effects
of LP on aldehydes deserve further attention.

5. Conclusions

Potassium sorbate applied at 1 g/kg of fresh forage mass is an effective additive for reducing production of ethanol and
ethyl esters in corn silage. A combination of potassium sorbate and L. plantarum may provide additional reductions in other
compounds, but whether these relative reductions persist for other silages must be determined. Evaluation of the response
to lower doses of potassium sorbate would be useful, as would additional quantication of effects on esters and aldehydes.
Lastly, quantication of effects of additives on VOC production and emission at the farm scale remain important research

Conict of interest

We conrm that there are no known conicts of interest associated with this publication.


We thank Mariele Agarussi, Lilian Oliveira, and Jonathan Lim for assistance during the study. We also appreciate the
help of the farm crew of the University of Delaware in growing and harvesting the corn plants used in this experiment.
We thank Shannon Ingram and Walter Mulbry (USDA Agricultural Research Service, Beltsville, MD) for providing access to
the gas chromatograph used for VOC analysis. Lastly, comments from two anonymous reviewers helped us improve this


AOAC, 1995. Ofcial Methods of Analysis, 16th ed. Association of Ofcial Analytical Chemists, Arlington, VA, USA.
Bernardes, T.F., De Oliveira, I.L., Lara, M.A.S., Casagrande, D.R., vila, C.L.S., Pereira, O.G., 2014. Effects of potassium sorbate and sodium benzoate at two
application rates on fermentation and aerobic stability of maize silage. Grass Forage Sci., http://dx.doi.org/10.1111/gfs.12133.
Carter, W.P., 2009. Development of the SAPRC-07 chemical mechanism and updated ozone reactivity scales. In: Tech. Rep. Final Report to the California
Air Resources Board Contract No. 03e318. Center for Environmental Research and Technology, College of Engineering, University of California,
http://www.engr.ucr.edu/carter/SAPRC/saprc07.pdf (accessed 17.09.14).
Calvar, N., Gonzalez, B., Dominguez, A., 2007. Esterication of acetic acid with ethanol: reaction kinetics and operation in a packed bed reactive distillation
column. Chem. Eng. Proc. 46, 13171323.
Chmelova, S., Triska, J., Ruzickova, K., Kalac, P., 2009. Determination of aliphatic aldehydes in maize and grass silages using on-bre derivatisation with
O-(2,3,4,5,6-pentauoro)benzylhydroxylamine. Anim. Feed Sci. Technol. 152, 152160.
S.D. Hafner et al. / Animal Feed Science and Technology 208 (2015) 7985 85

Daniel, J.L.P., Amaral, R.C., Goulart, R.S., Zopollatto, M., Santos, V.P., Toledo Filho, S.G., Cabezas-Garcia, E.H., Lima, J.R., Santos, M.C., Nussio, L.G., 2013.
Short-term effects of silage volatile compounds on feed intake and digestion in beef cattle. J. Anim. Sci. 91 (5), 23212331.
Gerlach, K., Ro, F., Wei, K., Bscher, W., Sdekum, K.-H., 2013. Changes in maize silage fermentation products during aerobic deterioration and effects
on dry matter intake by goats. Agric. Food Sci. 22, 168181.
Hafner, S.D., Franco, R.B., Kung, L., Rotz, C.A., Mitloehner, F., 2014. Potassium sorbate reduces production of ethanol and 2 esters in corn silage. J. Dairy Sci.
97, 78707878.
Hafner, S.D., Howard, C., Muck, R.E., Franco, R.B., Montes, F., Green, P.G., Mitloehner, F., Trabue, S.L., Rotz, C.A., 2013. Emission of volatile organic
compounds from silage: compounds, sources, and implications. Atmos. Environ. 77, 827839.
Hafner, S.D., Montes, F., Rotz, C.A., 2012. A mass transfer model for VOC emission from silage. Atmos. Environ. 54, 134140.
Hothorn, T., Bretz, F., Westfall, P., 2008. Simultaneous inference in general parametric models. Biom. J. 50, 346363.
Howard, C., Kumar, A., Malkina, I., Mitloehner, F., Green, P., Flocchini, R., Kleeman, M., 2010. Reactive organic gas emissions from livestock feed contribute
signicantly to ozone production in central California. Environ. Sci. Technol. 44, 23092314.
Hu, J., Howard, C.J., Mitloehner, F., Green, P.G., Kleeman, M.J., 2012. Mobile source and livestock feed contributions to regional ozone formation in central
California. Environ. Sci. Technol. 46, 27812789.
Kibe, K., Ewart, J.M., McDonald, P., 1977. Chemical studies with silage microorganisms in articial media and sterile herbages. J. Sci. Food Agric. 28,
Kleinschmit, D., Schmidt, R., Kung Jr., L., 2005. The effects of various antifungal additives on the fermentation and aerobic stability of corn silage. J. Dairy
Sci. 88, 21302139.
Krishnamoorthy, U., Muscato, T.V., Sniffen, C.J., Van Soest, P.J., 1982. Borate phosphate procedure as detailed in nitrogen fractions in selected feedstuffs. J.
Dairy Sci. 65, 217225.
Langin, D., Nguyen, P., Dumon, H., Malek, A., 1989. Aldehydes and ketones in silages quantitative-determination by high-performance
liquid-chromatography. Ann. Rech. Vet. 20, 119127.
Muck, R.E., Dickerson, J.T., 1988. Storage temperature effects on proteolysis in alfalfa silage. Trans. ASAE 31, 10051009.
Nelson, N., 1944. A photometric adaptation of the Somogyi method for the determination of glucose. J. Biol. Chem. 15, 375380.
R Core Team, R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna, Austria.
Raun, B., Kristensen, N., 2010. Propanol in maize silage at Danish dairy farms. Acta Agric. Scand. 60, 5359.
Simons, R.M., 1993. Esterication. In: McKetta, J.J.J. (Ed.), Chemical Processing Handbook. CRC Press, Boca Raton, FL, pp. 637658.
Teller, R.S., Schmidt, R.J., Whitlow, L.W., Kung Jr., L., 2012. Effect of physical damage to ears of corn before harvest and treatment with various additives on
the concentration of mycotoxins, silage fermentation, and aerobic stability of corn silage. J. Dairy Sci. 95, 14281436.
Van Soest, P.J., Robertson, J.B., Lewis, B.A., 1991. Methods for dietary ber, neutral detergent ber, and nonstarch polysaccharides in relation to animal
nutrition. J. Dairy Sci. 74, 35833597.
Weatherburn, M.W., 1967. Phenol-hypochlorite reaction for determination of ammonia. Anal. Chem. 39, 971974.
Wei, K., Auerbach, H., 2012. The effect of different types of chemical silage additives on DM losses, fermentation pattern, volatile organic compounds
(VOC) and aerobic stability of maize silage. In: Proc. XVIth Intl. Silage Conf., Hmeenlinna, Finland. MTT Agrifood Research Finland, University of
Helsinki, Helsinki, Finland, pp. 360361.
Wei, K., Auerbach, H., 2013. Novel results on the formation of volatile organic compounds (VOC) in silages. In: Proc. 22nd Intl. Grassl. Congr., Sydney,
New South Wales, Australia. New South Wales Department of Primary Industry, Orange, New South Wales, Australia, pp. 721724.
Wei, K., Kalzendorf, C., Sittlau, J., Auerbach, H.,2009. Novel results on the occurrence of volatile compounds in maize silages. In: Proceedings of the XVth
International Silage Conference. U.S. Dairy Forage Research Center, USDA-Agricultural Research Service, Madison, WI.
Zar, J.H., 1999. Biostatistical Analysis, 4th ed. Prentice Hall, Upper Saddle River, NJ.