Conversion of Thin Stillage Compounds Using Endemic Bacteria

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Conversion of Thin Stillage Compounds using Endemic Bacteria Augmented with Lactobacillus panis PM1B Kornsulee Ratanapariyanuch, Youn Young Shim, and Martin J.T. Reaney J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b00643 • Publication Date (Web): 04 Oct 2016 Downloaded from http://pubs.acs.org on October 7, 2016

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Journal of Agricultural and Food Chemistry

Conversion of Thin Stillage Compounds using Endemic Bacteria Augmented with Lactobacillus panis PM1B Kornsulee Ratanapariyanuch,† Youn Young Shim,*,†,‡ and Martin J. T. Reaney*,†,‡,§

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†Department

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Saskatchewan S7N 5A8, Canada

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of Plant Sciences, University of Saskatchewan, 51 Campus Drive, Saskatoon,

‡

Prairie Tide Chemicals Inc., 102 Melville Street, Saskatoon, Saskatchewan S7J 0R1, Canada

§

Guangdong Saskatchewan Oilseed Joint Laboratory, Department of Food Science and Engineering,

Jinan University, 601 Huangpu Avenue West, Guangzhou, Guangdong 510632, China

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CORRESPONDING AUTHOR INFORMATION

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E-mail address: [email protected] (YYS), [email protected] (MJTR) 1 ACS Paragon Plus Environment


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ABSTRACT

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A consortium of organisms endemic in wheat-based thin stillage (W-TS) obtained from a commercial

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ethanol production converts glycerol to 1,3-propanediol (1,3-PD) and lactic acid to acetic acid. We

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sought to improve conditions for 1,3-PD and acetic acid production to be used in future studies of

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industrial isolation of these compounds from two-stage fermentation. Occasionally stillage fermentation

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proceeded slowly but an inoculum of Lactobacillus panis PM1B augmented both fermentation rate and

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extent. Fermentation rate and product yield were enhanced by adjusting pH to 5 daily, adding glucose

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and glycerol (molar ratio 0.1:1), adding freeze-dried W-TS, and adding vitamins (B2, B3, and B12). 1,3-

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PD and 3-HPA did not inhibit 1,3-PD production during fermentation. Moreover, agitation did not

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improve fermentation rate or extent. Corn sugar was a suitable substitute for glucose. Fermentation was

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performed at both 20 and 150 L, with 1,3-PD production of 2% (w/v, 20 g/L) being routinely achieved

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or exceeded.

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Keywords: Thin stillage; Fermentation; Lactobacillus panis PM1B; Endemic micro-organism; 1,3-

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propanediol; Glycerol

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2 ACS Paragon Plus Environment

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INTRODUCTION

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Thin stillage (TS) is a liquid co-product of the bioethanol industry. Wheat-based thin stillage (W-TS)

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contains organic solutes from bacteria, plant, and yeast metabolism as well as protein, and salts.

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Glycerol is a major W-TS organic solute1,2 and W-TS is a potential commercial source. However, crude

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glycerol is inexpensive3 and purification of this relatively dilute compound from W-TS may not be

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economically feasible. Recently, Reaney et al.4 and Khan et al.5 reported that Lactobacillus panis

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PM1B, isolated from W-TS, converted glycerol to 1,3-propanediol (1,3-PD) and lactic acid to acetic

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acid. In this same fermentation, lactic acid is converted to acetic acid by the metabolic action of

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lactobacilli. The acetic acid boiling point is lower than that of lactic acid while the 1,3-PD boiling point

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is lower than that of glycerol. Therefore, distillation could potentially be used to recover 1,3-PD and

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acetic acid from W-TS residues at a lower production cost.

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It is recognized that W-TS contains many strains of bacteria other than L. panis PM1B,6 some of

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which might also convert glycerol to 1,3-PD. It is known that many different bacteria have metabolic

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pathways for conversion of glycerol to 1,3-PD, including: L. diolivorans, L. reuteri, Clostridium

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butyricum DSM 5431, C. butyricum CNCM 1211, C. butyricum VPI 3266, Klebsiella oxytoca LDH3, K.

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pneumoniae ATCC 15380, and K. pneumoniae M5al.7–14 Some of these bacterial cultures can produce

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1,3-PD ranging from 13.08 to 84.57 g/L, but are fastidious or strict anaerobes, requiring rigorously

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controlled fermentation conditions and/or costly nutrients. Moreover, several of the genera contain

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potentially pathogenic organisms, and isolates that might also produce toxins.

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Previous studies of L. panis PM1B showed conversion of glycerol and lactic acid to 1,3-PD and

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acetic acid, respectively. However, most of those studies were conducted primarily in de Man, Rogosa

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and Sharpe (MRS) medium and variants of MRS.5,15,16 Studies with controlled fermentation conditions

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with L. panis PM1B conducted by Kang et al.16 were important in elucidating fundamental biochemical

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pathways, but will likely not reflect fermentation performance in stillage media. As expected, the 3 ACS Paragon Plus Environment


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composition of both organic and inorganic solutes in MRS media are unlike W-TS solutes.16,17 Many

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MRS organic solutes could significantly modify bacterial metabolism and metabolic responses.

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Bacterial growth media, such as MRS, are supplemented with extracts, detergents, essential elements,

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proteins, vitamins, and buffers. Each of these supplements contributes to suitability of the medium for

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growth of a wider range of micro-organisms while sodium acetate is added to increase specificity for

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lactobacilli. Growth of lactobacilli and enhancement of metabolic pathways that convert glycerol and

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lactic acid in W-TS based media may require similar supplementation, but there has been no attempt to

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optimize W-TS medium for 1,3-PD and acetic acid production from glycerol and lactic acid.

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Glucose provides a large component of carbon available in MRS medium, however, this may be

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substituted.5,15 MRS contains several other undefined carbon sources, such as meat and yeast extracts.18

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Even though meat and yeast extracts are predominantly included to act as nitrogen sources, carbon is

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available from these media components. Stillage is also composed of an array of potential carbon

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sources,19 but with depleted glucose levels due to prior metabolism by yeast. Carbon sources used in

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Lactobacillus fermentations can have a large effect on metabolism.5,15 It is, therefore, uncertain whether

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metabolic studies of organisms in MRS can be useful in informing how the same organisms will grow in

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and metabolise substrates in W-TS. Moreover, W-TS solutes, nutrients, and adhesion sites are likely to

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influence growth and metabolism of lactobacilli, including glycerol metabolism. Active bacterial

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metabolic pathways expressed when growing in MRS likely differ greatly from those used by the same

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organisms grown in W-TS. Additionally, stillage contains colloids and particulate solids that could have

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multiple effects on fermentation that are not comparable in MRS media. Conversely, stillage solids are

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likely to provide surfaces for bacterial habitat, enabling biofilm formation that might improve cell

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division, abundance, and metabolic efficiency. The impact of solids present in W-TS on Lactobacillus

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growth and metabolism during fermentation is not known. While other researchers were not aware of

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the impact of W-TS particles on fermentation,17 the particles were not removed from W-TS prior to use

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as a fermentation media in this research. 4 ACS Paragon Plus Environment

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Kang et al.17 utilized genetically engineered L. panis PM1B while in this research, we utilized W-TS

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endemic organisms augmented with L. panis PM1B. Differences between genetically engineered L.

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panis PM1B and endemic organisms augmented with L. panis PM1B could lead to different results. In

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addition, centrifuged W-TS was sterilized prior to inoculation with L. panis PM1B.17 Ethanol

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production facilities are typically large. In these commercial fermentation environments, it may be

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neither practical nor economical to impose sterile conditions on millions of liters of solution each day.

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Therefore, we chose to study fermentation in the presence of endemic cultures. Fermentation with

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endemic cultures is widely used in food production. Traditional sourdough breads, and yoghurt are

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examples.20 Kang et al.17 reported that after sterilizing centrifuged W-TS, the initial pH of W-TS was

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6.5.17 In our research, the initial pH of non-sterile W-TS was approximately 4.0. This indicated that heat

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from sterilization might alter the buffer system of W-TS.

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Fermentation conducted in this research used corn sugar, largely α-D-glucose,21 instead of glucose as

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had been used by Grahame et al.15 The price of refined glucose is too high to be economical for an

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industrial carbon source while corn sugar is more economical for such fermentations. Kang et al.17

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utilized a glucose to glycerol molar ratio of 0.37 M/M. The quantity of glucose utilized in their

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fermentations might be too high and could be an obstacle for industry. Our research describes

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improvement of fermentation conditions for efficient production of 1,3-PD and acetic acid. Parameters

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studied include glucose concentration, addition of freeze-dried W-TS, vitamins, pH adjustment, and

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substrate substitution. In addition, fermentation scale was increased in stages from 50 mL to 150 L.

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MATERIALS AND METHODS

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Wheat distiller’s solubles (W-DS) sample was obtained from Terra Grain Fuels (Belle Plaine, SK,

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Canada) and stored at 4 °C. Pound-Maker Agventures Ltd. (Lanigan, SK, Canada) kindly provided W-

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TS samples and the samples were stored at 4 °C. Hereafter, the samples are called W-TS1, W-TS2, W-

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TS3, and W-TS4, respectively. Normally, TS is concentrated to distillers’ solubles (DS) by 5 ACS Paragon Plus Environment


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evaporation.22 Typically, W-DS was more stable than W-TS and, therefore, was used as a concentrate

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that could be diluted for specific studies where the same medium was needed over a long period of time.

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Lactobacillus panis PM1B was isolated previously by Reaney et al.4,5 and available through the

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International Depository Authority of Canada; accession number 180310-01.

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Analytical Methods.

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Protein Content. The Kjeldahl method (AOAC 981.10)23 was used to determine assay nitrogen

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content (total Kjeldahl nitrogen, TKN). Protein content was calculated from TKN after subtracting

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nitrogen contributed from non-protein compounds [glycerylphosphorylcholine (GPC) and betaine].2 The

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TKN was then multiplied by a conversion factor of 5.7,24 the same factor used for wheat as it was

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utilized as a raw material for ethanol production in Pound-Maker Agventures Ltd.

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Moisture Content. Moisture content was determined by heating samples at 100–102 °C for 16–18 h in an oven and determining mass lost as a portion of initial mass according to method AOAC 950.46.23

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Nuclear Magnetic Resonance Spectroscopy. Double pulse field gradient spin echo (DPFGSE-

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NMR) was conducted according to Ratanapariyanuch et al.2 Samples were centrifuged (Spectrafuge™

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24D, Labnet International Inc., Edison, NJ, USA) at 9,200 × g for 10 min prior to analysis. After

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centrifugation, supernatant was filtered through a syringe filter (25 mm syringe filter with 0.45 µm

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PTFE membrane, VWR International, West Chester, PA, USA). Proton NMR spectra were recorded at

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500 MHz (AMX 500-MHz, NMR Bruker, Mississauga, ON, Canada) with 16 scans per spectrum using

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a DPFGSE-NMR pulse sequence. NMR data collection and analyses were conducted with TopSpin™

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3.2 software (Bruker BioSpin GmbH, Billerica, MA, USA). Deuterium oxide (Cambridge Isotope

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Laboratories Inc., Andover, MA, USA) and dimethylformamide (EMD Chemicals Inc., Gibbstown, NJ,

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USA) were used as solvent and internal standard, respectively.

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Lactobacillus Enumeration. Lactobacilli were enumerated by the drop plate technique25 using

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Lactobacillus Heteroferm screening broth (HiMedia Laboratories Pvt. Ltd., Mumbai, India) containing 6 ACS Paragon Plus Environment

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2% agar, and normalized to colony forming units (CFU) per milliliter of sample (CFU/mL). Agar plates

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were incubated at 37 °C in the dark for 72 h in a candle jar to maintain a low oxygen environment

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conducive to growth of Lactobacillus species.

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Glucose Content. Samples were centrifuged at 9,200 × g for 10 min and supernatant filtered through

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0.45 µm syringe filters prior to analysis. Filtrates were diluted with 0.5 M phosphate buffer (pH 8) to

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dilute filtrates to a range of glucose concentrations suited for analysis by glucose hexokinase assay kits

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(Sigma-Aldrich, St. Louis, MO, USA) following the manufacturer’s guidelines.

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Fermentation Improvement.

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A fuel ethanol distillery in Saskatchewan provided W-DS for this study. It was previously found that

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over time, 1,3-PD and acetic acid spontaneously increased in concentration in W-DS as a result of

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metabolism by endemic micro-organisms.

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The 1,3-PD producing organism, L. panis PM1B4,5, was used in this study to assess effects of

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augmentation of endemic microbial conversion of glycerol to 1,3-PD in W-TS. L. panis PM1B cultures

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stored in double strength skim milk were grown on W-DS agar (500 mL W-DS, 500 mL water, 8 g

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yeast extract, 4 g glucose, pH 5.1 ± 0.1 adjusted with 1 M NaOH, 15 g agar, autoclaved at 121 °C for 15

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min).6 Agar plates were incubated at 37 °C in a candle jar for 72 h. Colonies of L. panis PM1B were

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used to inoculate starter culture to augment endemic bacteria present in 50 mL tubes of non-sterile W-

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TS to test effects of pH, glucose concentration, freeze-dried W-TS, 1,3-PD, vitamins, and agitation on

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production of 1,3-PD and associated co-products. In subsequent studies, L. panis PM1B in double

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strength skim milk (25 µL) was inoculated in filtered sterilized (0.22 µm) 25% W-DS containing 1.1 M

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of glycerol (25 mL) in a 25 mL sterilized centrifuge tube. The tube was incubated at 37 °C for 72 h.

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After incubation, tube contents were divided into sterile 2 mL micro-centrifuge tubes and stored at 80

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°C as culture stock. The L. panis PM1B stock was grown on W-DS agar. Agar plates were incubated at

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37 °C in a candle jar for 72 h. Colonies of L. panis PM1B were selected to inoculate starter cultures for 7 ACS Paragon Plus Environment


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studies of the effects of corn sugar, 3-hydroxypropionaldehyde (3-HPA), and pH and in 20 L

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fermentation vessels.

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For 50 mL fermentations (the smallest scale non-sterile culture), W-DS was diluted to 25% W-DS

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and centrifuged at 7,000 × g for 15 min at 4 °C. A dilution 1:3 with water was selected as this

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essentially reconstitutes the W-DS to a similar concentration of compounds found in W-TS. After

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centrifugation, supernatant samples were filtered through cotton. Hereafter, these samples are called

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“clarified 25% W-DS”. Clarified 25% W-DS was mixed with glycerol to a final concentration of 0.9 M

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and the solution (50 mL) was added to 50 mL sterile centrifuge tubes after which media were

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pasteurized by holding for 15 sec at 72 °C. The tube and contents were cooled prior to inoculation.

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Media were inoculated with 23 colonies of L. panis PM1B from the W-DS agar plates and incubated at

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37 °C for 48 h (Figure 1). Inoculant for 20 L scale fermentations was prepared in six bottles (500 mL)

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nearly filled with pasteurized clarified 25% W-DS and 0.9 M glycerol. After cooling, pasteurized media

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were inoculated with L. panis PM1B and incubated at 37 °C for 72 h (Figure 1).

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Fermentation in 50 mL Tubes. W-TS1 medium was mixed with 50 mL of starting culture to a final volume of 350 mL and divided into 50 mL sterilized centrifuge tubes (50 mL/tube).

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Effect of pH. The tubes were incubated at 37 °C (Figure 1). The pH of media (excluding controls)

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was adjusted to their perspective pH from 5.0–10.0 daily after approximately 46 h using 1 M NaOH and

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1 M HCl. Lactobacilli were enumerated at 0 h. After approximately 46 h of incubation, fermentations

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were sampled daily and subjected to NMR analysis.

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Effect of Glucose Concentration. Glucose was added to achieve final concentrations of 0.1, 0.2, 0.3,

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and 0.4 M and glycerol was added to a final concentration of 1 M. Cultures were incubated at 37 °C.

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After approximately 46 h, medium pH was adjusted to 5.0 and pH was readjusted to 5.0 every 24 h

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thereafter. When fermentation ceased, 1 g of glucose was added into 0.1 and 0.2 M of glucose

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treatments at 139 h of fermentation in an attempt to restart fermentation. Lactobacilli were enumerated


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as described above at 0 h. Fermentation media were sampled daily from each tube for analyses of

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organic solutes by NMR and glucose content.

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Effect of Freeze-dried W-TS, 1,3-PD, and Vitamins. In prior work, pH 5, glycerol (1 M), and glucose

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(0.1 M) aided fermentation. Therefore, media were adjusted to these optimized conditions for further

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work. It was hypothesized that organic solutes present in W-TS might act as micro-nutrients and aid

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metabolism and microbial growth. Furthermore, organic solutes might be heat labile. A concentrate,

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prepared to ensure retention of potentially heat labile organic solutes of W-TS, was made by freeze-

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drying W-TS (Stoppering Tray Dryer, 7948040, Labconco Inc., Kansas City, MO, USA). W-TS media

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were modified by addition of a range of compounds and additives. The increase of 1,3-PD and acetic

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acid was assessed and compared with a W-TS control medium [glucose (0.1 M) and glycerol (1.0 M)]

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with added 1,3-PD (1.0 g). Media were also modified by adding freeze-dried W-TS1 (0.5 g) or vitamins

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(B2, B3, and B12, 0.5 mg of each). After inoculation (approximately 46 h), medium pH was adjusted

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daily to 5.0 using either 1 M NaOH or 1 M HCl. Cultures were incubated at 37 °C. Lactobacilli were

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enumerated as described above at 0 h while glucose content and NMR analyses were conducted daily.

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After fermentation ceased (190 h), glycerol and lactic acid conversion in control media was no longer

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observed. In an attempt to restart fermentation, freeze-dried W-TS1 (0.5 g) was added to media

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supplemented with freeze dried W-TS added and control conditions. In addition, vitamins (0.5 mg each)

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were added to vitamin enriched culture media. The fermentation was continued until 233 h to determine

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effects of adding freeze-dried W-TS and vitamins.

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Effect of Agitation and Freeze-dried W-TS. A temperature-controlled benchtop shaker (Excella E24,

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New Brunswick Scientific Co., Inc., Edison, NJ, USA) was used for temperature controlled incubation

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and agitation. Freeze-dried W-TS1 (0.5 g) was introduced to agitated (200 rpm) and static cultures (0.1

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M glucose: 1.0 M glycerol) at 0 h. Medium pH was adjusted to 5.0 daily after approximately 46 h of

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fermentation except in condition 1. In that culture, medium pH was adjusted to 5.0 at 0 h and daily after.

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This culture was agitated and freeze-dried W-TS was included. The tubes were incubated at 37 °C. 9 ACS Paragon Plus Environment


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Lactobacilli were enumerated. Organic solutes and glucose content were determined from daily

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samples.

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Effect of 3-HPA, Corn Sugar, and pH. Four media were prepared to determine effects of 3-HPA,

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corn sugar, and pH on 1,3-PD production. A control medium (0.1 M glucose: 1 M glycerol + 0.5 g

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freeze-dried W-TS1), a similar medium with 3-HPA (0.3 g) added, a third medium similar to the control

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but substituting glucose with corn sugar (to produce 0.1 M glucose), and a control adjusted to pH 5.0

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daily starting at inoculation. For other media pH was adjusted to 5.0 daily after incubation for

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approximately 46 h. Cultures were incubated at 37 °C. W-TS solution was sampled daily for

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enumeration of lactobacilli, NMR analyses, and glucose content.

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Fermentation in a 25 L Vessel. Freeze-dried W-TS (2.48 kg) was prepared by drying W-TS2 at

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temperatures below 80 °C from 30.84 kg to 7.58 kg in a rotary evaporator (R-220) equipped with a

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vacuum controller (V-800), all from Büchi Labortechnik AG (Flawil, Switzerland) then drying the

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concentrate in a freeze dryer as described previously. Freeze-dried W-TS was used in media preparation

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as described below. A 25 L polyethylene pail and lid equipped with a gas trap (Wine Kitz, Sakatoon,

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SK, Canada) was used as a fermenter. W-TS2 was mixed with freeze-dried W-TS2 (200 g) and starting

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culture (total volume 3 L; Figure 1). Corn sugar (approximately 0.1 M glucose) and glycerol (final

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concentration 1 M) were also added. The fermentation medium volume was adjusted to 20 L and

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incubated at 25 ± 2 °C. Medium pH was adjusted to pH 5.0 daily. Medium was stirred daily using a

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Polytron homogenizer (45TE, Bronwill Scientific, Rochester, NY, USA) during pH adjustment and

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sampled afterwards. Lactobacillus enumeration, NMR analyses of organic solutes, and glucose content

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were conducted daily using these samples.

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Fermentation in a 210 L Vessel. Medium (W-TS2) was mixed with corn sugar and glycerol to final

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concentration 0.1 and 1.0 M, respectively. In addition, freeze-dried W-TS2 (200 g), and starting culture

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(3 L, Figure 1) were added. The medium volume was adjusted to 20 L in 25 L fermenters, which were

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then incubated at 25 ± 2 °C. Fermentation media were stirred using a Polytron homogenizer (described 10 ACS Paragon Plus Environment

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above) while adjusting pH to 5.0. After adjusting pH, fermentation medium were sampled for glucose

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content, Lactobacillus enumeration, and organic solute analysis. Fermentation continued until the 1,3-

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PD concentration reached approximately 6 and 15 g/L for replicate 1 and 2, respectively. After this,

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cultures were used to inoculate a 210 L fermenter equipped with a gas trap. W-TS3 was utilized as the

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fermentation medium and the experiment was repeated with W-TS4 for two 150 L fermentations.

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Medium was mixed with corn sugar (2.7 kg), freeze-dried W-TS2 (1.5 kg), and glycerol (12 kg).

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Fermentation medium volume was adjusted with W-TS to 150 L. The fermentation medium was mixed

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daily using a hand pump (SP-280P-V, Standard Pump Inc., Duluth, GA, USA) at a setting of 5 for 10

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min while adjusting pH to 5.0 during mixing. Fermentation medium was sampled daily after adjusting

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pH to determine glucose content, organic solutes, and enumerate bacteria.

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Statistical Analysis. Effects of agitation, glucose concentration, inhibitors, micro-nutrients, pH,

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and substrate substitution, were determined. The simultaneous study of all of these treatments was

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impractical with available facilities. The process was iterative with each successive round of

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fermentations building upon prior findings. Conditions in iterations that produced the highest

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concentration of 1,3-PD were used as the starting point for subsequent iterations. Nonetheless,

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additional response surface studies will be required to determine optimum conditions for large-scale

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fermentation. The largest fermentation scale possible using available equipment was 150 L. The nearest

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ethanol plant is 150 km from the research facility and it was impractical to transport more than 300 kg

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of stillage to the laboratory in a rental vehicle. Due to constraints of working with large samples, 20 and

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150 L fermentations were done in duplicate only.

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RESULTS AND DISCUSSION

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Characterization of W-TS. The protein and moisture contents and organic solutes varied among

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W-TS batches (Tables 1 and 2). Not all nitrogen present in W-DS and W-TS arises from protein.

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Typically, W-DS and W-TS contain betaine and GPC, which contribute to total nitrogen. Therefore, 11 ACS Paragon Plus Environment


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nitrogen content was corrected before estimating protein content. As expected, W-DS protein content

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was higher than that of W-TS while moisture content was lower. Meredith26 reported that DS had solid

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contents from 25 to 28% while Liu and Barrows27 noted that corn-based TS and DS had moisture

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contents of 90–95 and 50–75%, respectively. Organic solutes including 1,3-PD, acetic acid, betaine,

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ethanol, glycerol, GPC, isopropanol, lactic acid, phenethyl alcohol, and succinic acid were present in

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both W-DS and W-TS samples (Table 2). These compounds arise from grain metabolites and

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metabolism of grain metabolites by endemic micro-organisms.1,2 In addition, non-volatile compounds

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(e.g. glycerol) present in W-DS were more concentrated than in W-TS.

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Fermentation Improvement.

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Fermentation in 50 mL scale.

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Effect of pH on W-TS fermentation. Fermentation media pH was adjusted from 5.0–10.0. The growth

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of micro-organisms present in W-TS supplemented with L. panis PM1B lowered the pH of W-TS

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media, presumably due to production of lactic and acetic acids (Figures 2C and 2D) during normal

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metabolism of lactobacilli (data not shown). The highest production of both 1,3-PD and acetic acid was

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achieved when culture pH was adjusted to 5 (Figures 2B and 2D). Moreover, glycerol conversion was

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nearly complete with little glycerol remaining in the media at the end of fermentation at pH 5 while

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residual glycerol was present in cultures adjusted to pH 9, 10, and the unadjusted control (pH 3.4–3.9;

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Figure 2A). The highest residual glycerol and lowest 1,3-PD production were noted after fermentation at

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pH 10.0 (Figures 2A and 2B). These results contrast with observations of Grahame et al.15 They noted

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that when L. panis PM1B was grown in MRS medium at initial pH values of 9.0 and 10.0, 1,3-PD

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production was faster and cell density increased more slowly compared to other pH values tested. These

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responses may have been related to differences between MRS media and W-TS used or the use of

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endemic bacteria and not a pure strain of L. panis PM1B. The observation of slow production of 1,3-PD

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at higher pH is in agreement with Khan et al.5 who noted that the optimum pH for growth of L. panis

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PM1B was 4.5. The growth of L. panis PM1B was slower at higher pH in the present study and that of 12 ACS Paragon Plus Environment

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Khan et al.5 It should be noted, however, that Grahame et al.15 did not maintain the pH throughout their

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experiment, but only performed an initial pH adjustment, and pH likely decreased through fermentation.

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In addition, Kang et al.17 utilized sterilized centrifuged W-TS. They reported that the initial pH of

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sterilized centrifuged W-TS was 6.5 while the initial pH of non-sterile W-TS in this research was

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approximately 4.0 (Table 1). The difference of pH could be the result of heat from sterilization altering

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the composition of W-TS. Additionally, Grahame et al.15 and Kang et al.16 utilized modified versions of

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MRS medium as a base to prepare specific fermentation media rather than W-TS as was done in the

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present study. These choices may have led to differences observed with findings reported herein. Based

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on results of our initial test of optimal pH for improved production of 1,3-PD (Figure 2), subsequent

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experiments in this study used a pH 5.0 and 1 M glycerol to ensure an adequate supply of glycerol for

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conversion and higher production of 1,3-PD during fermentation.

298

Effect of Glucose Concentration on W-TS fermentation. Grahame et al.15 found that glucose was the

299

best carbon source tested for growth of L. panis PM1B in variations of MRS media that included

300

alternate sugars. Here, we tested the effects of glucose added to W-TS on glycerol and lactic acid

301

metabolism during fermentation in comparison to a control without added glucose. Concentrations up to

302

0.4 M glucose were tested in W-TS. The number of lactobacilli in the W-TS fermentation solution was

303

7 × 107 CFU/mL at 0 h. A glucose concentration of 0.1 M combined with 1 M of glycerol at pH 5

304

resulted in the greatest increase of 1,3-PD (11.9 g/L) and acetic acid when compared with other glucose

305

concentrations tested (Figures 3B and 3D). In addition, it was observed that production of 1,3-PD began

306

when glucose in the fermentation medium was nearly depleted (Figures 3B and 3E). This result was in

307

agreement with Grahame et al.15 who reported that under anaerobic conditions 1,3-PD production did

308

not occur until glucose was mostly depleted in late log to early stationary phase. According to Khan et

309

al.,5 1,3-PD production utilized NADH+H+ from glucose consumption to regenerate NAD+ for

310

continued glucose metabolisms under anaerobic condition. However, when glucose was depleted, 1,3-

311

PD accumulation still occurred. NADH+H+ for 1,3-PD accumulation after glucose depletion could arise 13 ACS Paragon Plus Environment


Page 14 of 38

312

from a pathway that converts lactic acid to acetic acid and, thereby, generates NADH+H+.28

313

Nevertheless, it should be noted that energy would be involved in converting lactic acid to acetic acid.

314

Higher starting concentrations of glucose (0.2, 0.3, and 0.4 M) did not yield greater concentrations of

315

1,3-PD or more effective use of glycerol (Figure 3). We hypothesize that glucose may have been in

316

excess for the reaction, and may have increased osmotic pressure leading to cell stress, lower bacterial

317

growth rates and 1,3-PD production (Figure 3B). In addition, it was observed that adding 1 g of glucose

318

at 139 h of fermentation did not improve 1,3-PD production, though glucose concentration decreased

319

and lactic acid was produced (Figure 3). These findings suggest that other essential nutrients for micro-

320

organisms might be depleted. Therefore, fermentation could not be restarted. Based on these findings,

321

0.1 M of glucose was used for subsequent fermentations in this study.

322

Effect of Freeze-dried W-TS, 1,3-PD, and Vitamins on W-TS fermentation. Normally, W-DS is

323

prepared by distillation of W-TS, which requires extensive exposure to elevated temperatures. While W-

324

DS is similar in composition to W-TS, heating during evaporation may degrade or inactivate important

325

labile nutrients and remove volatile substances. These might affect the compound conversion ability and

326

growth of endemic micro-organisms including L. panis PM1B. Freeze-dried W-TS was tested as a

327

potential source of concentrated nutrients to replace W-DS to determine if heat labile nutrients were lost

328

in preparing W-DS. According to Pflügl et al.,7 vitamins (B2, B3, and B12) might enhance 1,3-PD

329

production as vitamin B2 and B3 are involved in vitamin B12 biosynthesis. In addition, 1,3-PD might

330

also act as an end product inhibitor of its own production. Consequently, we explored the effects of

331

freeze-dried W-TS, vitamins, or 1,3-PD added to W-TS media. At the beginning of fermentation, the

332

inoculum was 7 × 107 CFU/mL. Adding either freeze-dried W-TS or vitamins enhanced 1,3-PD and

333

acetic acid production compared to controls (Figure 4). Sluggish growth and fermentation rates at the

334

end of fermentation might be due to exhaustion of essential nutrients and carbon source e.g. amino

335

acids, minerals and glucose. Freeze-dried W-TS was tested because vitamins are relatively expensive

336

and may be cost prohibitive for commercial use. Therefore, W-TS was dried under mild conditions as 14 ACS Paragon Plus Environment

Page 15 of 38


337

this might be preferred as a source of essential nutrients. Nevertheless, adding freeze-dried W-TS or

338

vitamins after 190 h fermentation neither improved 1,3-PD production nor restarted fermentation

339

(Figures 4A–4D). Addition of 1,3-PD did not inhibit production of 1,3-PD in W-TS (Figure 4B).

340

Therefore, it appears that end product inhibition by 1,3-PD does not block 1,3-PD production. In

341

addition, glycerol consumption still occurred after 1,3-PD accumulation reached its maximum. These

342

phenomena could be the result of endemic micro-organisms that utilize glycerol or other carbon sources.

343

Effect of Agitation and Freeze-dried W-TS on W-TS fermentation. Both agitated (0.5 g freeze-dried

344

W-TS; pH adjusted to 5.0 at 0 h) and static (0.5 g freeze-dried W-TS) cultures produced similar

345

amounts of 1,3-PD indicating that agitation had little effect on product yield (Figures 5A–5E). Agitation

346

during fermentation can aid in gas-liquid mass transfer29 and increase oxygen transfer from headspace to

347

fermentation media. This might impede 1,3-PD and acetic acid production according to Khan et al.5 who

348

observed 1,3-PD production only under anaerobic and micro-aerobic conditions. Furthermore, Oude

349

Elferink et al.30 also proposed a pathway for lactic acid degradation by L. buchneri under anaerobic

350

condition. One of the highest concentrations of 1,3-PD and acetic acid was observed in cultures that

351

were adjusted to pH 5.0 daily (Figures 5B and 5D). The greatest production of 1,3-PD and acetic acid

352

was noted when acids produced by micro-organisms were neutralized with NaOH. In addition, the

353

increase of Lactobacillus CFU was faster when pH was adjusted to 5 at 0 h along with agitation

354

compared to other conditions (Figure 5F). Based on these investigations, static conditions and addition

355

of freeze-dried W-TS were used in further studies.

356

Effect of 3-HPA, Corn Sugar, and pH on W-TS fermentation. Prior to increasing fermentation scale,

357

factors that might affect fermentation were tested including substitution of refined glucose with corn

358

sugar, adjustment of pH at time 0, and the effect of 3-HPA, a potentially toxic intermediate.31 Corn

359

sugar is produced by enzymatic hydrolysis of cornstarch and is a crude product containing mostly

360

glucose but also containing the disaccharide, maltose, and higher polysaccharides. Glucose is often used

361

in fermentation media for research purposes but corn sugar is a preferred carbon source for commercial 15 ACS Paragon Plus Environment


Page 16 of 38

362

fermentation due to lower raw material cost. As was expected corn sugar could replace laboratory

363

glucose without affecting conversion of glycerol to 1,3-PD or lactic to acetic acid (Figure 6). The CFU

364

number was also unaffected. In addition, there is no apparent effect of either maltose or polysaccharides

365

present in this carbon source. We noted above that pH 5 appeared to be superior for substrate

366

conversion. The effect of adjusting pH to 5.0 at 0 h was tested to compare with other treatments. It was

367

found that CFU increased more rapidly in cultures when pH was adjusted to 5.0 than for cultures grown

368

without this pH adjustment at 0 h of fermentation. Moreover, it was confirmed that metabolism was

369

more efficient when pH was adjusted to 5.0 at 0 h and daily thereafter. Therefore, corn sugar and pH

370

adjustment to 5.0 daily were employed for subsequent experiments. According to Saxena et al.,31 3-HPA

371

is toxic to bacteria and its presence during fermentation might slow cell growth or inhibit substrate

372

conversion. Adding 3-HPA to media affected neither culture growth nor increase of 1,3-PD.

373

Fermentation in a 25 L Vessel. The production of 1,3-PD increased to 40 and 36 g/L in replicates 1

374

and 2, respectively (Figures 7A and 7B). These were the greatest amounts of 1,3-PD observed in any of

375

the fermentations reported here and also more than double concentrations achieved by Kang et al.17 who

376

observed 16 g/L of 1,3-PD produced from fermentation. Kang et al.17 used sterilized centrifuged W-TS

377

inoculated with genetic engineered L. panis PM1B along with agitated condition, pH controlled (4.5),

378

and temperature 30 °C. In addition, the glucose to glycerol molar ratio was 0.37 M/M which was higher

379

than utilized in this research (0.1: 1). The differences in fermentation conditions could lead to different

380

results. Our findings also suggested that particles present in W-TS might aid compound conversion. It is

381

possible that the micro-organisms form a biofilm on the particles. Moreover, our findings in 25 L

382

fermentation vessels was further confirming as agitation did not accelerate fermentation as observed by

383

Kang et al.17 Furthermore, the high glucose to glycerol molar ratio employed in Kang et al.17 may not be

384

practical for industrial production as raw materials would contribute significantly to the cost of

385

production. Concentrations of acetic acid reported in this research in replicate 1 and 2 were 12 and 13

386

g/L, respectively. Fuchs et al.32 and Junker33 stated that surface aeration decreased when fermenter size 16 ACS Paragon Plus Environment

Page 17 of 38


387

increased. Larger fermenters often have lower headspace to fermenter volume ratios than smaller

388

fermenters. The 25 L fermenter had a lower ratio of surface area and headspace to culture volume

389

compared to 50 mL fermenter. The larger fermenter may have less oxygen that may lead to greater 1,3-

390

PD production. However, small amounts of oxygen introduced while stirring fermentation media using

391

a homogenizer might enhance growth of endemic flora and L. panis PM1B. The CFU in these

392

fermenters showed no clear trend during incubation (Figures 7C and 7D). Interestingly, when the

393

growth of micro-organisms reached stationary phase, glycerol consumption and 1,3-PD accumulation

394

still occurred. These could suggest that endemic micro-organisms might metabolize other forms of

395

carbohydrate or protein as energy sources. Fermentation in this experiment progressed at 25 °C while

396

Kang et al.17 conducted fermentation at 30 °C. Fermentation at 25 °C might benefit commercial

397

production, as energy input might not be required for heating these cultures.

398

Fermentation in a 210 L Vessel. For replicate 1, starting culture (20 L) with a concentration of 6 g/L

399

1,3-PD was utilized as an inoculum (Figure 8A). The concentration of 1,3-PD and acetic acid in 150 L

400

fermentation reached 23 and 11 g/L, in that order (Figure 8A). For repeated experiments, the

401

concentration of 1,3-PD in starting culture was approximately 15 g/L (Figure 8B). Production of 1,3-PD

402

(2%) and acetic acid was possible in a 150 L fermenter at 25 °C. This production was lower than that of

403

50 mL and 20 L scale fermentations but greater than achieved for cultures grown in MRS medium and

404

sterilized centrifuged medium by Grahame et al.,15 Kang et al.,16,17 Khan et al.,5 and Reaney et al.4 The

405

inoculation train for larger scale fermentation was greatly different than that of smaller fermentations.

406

This could lead to effects that might impact conversion and culture stability.34 With larger scale

407

fermentations, longer mixing times were required to increase media homogeneity and larger stagnant

408

regions could occur.33 This mixing likely introduces oxygen. Mixing efficiency is also reduced33 and

409

heat could be generated for longer mixing times.35 These factors can stress micro-organism35 leading to

410

decreased ability to effect desired conversions. In addition, a larger fermentation reactor has lower ratio

411

of surface area to volume compared to a small reactor. This would reduce heat transfer due to the 17 ACS Paragon Plus Environment


Page 18 of 38

412

reduction of surface to volume ratio.33 The CFU of 150 L fermentation and starting culture increased

413

after inoculation (Figure 9A) and became constant at about 60 h. A similar pattern was observed in 150

414

L fermentations and starting culture where CFU also increased then became constant after 60 h (Figure

415

9B). Nonetheless, longer incubation times did not improve fermentation yield. Therefore, shorter

416

incubation times of the 20 L starting culture could be preferred to achieve more rapid conversion.

417

Previous studies used genetically modified organisms to produced 1,3-PD. The examples of micro-

418

organisms were C. butyricum VPI 3266, C. butyricum F 2b, and K. pneumoniae M 5al, which produced

419

35.0, 43.5, and 58.8 g/L of 1,3-PD, respectively.3 Some of these micro-organisms, however, might

420

potentially be fastidious or pathogenic micro-organisms that produce toxins and/or require strictly

421

controlled fermentation conditions. In the current study, L. panis PM1B was utilized as it is a

422

predominant endemic micro-organism present in W-TS. Although the quantity of 1,3-PD produced (40

423

g/L) was lower than some of the above mentioned micro-organisms this has not yet been optimized.

424

In conclusion, W-TS contained organic solutes, for instance 1,3-PD, acetic acid, glycerol, and lactic

425

acid. Even though glycerol is a major compound present in W-TS it is inexpensive. In addition, a

426

complex process for purification of glycerol would be required. Therefore, it is likely that it would not

427

be profitable to recover and purify glycerol from W-TS. In addition, W-TS contained high boiling point

428

and hygroscopic solutes (glycerol and lactic acid) leading to obstacles for valuable compound (1,3-PD,

429

acetic acid, and GPC) extraction and recovery. Endemic flora especially L. panis PM1B could convert

430

glycerol and lactic acid to 1,3-PD and acetic acid, respectively. The boiling points of 1,3-PD and acetic

431

acid are lower than those of glycerol and lactic acid, respectively. Consequently, fermentation of W-TS

432

followed by isolation of 1,3-PD, acetic acid, and GPC could be a strategy for increasing the value of

433

commercial ethanol production. The effects of fermentation parameters of W-TS in the presence of

434

endemic micro-organisms supplemented with L. panis PM1B were studied to improve compound

435

conversion. We discovered that freeze-dried W-TS, pH 5 adjusted daily, ratio of glucose: glycerol 0.1:

436

1.0 (mol: mol), vitamins, and static conditions favoured conversion. The highest 1,3-PD production 18 ACS Paragon Plus Environment

Page 19 of 38


437

achieved in this research was 40 g/L which was higher than the research from Kang et al. 17 Their

438

fermentation conditions were different than conditions utilized in this research including W-TS

439

resource, agitation condition, fermentation temperature, genetic engineered L. panis PMB, glucose and

440

glycerol molar ratio, initial pH of W-TS prior to fermentation, the presence of particles, and the

441

presence of other micro-organisms. It was also noted that the intermediate, 3-HPA, and terminal

442

product, 1,3-PD did not block glycerol metabolism. Moreover, corn sugar could be utilized as a glucose

443

substitute and agitation was not required. Finally, fermentation scale was increased to 20 L and 150 L at

444

25 °C. At least 2% (20 g/L) of 1,3-PD was produced in all scaled up fermentations. Conditions that

445

lowered production costs such as culture at 25 °C, use of corn sugar as a carbon source, and minimal

446

agitation could be beneficial in this regard.

447

AUTHOR INFORMATION

448

Corresponding Authors

449

*Tel: +1 306 9665050 (YYS); +1 306 9665027 (MJTR). Fax: +1 306 9665015. E-mail:

450

[email protected] (YYS); [email protected] (MJTR).

451

Funding

452

This research was supported by the Strategic Research Program, Agricultural Development Funds of the

453

Saskatchewan Ministry of Agriculture (Grants No. 20080204 and 20140277), and Feeds Opportunities

454

from the Biofuels Industries Network.

455

Notes

456

The authors declare no competing financial interest.

457

ACKNOWLEDGEMENT

458

The authors acknowledge Pound-Maker Agventures Ltd. (Lanigan, SK, Canada) for kindly supply W-

459

TS and Terra Grain Fuels Inc. (Belle Plaine, SK, Canada) for W-DS. The authors thank Dr. Monique C. 19 ACS Paragon Plus Environment


Page 20 of 38

460

Haakensen (Contango Strategies Ltd., Saskatoon, SK, Canada) for her help in this manuscript, Mr.

461

Keith Rueve of Pound-Maker Agventures Ltd. for his help, and Dr. Shahram Emami of University of

462

Saskatchewan (Saskatoon, SK, Canada) for his kind assistance.


Page 21 of 38

463 464 465


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(2) Ratanapariyanuch, K.; Shen, J.; Jia, Y.; Tyler, R. T.; Shim, Y. Y.; Reaney, M. J. T. Rapid NMR

467

method for the quantification of organic compounds in thin stillage. J. Agric. Food Chem. 2011, 59,

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10454–10460.

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(3) Tan, H. W.; Aziz Abdul, A. R.; Aroua, M. K. Glycerol production and its applications as a raw material: A review. Renewable Sustainable Energy Rev. 2013, 27, 118–127. (4) Reaney, M. J. T.; Haakensen, C. M.; Korber, D.; Tanaka, T.; Ratanapariyanuch, K. Process for the conversion of glycerol to 1,3-propanediol. U.S. Patent Application 2013/0316417 A1. 2013.

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(5) Khan, N. H.; Kang, T. S.; Grahame, D. A. S.; Haakensen, M. C.; Ratanapariyanuch, K.; Reaney,

474

M. J. T.; Korber, D. R.; Tanaka, T. Isolation and characterization of novel 1,3-propanediol-producing

475

Lactobacillus panis PM1 from bioethanol thin stillage. Appl. Microbiol. Biotechnol. 2013, 7, 417–428.

476 477 478 479

(6) Ratanapariyanuch, K. Recovery of protein and organic compounds from secondary-fermented thin stillage. Ph.D. thesis. University of Saskatchewan. Saskatoon, SK, Canada, 2016. (7) Pflügl, S.; Marx, H.; Mattanovich, D.; Sauer, M. 1,3-Propanediol production from glycerol with Lactobacillus diolivorans. Bioresour. Technol. 2012, 119, 133–140.

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(8) Tobajas, M.; Mohedano, A. F.; Casas, J. A.; Rodríguez, J. J. Unstructured kinetic model for

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reuterin and 1,3‐propanediol production by Lactobacillus reuteri from glycerol/glucose cofermentation.

482

J. Chem. Technol. Biotechnol. 2009, 84, 675–680.

483 484 485 486

(9) Biebl, H.; Marten, S.; Hippe, H.; Deckwer, W.-D. Glycerol conversion to 1,3-propanediol by newly isolated clostridia. Appl. Microbiol. Biot. 1992, 36, 592–597. (10) Himmi, E. H.; Bories, A.; Barbirato, F. Nutrient requirements for glycerol conversion to 1,3propanediol by Clostridium butyricum. Bioresour. Technol. 1999, 67, 123–128.



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(11) Saint-Amans, S.; Perlot, P.; Goma, G.; Soucaille, P. High production of 1,3-propanediol from

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glycerol by Clostridium butyricum VPI 3266 in a simply controlled fed-batch system. Biotechnol. Lett.

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1994, 16, 831–836 (12) Yang, G.; Tian, J.; Li, J. Fermentation of 1,3-propanediol by a lactate deficient

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mutant of Klebsiella oxytoca under microaerobic conditions. Appl. Microbiol. Biotechnol. 2007, 73,

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1017–1024.

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(13) Tag, C. G. Mikrobielle Herstellung von 1,3-propandiol. Ph.D. thesis. University of Oldenburg. Oldenburg, Germany, 1990. (14) Cheng, K.-K.; Zhang, J.-A.; Liu, D.-H.; Sun, Y.; Liu, H.-J.; Yang, M.-D.; Xu, J.-M. Pilot-scale production of 1,3-propanediol using Klebsiella pneumoniae. Process Biochem. 2007, 42, 740–744.

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(15) Grahame, D. A. S.; Kang, T. S.; Khan, N. H.; Tanaka, T. Alkaline conditions stimulate the

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production of 1,3-propanediol in Lactobacillus panis PM1 through shifting metabolic pathways. World

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J. Microbiol. Biotechnol. 2013, 29, 1207–1215.

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(16) Kang, T. S.; Korber, D. R.; Tanaka, T. Glycerol and environmental factors: effects on 1,3‐

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propanediol production and NAD+ regeneration in Lactobacillus panis PM1. J. Appl. Microbiol. 2013,

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115, 1003–1011.

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(17) Kang, T. S.; Korber, D. R.; Tanaka, T. Bioconversion of glycerol to 1,3-propanediol in thin

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stillage-based media by engineered Lactobacillus panis PM1. J. Ind. Microbiol. Biotechnol. 2014, 41,

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629–635.

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(18) de Man, J. C.; Rogosa, M.; Sharpe, M. E. A medium for the cultivation of lactobacilli. J. Appl. Bacteriol. 1960, 23, 130–135.

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(19) Kim, Y.; Mosier, N. S.; Hendrickson, R.; Ezeji, T.; Blaschek, H.; Dien, B.; Cotta, M.; Dale, B.;

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Ladisch, M. R. Composition of corn dry-grind ethanol by-products: DDGS, wet cake, and thin stillage.

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Bioresour. Technol. 2008, 99, 5165–5176.


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(20) Nout, M. J. R. Upgrading traditional biotechnological processes, In: Applications of

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biotechnology to traditional fermented foods. National Academy Press, Washington, D.C., USA, 2015;

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pp 11–19.

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(21) U.S. Food and Drug Administration. Select Committee on GRAS Substances (SCOGS)

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Opinion:

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(dextrose),

corn

syrup,

invert

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http://www.fda.gov/Food/IngredientsPackagingLabeling/GRAS/SCOGS/ucm261263.htm,

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July 1, 2016.

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(22) Monceaux, D. A.; Kuehner, D. Dryhouse technologies and DDGS production. In: Ingledew, W.

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M.; Kelsall, D. R.; Austin, G. D.; Kluhspies, C. (Eds.), The alcohol textbook. 5th edn. Nottingham

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University Press. Nottingham, UK, 2009; pp 303–321.

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(23) AOAC. Official Methods of Analysis, 18th ed.; Horwitz, W., Latimer, G. W., Eds.; Association of Official Analytical Chemists: Gaithersburg, MD, 2006; methods 981.10 and 950.46. (24) Sosulski, F. W.; Imafidon, G. I. Amino acid composition and nitrogen-to-protein conversion factors for animal and plant foods. J. Agric. Food Chem. 1990, 38, 1351–1356.

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(25) Kirk, J. L.; Klironomos, J. N.; Lee, H.; Trevors, J. T. The effects of perennial ryegrass and

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alfalfa on microbial abundance and diversity in petroleum contaminated soil. Environ. Pollut. 2005,

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133, 455–465.

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(26) Meredith, J. Dryhouse design: Focusing on reliability and return on investment, In: Jacques, K.

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A., Lyons, T. P., Kelsall, D. R. (Eds.), The alcohol textbook. 4th edn. Nottingham University Press,

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Nottingham, UK, 2003; pp 363–376.

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(27) Liu, K.; Barrows, F. T. Methods to recover value-added coproducts from dry grind processing of grains into fuel ethanol. J. Agric. Food Chem. 2013, 61, 7325–7332.

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(28) Kang, T. S.; Korber, D. R.; Tanaka, T. Metabolic engineering of a glycerol-oxidative pathway in

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Lactobacillus panis PM1 for utilization of bioethanol thin stillage: potential to produce platform

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chemicals from glycerol. Appl. Environ. Microbiol. 2014, 80, 7631–7639. 23 ACS Paragon Plus Environment


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(29) Maier, U.; Losen, M.; Büchs, J. Advances in understanding and modeling the gas-liquid mass transfer in shake flaks. Biochem. Eng. J. 2004, 17, 155–167.

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(30) Oude Elferink, S. J. W. H.; Krooneman, J.; Gottschal, J. C.; Spoelstra, S. F.; Faber, F.; Driehuis,

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F. Anaerobic conversion of lactic acid to acetic acid and 1,2-propanediol by Lactobacillus buchneri.

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Appl. Environ. Microbiol. 2001, 67, 125–132.

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(31) Saxena, R. K.; Anand, P.; Saran, S.; Isar, J. Microbial production of 1,3-propanediol: Recent developments and emerging opportunities. Biotechnol. Adv. 2009, 27, 895–913. (32) Fuchs, R.; Ryu, D. D. Y.; Humphery, A. E. Effect of surface aeration on scale-up procedures for fermentation processes. Ind. Eng. Chem. Process. Des. Dev. 1971, 10, 190–196. (33) Junker, B. H. Scale-up methodologies for Escherichia coli and yeast fermentation processes. J. Biosci. Bioeng. 2004, 97, 347–364.

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(34) Okonkowski, J.; Kizer-Bentley, L.; Listner, K.; Robinson, D.; Chartrain, M. Development of a

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robust, versatile, and scalable inoculum train for the production of a DNA vaccine. Biotechnol. Prog.

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2005, 21, 1038–1047.

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(35) Schmidt, F. R. Optimization and scale up of industrial fermentation process. Appl. Microbiol. Biotechnol. 2005, 68, 425–435.


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FIGURE CAPTIONS

552

Figure 1. Flow chart for inocula preparation. Fifty mL and 500 mL of inocula were incubated 48 and 72

553

h, respectively at 37 °C.

554

Figure 2. The effect of pH on concentration of A) glycerol, B) 1,3-PD, C) lactic acid, and D) acetic acid

555

during fermentation with endemic bacterial populations augmented with L. panis PM1B inoculum.

556

Figure 3. The effect of glucose addition to W-TS on concentration of A) glycerol, B) 1,3-PD, C) lactic

557

acid, D) acetic acid, and E) glucose during fermentation with endemic bacterial populations augmented

558

with L. panis PM1B inoculum. Glucose concentration was not determined when its concentration was

559

lower than the assay detection limit. Glucose concentration 0.1 and 0.2 M conditions were analyzed

560

after 139 h fermentation.

561

Figure 4. The effect of 1,3-PD, freeze-dried W-TS, and vitamins on concentration of A) glycerol, B)

562

1,3-PD, C) lactic acid, D) acetic acid, and E) glucose during fermentation with endemic bacterial

563

populations augmented with L. panis PM1B inoculum. Glucose concentration was not determined when

564

its concentration was lower than the assay detection limit. Control, freeze-dried W-TS added, and

565

vitamin added conditions were analyzed after 190 h fermentation.

566

Figure 5. The effect of freeze-dried W-TS, agitation, and pH adjusting on the concentration of A)

567

glycerol, B) 1,3-PD, C) lactic acid, D) acetic acid, and E) glucose, and F) number of bacteria during

568

fermentation with endemic bacterial populations augmented with L. panis PM1B inoculum. Glucose

569

concentration was not determined when its concentration was lower than the assay detection limit.

570

Figure 6. Effects of 3-HPA, corn sugar, and adjusting pH on the concentrations of A) glycerol, B) 1,3-

571

PD, C) lactic acid, D) acetic acid, E) 3-HPA, and F) glucose, and G) number of bacteria during

572

fermentation with endemic bacterial populations augmented with L. panis PM1B inoculum. Glucose and

573

3-HPA concentrations were not determined when their concentrations were lower than the assay

574

detection limit.



Page 26 of 38

575

Figure 7. Media concentration of A) glycerol, lactic acid, 1,3-PD, acetic acid, 3-HPA, and glucose of

576

duplicate 1, B) glycerol, lactic acid, 1,3-PD, acetic acid, 3-HPA, and glucose of duplicate 2, C) number

577

of bacteria (CFU/mL) duplicate 1, and D) number of bacteria (CFU/mL) duplicate 2 during

578

fermentation with endemic bacterial populations augmented with L. panis PM1B inoculum. Glucose

579

concentration was not determined when its concentration was lower than the assay detection limit.

580

Figure 8. Media concentration of glycerol, lactic acid, glucose, 1,3-PD, acetic acid, and 3-HPA of A)

581

starting culture (left) and 150 L fermentation (right) and B) starting culture (left) and 150 L fermentation

582

(right) of repeated experiment during the fermentation with endemic bacteria populations augmented

583

with L. panis PM1B inoculum. Glucose concentration was not determined when its concentration was

584

lower than the assay detection limit.

585

Figure 9. Number of bacteria for cultures with augmented inoculation with L. panis PM1B (CFU/mL)

586

of starting culture and 150 L fermentation for two separate fermentations (A and B).


Page 27 of 38


Table 1. Protein and Moisture Contents of W-DS and W-TS Samplesa characteristic (%, w/w)

W-DS

W-TS1

W-TS2

W-TS3

W-TS4

total nitrogenb

1.71 ± 0.00

0.62 ± 0.00

0.58 ± 0.00

0.64 ± 0.04

0.52 ± 0.00

GPC nitrogenc

0.020 ± 0.000 0.005 ± 0.000 0.004 ± 0.000 0.006 ± 0.000 0.005 ± 0.000

betaine nitrogenc

0.045 ± 0.000 0.010 ± 0.000 0.008 ± 0.000 0.011 ± 0.000 0.010 ± 0.000

corrected proteind

9.38 ± 0.03

3.45 ± 0.01

3.24 ± 0.00

3.57 ± 0.20

2.89 ± 0.01

moisture

75.94 ± 0.00

92.92 ± 0.02

93.40 ± 0.00

91.64 ± 0.00

92.34 ± 0.22

aEach

value is presented as the mean ± standard deviation (SD, n = 2). bTotal nitrogen was determined

by the Kjeldahl method. cNitrogen contributed by GPC and betaine was determined by DPFGSE-NMR method. The nitrogen contributed by these materials to total nitrogen was subtracted prior to calculation of protein content. dCorrected protein was calculated using conversion factor 5.7 and expressed as crude protein. The pH of W-TS was pHs 3.4–3.9.



Page 28 of 38

Table 2. Concentration (g/L) of Organic Solutesa of W-DS and W-TS Samples

a

component

W-DS

W-TS1

W-TS2

W-TS3

W-TS4

1,3-PD

2.06 ± 0.01

0.52 ± 0.01

0.38 ± 0.00

0.69 ± 0.02

0.72 ± 0.00

acetic acid

2.35 ± 0.00

1.10 ± 0.05

1.07 ± 0.02

1.44 ± 0.05

1.53 ± 0.00

betaine

3.72 ± 0.00

0.83 ± 0.01

0.67 ± 0.00

0.88 ± 0.02

0.83 ± 0.01

ethanol

0.21 ± 0.00

0.13 ± 0.00

0.13 ± 0.00

0.26 ± 0.01

0.24 ± 0.02

glycerolb

28.07 ± 0.30

8.60 ± 0.19

7.84 ± 0.10

10.16 ± 0.37

8.73 ± 0.02

GPC

3.79 ± 0.04

0.98 ± 0.02

0.81 ± 0.01

1.10 ± 0.03

0.90 ± 0.02

isopropanol

1.67 ± 0.00

0.29 ± 0.00

0.26 ± 0.00

0.37 ± 0.01

0.35 ± 0.01

lactic acid

5.07 ± 0.08

3.61 ± 0.04

3.42 ± 0.05

4.84 ± 0.04

4.93 ± 0.00

phenethyl alcohol

0.58 ± 0.01

0.36 ± 0.01

0.30 ± 0.01

0.40 ± 0.01

0.36 ± 0.00

succinic acid

1.80 ± 0.01

0.69 ± 0.01

0.62 ± 0.00

0.73 ± 0.03

0.77 ± 0.01

Each value is presented as the mean ± SD (n = 2). bThe concentration of glycerol in W-DS and W-TS

samples may be affected by the presence of interfering resonances from carbohydrate and protein.


Page 29 of 38


CULTURE

INOCULUM

FERMENTATION

Sterile condition (37 °C, 72 h)

Pasteurization (72 °C, 15 sec) Incubation (37 °C)

Non-sterile condition 37 °C

2 ̶ 3 colonies

50 mL 20 ̶ 30 colonies/bottle

350 mL

B

20 L

C

25 °C

20 ̶ 30 colonies/bottle

Culture Culture plate

50 mL

25 °C

500 mL

500 mL

A

20 L

150 L

Figure 1.



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Figure 2.


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Figure 3.



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Figure 4.


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Figure 5.



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Figure 6. 34 ACS Paragon Plus Environment

Page 35 of 38


Figure 7.



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Figure 8.


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Figure 9.



Table of Contents Graphic Table of Contents Graphic 206x201mm (96 x 96 DPI)

ACS Paragon Plus Environment

Page 38 of 38

Conversion of Thin Stillage Compounds Using Endemic Bacteria

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