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REDUCING BACTERIAL CONTAMINATION IN FUEL ETHANOL FERMENTATIONS BY OZONE TREATMENT OF UNCOOKED CORN MASH Mary L. Rasmussen, Jacek A. Koziel, Jay-lin Jane, and Anthony L Pometto J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b00563 • Publication Date (Web): 12 May 2015 Downloaded from http://pubs.acs.org on May 18, 2015

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Journal of Agricultural and Food Chemistry

POET ground corn

POET corn slurry (pH 4.0) Urea (0.03% w/w)

Slurry (30 min, room temp.)

pH adjustment, if any (pH 4–5) Lactobacillus plantarum (ground corn only)

Ozonation/aeration Novozyme 50009 (0.11% v/w) Ethanol Red yeast (0.25% w/w)

SSF (stationary, 27°C, daily sampling)

Figure 1. Corn mash preparation, ozonation/aeration, and fermentation using POET ground corn and POET corn slurry.

ACS Paragon Plus Environment


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REDUCING BACTERIAL CONTAMINATION IN FUEL ETHANOL FERMENTATIONS BY OZONE TREATMENT OF UNCOOKED CORN MASH

Mary L. Rasmussena,b, Jacek A. Kozielc,a,d, Jay-lin Janed, Anthony L. Pometto IIIe,f a

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Department of Civil, Construction, and Environmental Engineering, Iowa State University, Ames, IA 50011

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b

Biorenewable Resources and Technology Program, Iowa State University, Ames, IA 50011

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c

Department of Agricultural and Biosystems Engineering, Iowa State University, Ames, IA 50011

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(ORCID #0000-0002-2387-0354)

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d

Department of Food Science and Human Nutrition, Iowa State University, Ames, IA 50011

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e

Department of Food, Nutrition, and Packaging Sciences, Clemson University, Clemson, SC

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29634 f

Corresponding author: Professor Anthony L. Pometto III, 223 Poole Agricultural Center, Clemson SC 29634-0316, Phone 864-656-4382; Fax 864-656-0331; E-mail [email protected]

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1 ACS Paragon Plus Environment

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Abstract

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Ozonation of uncooked corn mash from the POET BPXTM process was investigated as a

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potential disinfection method for reducing bacterial contamination prior to ethanol fermentation.

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Corn mash (200 g) was prepared using POET ground corn and POET corn slurry and was

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ozonated in 250-mL polypropylene bottles. Lactic and acetic acid levels were monitored daily

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during the fermentation of ozonated, aerated, and non-treated corn mash samples to evaluate

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bacterial activity. Glycerol and ethanol contents of fermentation samples were checked daily to

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assess yeast activity. No yeast supplementation, no addition of other antimicrobial agents (such

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as antibiotics), and spiking with a common lactic acid bacterium found in corn ethanol plants,

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Lactobacillus plantarum, amplified the treatment effects.

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The laboratory-scale ozone dosages ranged from 26–188 mg/L, with very low estimated

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costs of $0.0008 to $0.006 per gallon ($0.21 to $1.6 per m3) ethanol. Ozonation was found to

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decrease the initial pH of ground corn mash samples, which could reduce the sulfuric acid

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required to adjust the pH prior to ethanol fermentation. Lactic and acetic acid levels tended to be

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lower for samples subjected to increasing ozone dosages, indicating less bacterial activity. The

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lower ozone dosages in the range applied achieved higher ethanol yields. Preliminary

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experiments on ozonating POET corn slurry at low ozone dosages were not as effective as using

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POET ground corn, possibly because corn slurry samples contained recycled antimicrobials from

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the backset. The data suggest additional dissolved and suspended organic materials from the

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backset consumed the ozone or shielded the bacteria.

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Keywords: ozonation; no-cook; BPXTM process; corn mash; fuel ethanol; bacteria; dry-grind

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ethanol; lactic acid



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Introduction

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POET leads the fuel ethanol industry in streamlining corn dry milling to improve ethanol

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production efficiency, as well as their feed coproduct, Dakota Gold BPXTM distiller grains. The

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efficient BPXTM, or no-cook, process involves raw starch hydrolysis that, like the conventional

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process, adds enzymes to convert starch to sugars and adds yeast to ferment sugars to ethanol,

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but without the cooking step. 1 The BPXTM process is now used by 24 of the 27 POET

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biorefineries. POET produces over 1.5×109 gal (5.7×106 m3) of ethanol annually in a network

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spanning seven Midwestern states.

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The POET BPXTM process for dry-grind corn ethanol production has important

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advantages over the conventional process. The energy-intensive liquefaction and cooking steps

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are omitted, thus reducing plant energy requirements by 8–15%. 1 The BPXTM process achieves

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higher ethanol yields by improving starch accessibility, lowers volatile organic carbon (VOC)

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emissions, cuts cooling water needs, and enhances the nutrient quality, flowability and anti-

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caking properties of the distillers dried grains with solubles (DDGS) coproduct. Amino acid

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digestibility in DDGS samples varies considerably among conventional ethanol facilities,

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attributable to heat damage from corn mash cooking and DDGS drying. 2-5 Heat damage is

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responsible for the lower amino acid digestibility in DDGS than in corn grain. 4, 6 Removing the

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cooking step, therefore, improves the feed quality of Dakota Gold BPXTM DDGS over

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conventional DDGS by reducing heat damage of the protein. 7

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Unlike most beverage alcohols, fuel ethanol fermentations are not conducted under pure

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culture conditions. 8 High-temperature jet cooking (90–165°C) in the conventional process not

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only gelatinizes the corn starch, it helps reduce contamination and yield losses caused by

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unwanted lactic acid bacteria (LAB). 9 Contamination in fuel ethanol fermentations originates


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from bacteria present on the corn from the fields. 10 Bacteria compete with yeast for nutrients –

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glucose, trace minerals, vitamins, and free amino nitrogen – and lower ethanol yields by

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diverting glucose to lactic and acetic acids, which are inhibitory compounds to yeast. 11-13 In

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addition to yield losses by chronic LAB contamination, acute infections arise unpredictably and

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can result in plant shut-downs for cleaning – an expensive loss in productivity. 8, 13, 14

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Gram-positive and gram-negative bacterial isolates from fuel ethanol production include

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species of Pediococcus, Enterococcus, Acetobacter, Gluconobacter, Clostridium, and

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Lactobacillus. 8, 15 Lactobacilli sp. are the most prevalent in the industry and are ubiquitous in

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nature. In a study of two commercial dry grind ethanol plants by Skinner and Leathers, 8 the

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genus Lactobacillus was isolated most frequently, representing 38 and 77% of total bacterial

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isolates from the first and second plants, respectively. Gram-positive LAB are ethanol-tolerant,

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grow faster than yeast, and produce organic acids which inhibit yeast.

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During this research bacterial growth was controlled at POET biorefineries potentially by

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dosing with antibiotic and antimicrobial agent and by operation at lower pH levels (pH~ 4.2).

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Conventional ethanol plants, despite jet cooking, also add antibiotics to fight chronic or acute

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bacterial infections. 8, 16-18 If no antibiotics are used, it is common for a facility to lose 1–5% of

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its potential ethanol yield. 10 Rising concerns about the use of antibiotics and the emergence of

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antibiotic-resistant bacteria is creating a demand for alternative methods of controlling bacteria.

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Cleaning and sanitation are important microbial control measures in the corn ethanol

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industry. Infections in process tanks and continuous yeast propagators, as well as resistant

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biofilms within the process train, can continually reintroduce bacterial contaminants into the

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fermentors. 8, 19 Large yeast inoculums (≥2% [v/v]) help control contamination during



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fermentations; however, yeast growth and fermentation rates are still reduced by high lactobacilli

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concentrations. 20

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Penicillin and virginiamycin are the antibiotics available commercially for fuel ethanol

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production. 15, 21 Virginiamycin, produced by Streptomyces virginiae, has had limited use in

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human medicine but extensive use as a growth-promoting additive in animal feed. 22 The

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recommended dose of virginiamycin in fuel ethanol fermentations is 0.25–2.0 ppm. 23 The use

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of antibiotics has limitations. Commercial antibiotic applications are essentially selection

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experiments for resistant microorganisms. Hynes et al. 23 observed reductions in the

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effectiveness of virginiamycin after extended fermentations, possibly resulting from its

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breakdown by lactobacilli. 24 Lactobacillus sp. with higher tolerance to virginiamycin have been

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isolated from fuel ethanol plants that dose with virginiamycin. Moreover, bacterial isolates have

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emerged with resistance to both virginiamycin and penicillin. 15, 25

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Hop acids offer a natural alternative to antibiotics for fuel ethanol production. 10 Many

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of the organic acids found in hops exhibit antimicrobial activities. Hop-compounds inhibit

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growth by disrupting the trans-membrane pH gradient of microbes. 26, 27 Hops have long been

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used in the brewing industry to contribute to the flavor of beer, by adding bitterness and aroma,

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and to improve product shelf-life. IsoStabTM, a liquid hop extract, is a commercially-available

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product for fuel ethanol applications. U.S. producers using IsoStabTM have obtained higher

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ethanol yields with less fluctuation.

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A variety of disinfectants have been investigated on lab scale for fuel ethanol

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fermentations, including hydrogen peroxide, chlorine dioxide, potassium metabisulfite, 3,4,4’-

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trichlorocarbanilide, and lactate with a lactate-tolerate yeast strain. 8, 28-33 Bacteria were

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inhibited over yeast by these agents, but the effectiveness depended on the bacterial strain. 8


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Chlorine dioxide and ozonation are common alternatives to chlorination for disinfection of

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drinking water. Meneghin et al. 30 evaluated the effect of chlorine dioxide against bacteria

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prevalent in ethanol fermentations (e.g., Lactobacillus plantarum and L. fermentum). The

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minimum inhibitory concentrations (MIC) for chlorine dioxide on the bacteria and on the yeast

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inoculum were determined using nutrient media; the effectiveness in nutrient media is likely

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different from that in the high suspended-solids corn mash at fuel ethanol plants. The MICs

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ranged from 10–125 ppm for the bacterial strains tested; L. plantarum had the highest MIC of

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125 ppm. Yeast growth was also affected at chlorine dioxide concentrations over 50 ppm.

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Ozone is a microbial inhibitor that is considered an alternative to chlorine and hydrogen

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peroxide in food applications. 34 The rate of microbial inactivation is affected by the several

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environmental factors (concentration of organic compounds, the physiological state of the

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microorganism, pH of the medium, temperature). 35 Thus it has had mixed success in the food

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industry. Garcia-Cubero et al., 36 illustrated the benefit of pretreatment of straw with ozone

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resulted in a reduction in acid insoluble lignin (Klason Lignin) and improved enzymatic

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hydrolysis of cellulose and hemicelluloses for biofuel production. Freitas-Silva et al., 37

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demonstrated mycotoxin degradation by ozone use in corn on the cob, corn kernels, and corn and

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rice powder. Miller et al., 38 reviewed the advantages and disadvantages of ozonation use for

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fruits and vegetables preservation while Gerrity and Snyder 39 review its use in water reuse

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applications. Thus, the application of ozone in dry grind corn ethanol production is needed.

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Research Objectives

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In this research ozone was used in place of antibiotics (e.g., Lactrol® and IsoStabTM) for

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the control of bacterial contamination in ethanol (yeast) fermentations. Preliminary experiments

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were subsequently performed using corn slurry to evaluate the effectiveness of ozonation for



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reducing bacterial contamination under conditions more similar to plant operation, including the

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contributions of recycled water sources.


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Materials and Methods

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Corn mash preparation

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Ground corn and corn slurry were obtained from POET Biorefining in Jewell, IA in the

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fall and spring of 2007 and 2008. The corn slurry obtained from POET contains the ground corn

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in a slurry with recycled water streams, such as backset. Figure1 illustrates the corn mash

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preparation, ozone treatment, and simultaneous saccharification and fermentation (SSF)

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procedures. Corn mash was prepared in 250-mL polypropylene (PP) bottles with 35% (w/w)

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ground corn (200 g mash per bottle) and deionized water or with 200 g corn slurry per bottle.

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Urea was added (0.03% [w/w]) as a N source. The final corn mash volume was 186 mL per

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bottle. Bottles were shaken for 30 min at 250 rpm and room temperature to slurry contents. The

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pH was adjusted to 5.0 in select experiments. In preliminary work (pH 4.2), all controls with and

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without Lactrol® and/or IsoStabTM added had similar values for lactic and acetic acids, glycerol,

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and ethanol, which indicated minimal bacterial contamination even without ozonation (results

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not shown). Therefore, no antibiotics or other antimicrobials (i.e., Lactrol® or IsoStabTM) were

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added in these experiments to exemplify treatment effects of ozone. The POET corn slurry

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experiments, however, potentially contained recycled antimicrobials contributed in the backset

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from previous fermentations at the plant. Select samples were also LAB-spiked and/or not yeast-

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supplemented to amplify the differences in treatment effects.

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Microorganisms

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Select corn mash samples were spiked with Lactobacillus plantarum, a lactic acid

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bacterium, obtained from the American Type Culture Collection (ATCC 14917, Rockville, MD)

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prior to ozone treatment. Stock cultures of L. plantarum were freeze-dried and stored previously

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in sterile skim milk at 4 °C. Fermentis Ethanol Red (Lesaffre Yeast Corporation, Milwaukee,



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WI), containing the yeast Saccharomyces cerevisiae, was added (0.5 g) after ozone treatment for

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ethanol fermentation.

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Ozonation

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The laboratory-scale installation used to apply the ozone treatment is shown in Figure 2.

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Ozone gas was generated from oxygen supplied by a compressed-air gas cylinder using a corona-

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discharge ozone generator (TOG C2B, Trigon, Glasgow, Scotland). Moisture and hydrocarbon

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traps (Restek, State College, PA) were placed between the ozone generator and compressed-air

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cylinder to remove impurities in the feed gas. A mass flow controller (GLC17, Aalborg,

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Orangeburg, NY) was used to control the gas flow rate.

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Ozone gas was bubbled into the corn mash samples (200 g) in 250-mL PP bottles with a

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flow rate of 500 mL/min. The ozone concentration in the gas phase was measured by the

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potassium iodide titration method. 40 The ozone dose, or consumption, was calculated based on

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ozone concentrations in the influent and effluent gas from the bottle over time. Corn mash

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samples were ozonated and residual ozone in off-gas measured by titration after varying

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ozonation times. This setup was used to determine the effects of different ozone dosages on

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ethanol fermentation of corn mash samples prepared from ground corn or corn slurry. Aeration

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treatment, as a control, was also performed using this equipment. The aeration and ozonation

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procedures were the same (e.g., similar gas flow rate), but with the ozone generator turned off.

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Ethanol fermentation

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Following ozonation, raw starch hydrolyzing enzyme (Novozyme 50009, 0.11% [v/w])

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and Ethanol Red (0.25% [w/w]) were added to ozonated, aerated, and non-treated corn mash

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(Figure 1). Bottles were shaken for 2 min at 250 rpm to mix contents and incubated without


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shaking at 27 °C (POET incubation temperature) until ethanol levels reached a plateau – up to

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168 h. SSF samples were HPLC-analyzed on a daily basis.

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Sample analyses

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The ethanol, glycerol, lactic acid, and acetic acid contents of corn mash and SSF samples

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were quantified using a Waters high pressure liquid chromatograph (HPLC) (Millipore

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Corporation, Milford, MA). The HPLC system was equipped with a Waters Model 401

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refractive index detector, column heater, automatic sampler, and computer controller. The

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Aminex HPX-8711 column (300×7.8 mm, Bio-Rad Chemical Division, Richmond, CA)

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separated sample constituents with a mobile phase of 0.012 N sulfuric acid, flow rate of 0.8

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mL/min, injection volume of 20 µL, and column temperature of 65 °C. 41

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Experiment sets

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A pH experiment was conducted prior to fermentation. Aeration treatment was included

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to confirm that ozonation, rather than aeration and mixing, lowered the initial corn mash pH

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prior to fermentation (0 h).

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The ethanol fermentation experiments reported in this publication are divided into three sets – two with POET ground corn and one with POET corn slurry – as outlined in Table 1.

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POET ground corn (Experiment sets 1 & 2)

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Two sets of fermentation experiments with ozone-treated corn mash prepared from

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ground corn were performed. The first set included spiking with L. plantarum before ozone

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treatment (0–188 mg/L) of corn mash; no yeast and no other antimicrobial agents were added to

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select bottles to further amplify the treatment effects. The second set included ozonation and

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aeration treatments of corn mash prior to ethanol fermentation. Select bottles were spiked with

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L. plantarum. Fermentation experiments were conducted with aeration treatment as a control to



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determine whether lower lactic acid contents in daily samples resulted from ozonation as a

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disinfect/oxidant or from possible stimulatory effects of aeration on the yeast.

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POET corn slurry (Experiment set 3)

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A preliminary set of fermentation experiments was conducted with POET corn slurry to

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assess the effectiveness of ozonation for reducing bacterial contamination under conditions more

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similar to plant operation, including contributions of recycled water streams. The recycled

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backset potentially contributed other antimicrobials in these experiments. Corn mash samples

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were prepared with/without the addition of yeast, with the slurry initial pH of 4.0 and adjusted to

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pH 5.0, and with/without ozone treatment, as shown in Table 1.

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Statistical Analysis All treatments were performed in replicates of three. Statistical analysis was carried out

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using the Statistical Analysis System package (SAS Computer Program Version 9.1.2, SAS

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Institute Inc., Cary, NC). Student’s t-test analyses were performed on HPLC data sets to

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determine statistical significance with a p value of 0.05.


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Results and Discussion

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Ozone dosages

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The results of ozonating corn mash samples for various treatment times in order to

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determine ozone consumption (i.e., ozone dosage) are illustrated in Figure 3. Ozone

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consumption is the difference between the ozone applied to the corn mash, f(x), and the unused

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ozone exiting the headspace of the treated corn mash over time (off-gas), g(x); it equals the green

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area in Figure 3. The following fitted equations were used to calculate ozone

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consumption/dosage:

229 230 231 232 233 234 235 236 237 238 239 240

Applied ozone (mg/min) Off-gas ozone (mg/min) Off-gas ozone (mg/min)

= f(x) = 0.996 = g(x) = 0.00319x+0.000538x2 = g(x) = 0.884

0 ≤ t ≤ 37.5 min 37.5 min ≤ t

(1) (2) (3)

௧

௧

= 0.996t-0.00159t2-0.000179t3

0 ≤ t ≤ 37.5 min

(4)

௧

௧

= 0.112*(t-37.5) + 25.7

37.5 min ≤ t

(5)

‫׬‬଴ ݂ሺ‫ݔ‬ሻ݀‫ ݔ‬− ‫׬‬଴ ݃ሺ‫ݔ‬ሻ݀‫ݔ‬ ‫׬‬଴ ݂ሺ‫ݔ‬ሻ݀‫ ݔ‬− ‫׬‬଴ ݃ሺ‫ݔ‬ሻ݀‫ݔ‬

The ozone dosages computed for each treatment time are provided in Table 2. In our

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preliminary work, the additional dosages of 26 and 75 mg O3/L were tested. The higher dosages

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(127, 152, and 188 mg/L) were chosen for replicate experiments, as reported in this publication.

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Ozone cost estimates

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The ozone cost estimates include operating costs to purchase electricity for the ozone

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generator and to prepare air for the ozonation. The costs of electricity and air are calculated as

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follows:

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Electricity consumption on typical ozone generators: 10 kWh/kg ozone Electricity: $0.07/kWh, 2009 average for industrial consumers 40 Electricity consumption for air feed drying: approximately 2 kWh/kg ozone Total electricity consumption and cost: 12 kWh/kg and $0.84/kg, respectively 12 ACS Paragon Plus Environment

(6) (7) (8) (9)


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Capital costs: since the ozone dosage has not yet been optimized in relation to corn mash

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formulations in industry, including recycled water sources, the required ozone generator size for

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a typical plant cannot be determined. As a rough guide, the amortization costs will equal the

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energy costs. The cost of ozone, therefore, can be expected to amount to roughly $1.68/kg. This

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cost still needs to be determined to a greater degree of accuracy. Table 2 presents the estimated

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ozonation costs based on the ozone dosages applied in experiments to date.

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Initial pH experiment

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The initial pH of corn mash prepared using ground corn, prior to fermentation (0 h), was

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adjusted to 4.2 or 5.0. The pH of corn mash samples tended to decrease with increasing ozone

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treatment times/dosages (Figure 4). The pH of ozonated samples (initial pH 5.0) started to

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decrease after 15 min of ozonation (75 mg O3/L), reaching an average pH of 4.8 in 60 min (152

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mg O3/L). With an initial pH of 4.2, the corn mash pH was also reduced by 0.2 pH units to 4.0

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after 60 min of ozonation. Aeration using the same procedure, but no ozone generated, did not

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affect the corn mash pH. This finding shows that the ozone or ozone byproducts, rather than

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aeration and additional mixing, were responsible for the corn mash pH reductions observed in all

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experiments using ground corn.

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Corn mash samples were analyzed on the HPLC before and after treatment to detect any

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changes in concentrations, in particular for organic acids. No consistent changes were observed

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for ozonated and aerated samples. A slight increase in acetic acid of 0.35 g/L occurred in one

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ozonated sample, but not in replicate samples. There were no changes in acetic acid in aerated

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samples. No lactic acid was detected in any samples before or after ozonation/aeration.

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Ground corn (Experiment sets 1 & 2)

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Effects of ozonation on pH and lactic acid contents


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Experiment set 1 (Figure 5): the daily pH values of ozonated and non-ozonated corn

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mash samples decreased the first 24–48 h of fermentation only. The exceptions were samples

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with higher ozone dosages (152 and 188 mg/L) and no yeast added; the pH of these samples

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started decreasing after 24–48 h of fermentation and stabilizing by 96–120 h. All pH trends

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corresponded with increases in lactic acid contents. Higher daily pH values, and less lactic acid,

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were observed in samples subjected to higher ozone dosages.

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Statistical analysis was conducted to compare the significance (p

Reducing Bacterial Contamination in Fuel Ethanol ... - ACS Publications

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