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Production of Protein Concentrate and 1,3Propanediol by Wheat-based Thin Stillage Fermentation Kornsulee Ratanapariyanuch, Youn Young Shim, Shahram Emami, and Martin John Tarsisius Reaney J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 28 Apr 2017 Downloaded from http://pubs.acs.org on May 1, 2017

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Journal of Agricultural and Food Chemistry

Production of Protein Concentrate and 1,3-Propanediol by Wheatbased Thin Stillage Fermentation

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Kornsulee Ratanapariyanuch,† Youn Young Shim,*,†,‡,§ Shahram Emami,† Martin J. T.

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Reaney*,†,‡,§

6 7

†

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S7N 5A8, Canada

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‡

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§

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

Department of Plant Sciences, University of Saskatchewan, 51 Campus Drive, Saskatoon, Saskatchewan Prairie Tide Chemicals Inc., 102 Melville Street, Saskatoon, Saskatchewan S7J 0R1, Canada Guangdong Saskatchewan Oilseed Joint Laboratory, Department of Food Science and Engineering, Jinan

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CORRESPONDING AUTHOR INFORMATION

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Fax: +1 306 9665015. E-mail address: [email protected] (YYS), [email protected]

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(MJTR) 1 ACS Paragon Plus Environment


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ABSTRACT

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Fermentation of wheat with yeast produces thin stillage (W-TS) and distiller’s wet grains. A subsequent

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fermentation of W-TS (two-stage fermentation, TSF) with endemic bacteria at 25 and 37 °C decreased

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glycerol and lactic acid concentrations while 1,3-propanediol (1,3-PD) and acetic acid accumulated with

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greater 1,3-PD and acetic acid produced at 37 °C. During TSF, W-TS colloids coagulated and floated in

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the fermentation medium producing separable liquid and slurry fractions. The predominant endemic

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bacteria in W-TS were Lactobacillus panis, L. gallinarum, and L. helveticus and this makeup did not

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change substantially as fermentation progressed. As nutrients were exhausted, floating particles

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precipitated. Protein contents of slurry and clarified liquid increased and decreased, respectively, as TSF

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progressed. The liquid was easily filtered through an ultrafiltration membrane. These results suggested

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that TSF is a novel method for W-TS clarification and production of protein concentrates and 1,3-PD

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from W-TS.

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Keywords: Anoxic gas flotation; clarification; wheat-based thin stillage; two-stage fermentation

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INTRODUCTION

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The production of ethanol by yeast fermentation followed by distillation produces thin stillage (TS), which

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is composed of organic solutes, suspended particles, and inorganic salts.1−3 Typically, wheat-based thin

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stillage (W-TS) contains colloids, microorganisms, inorganic and organic solutes, particulate matter,

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polysaccharides, and proteins.2 Organic solutes present in W-TS included 1,3-propanediol (1,3-PD),

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acetic acid, glycerol, alpha-glycerylphosphorylcholine (GPC), and lactic acid.3 These compounds are

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potentially valuable without modification or as precursors for additional processing. 1,3-PD may be used

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to replace ethylene glycol4 or as an intermediate chemical for synthesis of polyamides, polyesters,

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polyethers, and polyurethanes.5,6 Acetic acid might be utilized as a food ingredient, a precursor for

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production of polyvinyl acetate for synthetic fibers, or vinegar.7,8 GPC is a cholinergic substance that

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releases choline when consumed. Cholinergic compounds can be used to mitigate the effects of Alzheimer

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disease9 and transient ischemic attacks.10 GPC can also be esterified with fatty acids for the synthesis of

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lecithin and lysolecithin.

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Microorganisms present in W-TS include bacteria, fungi, and yeast.2 Some of these microorganisms,

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specifically Lactobacillus panis PM1B, may be used to conduct a second fermentation of W-TS.11−13 The

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modified two-stage fermentation (TSF) is, potentially, a novel intermediate process for adding value to

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W-TS. Recovery of soluble organic compounds from W-TS is a challenging step14 due to the high

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concentration of particulates and high boiling point and hygroscopic solutes. The complexity of W-TS

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limits options for developing an inexpensive enrichment process.

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Inorganic solutes, particles, and soluble protein and non-protein biopolymers remain in W-TS after

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ethanol fermentation.2 TSF occurs when endemic flora including L. panis PM1B, an organism discovered

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in W-TS, are allowed to proliferate in W-TS. L. panis PM1B converted glycerol to 1,3-PD and lactic acid

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to acetic acid.11−13,15 W-TS solutes, therefore, are converted by TSF to less hygroscopic forms that have

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lower boiling points. Conversion of these solutes might enable approaches for simplified processing to

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recover W-TS compounds. 3 ACS Paragon Plus Environment


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TSF also produces CO2, an anoxic gas, from Lactobacillus metabolism.5,11 Others have used anoxic

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gas generated by anaerobic fermentation to clarify dairy manure and sewage sludge in a process known

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as anoxic gas flotation (AGF). AGF can concentrate and return bacteria, organic acids, protein, and

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undigested substances to anaerobic digesters.16−18 Typically, gas bubbles in water have negative surface

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charges that repel adjacent bubbles due to electrostatic forces. Positively charged particles bind to bubble

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surfaces and reduce the net charge. In addition, van der Waals, hydrodynamic retardation, and

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hydrophobic forces are also associated with bubble and particle interactions.19 These phenomena enhance

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colloid coagulation.20 Colloids or particles present in W-TS could adhere to anoxic gas bubbles produced

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by lactobacilli. Particles adhering to bubble surfaces might be aggregated and float in fermentation

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medium as TSF progresses. Furthermore, lactobacilli also produce exopolysaccharides (EPSs) that might

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affect colloid stability and clear solutions as TSF progresses. EPSs are produced by L. casei CG11 grown

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in a basal medium,21 L. plantarum EP56,22 L. sanfranciscensis in sour dough,23 and lactic acid-producing

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bacteria present in sour dough.24 Therefore, it is possible that endemic flora present in W-TS specifically

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Lactobacillus species produce EPS during TSF. It is not known if AGF and EPS production might act

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synergistically to aggregate, coagulate, and separate W-TS particles from colloids during fermentation.

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In addition, the use of TSF to clarify W-TS has not been reported. The objective of this study was to study

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the effect of TSF for W-TS clarification and subsequent production of a W-TS protein concentrate and

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1,3-PD. Once the stillage is clarified, numerous solution processes might be devised to separate useful

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compounds from solution and enrich protein particles.

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MATERIALS AND METHODS

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W-TS samples were collected approximately 350 L from Pound-Maker Agventures Ltd. (Lanigan, SK,

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Canada) on different dates, hereafter, called W-TS1, W-TS2, W-TS3, W-TS4, and W-TS5, respectively.

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W-TS samples were stored in 10 L containers at 4 °C until utilized.


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Analytical Methods.

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Protein and Moisture Contents. Sample protein contents were determined using the Kjeldahl method

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(AOAC 981.10).25 Nitrogen content present in non-protein compounds (GPC and betaine) was subtracted

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from total nitrogen content prior to calculate protein content.3 A conversion factor of 5.7 was multiplied

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by nitrogen to estimate the protein content,26 referred to as corrected protein. Moisture content was

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determined by oven drying samples at 100 ± 2 °C for 16−18 h to reach a constant weight (AOAC 950.46

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B.a.)25 as described by Ratanapariyanuch.2 Samples were cooled to room temperature in a desiccator for

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at least 1 h prior to weighing.

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Nuclear Magnetic Resonance (NMR) Spectroscopy. Double pulse field gradient spin echo NMR

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(DPFGSE-NMR) was conducted to quantify organic compounds according to a modification of the

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method of Ratanapariyanuch et al.3 Samples of W-TS, liquid I, and slurry I were centrifuged (Spectrafuge

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24D, Labnet International Inc., Edison, NJ, United States) at 9200g 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 PTFE

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membrane, VWR International, West Chester, PA, United States). Proton NMR spectra of filtrates were

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recorded on a Bruker Avance 500 MHz NMR spectrometer (Bruker BioSpin, Rheinstetten, Germany)

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with 16 scans per spectrum using a DPFGSE-NMR pulse sequence. NMR data collection and analysis

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were conducted with TopSpin 3.2 software (Bruker BioSpin GmbH, Billerica, MA, United States).

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Deuterium oxide (Cambridge Isotope Laboratories, Inc., Andover, MA, United States) and

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dimethylformamide (EMD Chemicals Inc., Gibbstown, NJ, United States) were used as solvent and

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internal standard, respectively.

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16S Ribosome Sequencing for Taxonomic Classification. The taxonomic classification of microbial

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populations was determined using 16S ribosome sequencing27 for slurry microorganism populations

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(Figure 1) of small-scale fermentation experiments and studies of the effects of temperature on

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fermentation rate. Analysis was conducted at Contango Strategies Ltd. (Saskatoon, SK, Canada). The

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Greengenes database (version 13-8) was searched to confirm taxonomic classifications. 5 ACS Paragon Plus Environment


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Fermentation Procedures.

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Small-scale Fermentation. W-TS1 and W-TS2 were utilized as fermentation media for replicate 1 and

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2, respectively. Fermentation was conducted in two thirty-liter semi-transparent polypropylene plastic

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pails (described as fermenter 1 and fermenter 2) with lids equipped with fermentation gas traps. Twenty-

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five liters of W-TS was added to each vessel where fermentation was allowed to progress at 25 °C until

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gas evolution ceased and particles precipitated. Slurry I and liquid I in both fermenters were sampled daily

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to determine protein and moisture contents. Organic solutes were determined by DPFGSE-NMR analysis.

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The volume of liquid I was recorded each day. Slurry I samples from replicate 2 at 0, 46, and 94 h were

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collected and stored at −80 °C for 16S ribosome sequencing. Liquid I samples from small-scale TSF at

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25 °C were filtered with regenerated cellulose membranes (10 kDa molecular weight cut-off; MWCO;

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PL type; Millipore Corp., Bedford, MA, United States) at 380 kPa in a stirred ultrafiltration cell (8010,

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Millipore Corp., Bedford, MA, United States) through an effective membrane area of 4.1 cm2 according

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to Ratanapariyanuch.14 Membranes were prepared by washing according to manufacturer’s guidelines to

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remove preservative deposited on the membrane. Elastomer tubing attached to the stirred cell outlet was

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inserted into a 10 mL graduate cylinder that was covered with flexible film (Parafilm M, Bemis Company

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Inc., Neenah, WI, United States) to limit liquid I filtrate evaporation. Liquid I (10 mL) was added to the

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stirred cell. Agitation speed was maintained at 600 rpm. Filtrate volume was recorded every 10 min and

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filtration proceeded until the solution volume was reduced from 10 mL to 1.0 mL and filtrate volume

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reached approximately 9.0 mL. Flux and solution volume concentration were calculated using Equations

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1 and 2, respectively.

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Solution flux from step 2 = L/M2/h

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Where, L = filtrate volume, M2 = membrane surface area, h = time of filtration in hours

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Volume concentration =

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Filtrate protein contents were estimated using the Bradford protein assay28 with bovine serum albumin as

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a standard.

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(2)


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Temperature Effects on Fermentation. Fermentation was compared at two incubation temperatures

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(25 and 37 °C). W-TS4 (25 L) was utilized as a fermentation medium for determining the effects of

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temperature on fermentation at 25 and 37 °C for replicate 1 and W-TS5 was used as fermentation medium

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for replicate 2. Fermentation was conducted in 30 L semi-transparent polypropylene plastic pails equipped

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with lids and gas traps at 25 and 37 °C until fermentation ceased. Slurry I and liquid I from fermentation

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media were sampled daily (Figure 1). Protein content, moisture content, and the concentration of organic

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solutes of liquid and slurry were determined as previously described. The volume of liquid I was recorded

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daily. Microbial populations of slurry I at 0, 47, and 101 h of fermentation replicate 1 were characterized

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using 16S ribosome sequencing as described above.

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Replications of Small-Scale Fermentation. W-TS2 and W-TS3 were employed as fermentation media

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for replicate 1 and replicate 2. Twelve thirty-liter transparent polypropylene plastic pails with lids

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equipped with gas traps were utilized as fermenters for each replicate. Medium (25 L in each vessel) was

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fermented at 25 °C until gas evolution ceased and slurry I precipitated. Liquid I and slurry I from

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replications of small-scale TSF were sampled each day from two fermenters indicated as fermenter 1 and

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fermenter 2 as representative of the twelve fermenters for each replicate. Protein content, moisture

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content, and organic solute content were determined on samples. The volume of liquid I was recorded

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daily.

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Statistical Analysis. Data was obtained from analysis of duplicate samples of liquid I, slurry I, and

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ultrafiltration filtrate. Means comparisons were made by analysis of variance (ANOVA) and Duncan’s

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multiple-range test using SPSS statistical software (SPSS 21.0, IBM Corp., Armonk, NY, United States).

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RESULTS AND DISCUSSION

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Characterization of W-TS. W-TS protein (38−43% w/w, db) and moisture contents (91−94%, w/w)

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varied with sample collection date (Table 1). This result agreed with other studies of W-TS, which

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indicated W-TS protein at 36.6 and 45.7% (w/w, db).29,30 TS (corn) moisture content was approximately 7 ACS Paragon Plus Environment


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90−95% (w/w).31 Wheat endosperm and endemic TS microorganisms might contribute to the observed

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protein. W-TS contained organic compounds including 1,3-PD, acetic acid, betaine, ethanol, glycerol,

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GPC, isopropanol, lactic acid, phenethyl alcohol, and succinic acid. The organic compound concentrations

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differed on each sample collection date (Table 2). In addition, glycerol and lactic acid were major W-TS

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organic solutes. These organic solutes are products of microorganisms and wheat and are similar to

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compounds reported previously.2,3

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TSF of W-TS.

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Small-Scale TSF at 25 °C. Proton NMR analysis showed that as fermentation progressed glycerol and

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lactic acid concentrations decreased with a simultaneous increase in 1,3-PD and acetic acid concentrations

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(Figure 2 and Figure S1 of the Supporting Information). It should be noted that the decline of lactic acid

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concentration was not substantial. It is possible that lactic acid was produced from metabolism and was

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also converted to acetic acid. Lactic acid accumulated if the rate of synthesis was greater than the rate of

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catabolism to acetic acid. These changes were associated with metabolism by W-TS endemic flora,

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especially lactobacilli like L. panis PM1B, that consumes glycerol and lactic acid to produce 1,3-PD and

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acetic acid, respectively.11,12,15 Moreover, TSF can proceed at 25 °C enabling fermentation to be

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conducted in unheated fermenters. The concentrations of glycerol and lactic acid in slurry I tended to

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decrease more rapidly than in liquid I while 1,3-PD and acetic acid concentrations in slurry I increased

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more rapidly than in liquid I suggesting that metabolic activity in the slurry was likely greater than in the

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liquid. Slurry formation might have been aided by EPS formed by fermentation organisms including

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lactobacilli.21,22,24 Though not investigated here, EPS production might stabilize bacteria adhesion to

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particle surfaces and allow biofilm formation.32,33 Biofilms are conducive to metabolism. EPS produced

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by bacteria plays numerous roles including: acting as adhesives for interactions with other bacteria,

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surfaces, or substrates, hiding bacteria surfaces, protective agents against adverse environmental

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conditions, signalling molecules, structure stabilizer in biofilms, and substances for bacteria aggregation 8 ACS Paragon Plus Environment

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in rhizosphere communities.34 The discovery that the metabolism in slurry I was more rapid than that in

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liquid I might be utilized to improve conversion of glycerol and lactic acid to 1,3-PD and acetic acid,

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respectively by adding substrates e.g. glycerol, carbohydrates, and nutrients into slurry I to increase

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concentrations of 1,3-PD and acetic acid. In addition, slurry I could be utilized as the inoculum for the

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following fermentation.

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Ribosome sequencing (16S) revealed that 99% of slurry I sequences were from members of the genus

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Lactobacillus. Other bacteria, including members of Acetobacteraceae, Bifidobacteriaceae, and

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unidentified bacteria, contributed approximately 1% of sequences (Figure 3). Two lactobacilli species, L.

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panis (approximately 51%) and L. helveticus (approximately 41%), in slurry I accounted for

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approximately 91% of all sequences. L. panis and L. helveticus have been identified and used in

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production of sour dough breads and Swiss cheese, respectively.24,35−38 Bacterial taxonomic classification

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indicates that slurry I might be considered a potential source of probiotic organisms but this would require

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further investigations.39,40 In addition, genes encoding glycerol dehydratase and 1,3-PD oxidoreductase

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should be investigated in other lactobacilli present in slurry I to determine the ability of those lactobacilli

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to facilitate 1,3-PD production. Conversion of glycerol and lactic acid observed in the replications of

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small-scale fermentation indicated similar metabolic action (Figures S2 and S3 of the Supporting

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Information).

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Fermentation at 25 and 37 °C. The effect of fermentation temperature was determined at 25 and 37 °C.

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At both temperatures, the concentrations of glycerol and lactic acid decreased while the concentrations of

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1,3-PD and acetic acid increased. These conversions appeared to occur more rapidly in slurry I than in

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liquid I (Figure 4 and Figure S4 of the Supporting Information). It should be noted that the concentration

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of lactic acid increased at the beginning of fermentation and decreased thereafter. This phenomenon could

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be explained by more rapid production of lactic acid than lactic acid catabolism early in fermentation.

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Later in fermentation, the rate of conversion of lactic acid to other products exceeded its production.

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Glycerol conversion to 1,3-PD at 37 °C was much faster than at 25 °C with full conversion occurring in 9 ACS Paragon Plus Environment


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half the time (50−60 h earlier). Khan et al.15 reported that the optimum temperature for L. panis PM1B

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growth and metabolism in de Man, Rogosa and Sharpe (MRS) medium is 32−37 °C. In addition, 3-

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hydroxypionaldehyde (3-HPA), the product of glycerol dehydratase that is converted to 1,3-PD, was

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mostly observed at 25 °C (Figure 4 and Figure S4 of the Supporting Information). There was no indication

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that 3-HPA impeded fermentation. This finding is in agreement with our observation that adding 3-HPA

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did not inhibit either 1,3-PD or acetic acid accumulation.13 Rapid fermentation could benefit industrial

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scale fermentation by increasing 1,3-PD and acetic acid yield while decreasing fermentation time.

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Commercial fermenter volumes could be decreased by half and associated capital costs could be

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decreased. It is unlikely that heating the stillage would be a major cost as stillage exits the ethanol

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distillation unit at temperatures in excess of 37 °C. Taxonomic classification based on 16S ribosome

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sequences indicated that major microbial populations in slurry I from fermentation at 25 and 37 °C were

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comprised of lactobacilli which accounted for more than 93% of microorganisms (Figures 5 and 6). In

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addition, L. panis (28−38%) and L. gallinarum (29−39%) were major slurry I Lactobacillus species. L.

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gallinarum is a microorganism found in dairy products41 and potentially could survive in gastrointestinal

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tract and impact intestinal microbial metabolism.42 Therefore, L. gallinarum could have utility as a

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probiotic lactobacillus.43,44 However, it should be noted that the different species of lactobacillus

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discovered in small-scale TSF at 25 °C could be related to the batch of W-TS.

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Progress of Small-Scale Fermentation. Early in TSF, slurry I settled first and liquid I separated due

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to the greater slurry density (Figure 7A). As fermentation proceeded, slurry I floated and separated

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producing a clearer solution (Figure 7B). Finally, when fermentation ceased, slurry I sank to the fermenter

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bottom (Figure 7C). Based on the predominance of lactobacilli in slurry I, it would be expected that these

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organisms would produce CO2 (anoxic gas).5,11,45 At the beginning of fermentation, CO2 production was

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insufficient to separate slurry from W-TS. Therefore, liquid I and slurry I separated partially due to

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gravity. However, as fermentation progressed lactobacilli produced sufficient CO2 to assist coagulation,

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which was evident as slurry I layer floated in the fermentation medium. Burke18 stated that anoxic gas 10 ACS Paragon Plus Environment

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produced from AGF process under anaerobic condition concentrated, floated, and returned bacteria,

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enzyme, organic acids, protein, and undigested substrates. Once floated, these materials could be skimmed

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and returned to the anaerobic digester.18 As the duration of fermentation increased, nutrients consumed

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by lactobacilli were reduced until CO2 production and fermentation ceased. The AGF ceased and slurry I

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precipitated in the fermenter.

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Solids floated after 45 and 22 h of fermentation in replicate 1 and 2, respectively. Fermentation ceased

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at 172 and 94 h in replicates 1 and 2, in that order. For slurry I, as fermentation progressed moisture and

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protein contents decreased and increased, respectively (Figure 8 and Figure S5 of the Supporting

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Information). The opposite trend occurred with liquid I. At the end of TSF the protein content of slurry I

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was approximately 50% (w/w, db) compared to approximately 40% (w/w, db) for W-TS at 0 h. The higher

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protein concentration observed in slurry I indicated a greater portion of protein present in the fermentation

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medium at 0 h aggregated into slurry I. Aggregation might involve simple coalescence of protein rich

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particles or might involve microbial growth during fermentation and protein metabolism in solution and

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on particles. If slurry I protein content reflects a high bacterial uptake and incorporation by endemic L.

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panis and L. helveticus (Figure 3), it is possible that feed arising from slurry I produced from TSF could

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be considered to be a protein concentrate and it might have probiotic organisms when included in animal

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feed.39 Lactobacilli present in wet wheat distillers’ grain have potential use as probiotics and L. helveticus

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in distillers’ grain inhibited Campylobacter jejuni invasion of human intestinal epithelial cell cultures.40

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The volume of liquid I increased as fermentation time increased. In total, approximately 28 ̶ 40% of

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clarified liquid or 7−10 L of liquid I were obtained from 25 L of fermentation medium. The volume of

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low turbidity solution released was substantially greater than produced by additives, centrifugation,

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filtration, and size exclusion in earlier studies.14 This confirmed the potential for using TSF for W-TS

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clarification without using additives. TSF could lower processing costs for recovering materials from W-

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TS. Two actions of bacterial growth may aid in this separation and density increase. First, lactobacilli

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produce bubbles of anoxic gas, CO2, when metabolizing glucose and other carbohydrates, bubbles attach 11 ACS Paragon Plus Environment


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to suspended particles to form aggregates that float to the fermenter surface (Figure 9). Bubbles not only

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induce particle aggregation but can also be used with coagulants to collapse solution colloids.19 Second,

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many Lactobacillus sp. also produce EPS.34 If they are present, bacterial EPS could change media

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rheological properties and this might act as a stabilizer or stimulate preferential microbial growth46 though

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it was not investigated here. However, the EPS production by L. panis PM1B and other strains in thin

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stillage has not been established.

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Ultrafiltration of W-TS and Liquid I. The flux and volume concentration through ultrafiltration

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membranes were determined for unfermented W-TS (0 h) and samples of liquid I collected as

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fermentation progressed (Figure 10 and Figure S6 of the Supporting Information). The transmembrane

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flux of unfermented W-TS (31 L/M2/h) was initially high but decreased rapidly as filtration progressed.

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Colloids present in W-TS prevented effective volume concentration. This finding was in good agreement

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with Porter47 who noted that colloids or macromolecules that deposited on membranes led to reversible

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flux losses. The transmembrane flux of liquid I was substantially lower than W-TS flux, and flux remained

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constant during volume concentration. As most of liquid I passed through the membrane, liquid I is mostly

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a solution and not a colloid. Liquid I, approximately 3−5% w/w db (Figure 8 and Figure S5 of the

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Supporting Information), contained small solutes and might also contain macromolecules. Liquid I filtrate

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had a low protein content (less than 0.5 g/L) (Table 3 and Table S1 of the Supporting Information). It is

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possible that protein concentration was as low as indicated or that proteins present did not bind Coomassie

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Blue dye. Protein content, determined by dye binding, cannot be directly compared with protein content

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determined by nitrogen (Kjeldahl protein assay). The dye binds weakly with histidine, lysine,

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phenylalanine, tryptophan, and tyrosine residues. In addition, the Bradford assay is insensitive to peptides

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with masses below 3−5 kDa. Amino acids and small peptides, therefore, would not bind with the dye.48

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Protein and Moisture Content of TSF Products. The separation of slurry I and liquid I from

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fermentation at 25 and 37 °C occurred due to gravity, anoxic gas, and possibly EPS produced from

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lactobacilli as previous described. It was noticed that anoxic gas flotation did not occur when fermenting 12 ACS Paragon Plus Environment

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W-TS at 37 °C which may be due to higher solubility of anoxic gas when temperature increases. Protein

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and moisture contents of slurry I indicate that as fermentation progressed, protein content and moisture

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content increased and decreased, respectively. However, the results were opposite with liquid I (Figures

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11 and 12 and Figures S7 and S8 of the Supporting Information). Aggregation was likely induced by

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anoxic gas and EPS as previously described. In addition, the quantity of liquid I from fermentation at 25

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and 37 °C was approximately 8−14 L accounting for 32−56% liquid-solid separation. Moreover, protein

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content at the end of TSF increased to approximately 50% (w/w, db) with fermentation at 25 and 37 °C.

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These results are similar to those for protein and moisture contents (Figure 8 and Figure S5 of the

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Supporting Information) and quantity of liquid I from small-scale fermentation at 25 °C.

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Replications of Small-Scale TSF. Low turbidity solutions formed in all twelve fermenters (Figure 13

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and Figure S9 of the Supporting Information). AGF using autogenic gas likely aided in breaking colloids

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and clearing solution. While particles floated during the early stages of fermentation, settling occurred

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after approximately 46 and 100 h for replicate 1 and 2, respectively. Settling was completed after 94 and

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167 h for replicates 1 and 2 (out of twelve fermenters), in that order, confirming that fermentation had

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ceased. At the end of TSF, there were 67 L (22%) and 83 L (28%) of liquid I from replicate 1 and replicate

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2, respectively, from 300 L of fermentation medium. The protein content of slurry I was higher than that

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of liquid I (Figure 8 and Figure S5 of the Supporting Information). In addition, liquid I had higher moisture

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content than slurry I. At the end of TSF, slurry I protein content increased (45−47%, w/w, db) compared

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to original medium before fermentation (40−42%, w/w, db). Thus, most protein from the fermentation

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medium was recovered from W-TS in slurry I.

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In conclusion, W-TS is an aqueous colloid with organic solutes. Valuable compounds present in W-

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TS were 1,3-PD, acetic acid, and GPC. Unfortunately, these valuable compounds cannot be easily

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extracted from W-TS due to colloids remaining from yeast fermentation and high boiling and hygroscopic

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solutes. Colloids were converted to slurries during TSF using endemic lactobacilli populations. This

311

fermentation also converts glycerol and lactic acid (high boiling point and hygroscopic solutes) to 1,3-PD 13 ACS Paragon Plus Environment


Page 14 of 41

312

and acetic acid (lower boiling solutes), respectively while maintaining GPC content. W-TS was processed

313

using TSF at both 25 and 37 °C. Fermentation time was substantially reduced when fermenting W-TS at

314

37 °C. Though AGF was not observed clearly when fermenting W-TS at 37 °C, EPS produced by

315

lactobacilli could aid slurry formation. Fermentation at higher temperatures could save processing space

316

and, thereby, reduce the need for capital investment in large volume fermenters. Interestingly, 16S

317

ribosome sequencing of slurry I revealed that L. panis, L. helveticus, and L. gallinarum were the major

318

endemic microorganisms and lactobacilli accounted for more than 93% of microbial populations.

319

Fermentative coagulation was a novel and efficient method to clarify fermentation medium using anoxic

320

gas produced from endemic flora present in W-TS. Approximately 22−56% separation of liquid I and

321

slurry I from fermentation medium could be achieved using TSF. The liquid from TSF could be

322

ultrafiltered through a 10 kDa MWCO membrane in a high shear cell. Therefore, liquid from TSF is a

323

weak colloid or not colloidal, contains small solutes, and is largely free of particulate matter.

324

Consequently, it is suggested that TSF could be utilized as a preparation step for removing particles prior

325

to filtration. Slurry I from TSF product had a higher protein content (approximately 50%, w/w, db) than

326

that of W-TS (38−43%, w/w, db). Microbial growth during fermentation concentrated the protein. In

327

addition, approximately 93% of the microbial population in W-TS and slurry I were lactobacilli. Due to

328

its high protein content and microbial population, it might be possible to utilize slurry I as a protein source

329

in animal feed. Further studies will explore the use of commercial separation tools for separation of protein

330

rich slurries produced by TSF.

331

ASSOCIATED CONTENT

332

Supporting Information

333

Supplemental table: Protein content (g/L) of filtrate from ultrafiltration of liquid I from fermentation at

334

25 °C of small-scale fermentation replicate 2 at different fermentation time (Table S1). Supplemental

335

figures: Concentration of (A) glycerol, (B) 1,3-PD, (C) lactic acid, and (D) acetic acid from small-scale 14 ACS Paragon Plus Environment

Page 15 of 41


336

TSF obtained by DPFGSE-NMR analysis of replicate 2 (Figure S1), Concentration of (A) glycerol, (B)

337

1,3-PD, (C) lactic acid, and (D) acetic acid from replications of small-scale TSF obtained by DPFGSE-

338

NMR analysis of replicate 1 (Figure S2), Concentration of (A) glycerol, (B) 1,3-PD, (C) lactic acid, and

339

(D) acetic acid from replications of small-scale TSF obtained by DPFGSE-NMR analysis of replicate 2

340

(Figure S3), Concentration of (A) glycerol, (B) 1,3-PD, (C) lactic acid, (D) acetic acid, and (E) 3-HPA

341

when fermented at 25 and 37 °C in duplicate 25 L fermentations from DPFGSE-NMR analysis of replicate

342

2 (Figure S4), Moisture and protein contents of TSF products from fermentation at 25 °C of small-scale

343

fermentation where (A) moisture content of TSF products fermenter 1, (B) protein content of TSF

344

products fermenter 1, (C) moisture content of TSF products fermenter 2, and (D) protein content of TSF

345

products fermenter 2 replicate 2 (Figure S5), Transmembrane (10 kDa MWCO) flux of liquid I from (A)

346

fermenter 1 and (B) fermenter 2 replicate 2. Filtered volume of liquid I passed through a 10 kDa MWCO

347

membrane of liquid I from (C) fermenter 1, and (D) fermenter 2. The time in the legend presents the

348

fermentation time (Figure S6), Moisture and protein contents of TSF products from fermentation at 25 °C

349

where (A) moisture content of TSF products fermenter 1, (B) protein content of TSF products fermenter

350

1, (C) moisture content of TSF products fermenter 2, and (D) protein content of TSF products fermenter

351

2 replicate 2 (Figure S7), Moisture and protein contents of TSF products from fermentation at 37 °C where

352

(A) moisture content of TSF products fermenter 1, (B) protein content of TSF products fermenter 1, (C)

353

moisture content of TSF products fermenter 2, and (D) protein content of TSF products fermenter 2

354

replicate 2 (Figure S8), and Moisture and protein contents of replications of small-scale TSF of twelve

355

fermenters where (A) moisture content of TSF products fermenter 1, (B) protein content of TSF products

356

fermenter 1, (C) moisture content of TSF products fermenter 2, and (D) protein content of TSF products

357

fermenter 2 replicate 2 (Figure S9). This material is available free of charge via the Internet at

358

http://pubs.acs.org.



Page 16 of 41

359

AUTHOR INFORMATION

360

Corresponding Authors

361

*Tel: +1 306 9665050; Fax: +1 306 9665015; E-mail: [email protected].

362

*Tel: +1 306 9665027; E-mail: [email protected].

363

Funding

364

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

365

Saskatchewan Ministry of Agriculture (Grants 20080204 and 20140277), and Feeds Opportunities from

366

the Biofuels Industries Network.

367

Notes

368

The authors declare no competing financial interest.

369

ACKNOWLEDGEMENT

370

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

371

TS.


Page 17 of 41

372


REFERENCES

373

(1) Meredith, J. Dryhouse design: focusing on reliability and return on investment. In The alcohol

374

textbook, 4th ed.; Jacques, K. A., Lyons, T. P., Kelsall, D. R., Eds.; Nottingham University Press:

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Nottingham, UK, 2003; pp 363–376.

376 377

(2) Ratanapariyanuch, K. Protein extraction from mustard (Brassica juncea L. Czern.) meal using thin stillage. M.Sc. Thesis. University of Saskatchewan, Saskatoon, SK, Canada, 2009.

378

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

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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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(4) Liu, H.; Xu, Y.; Zheng, Z.; Liu, D. 1,3-Propanediol and its copolymers: research, development and industrialization. Biotechnol. J. 2010, 5, 1137–1148. (5) Biebl, H.; Menzel, K.; Zeng, A.-P.; Deckwer, W.-D. Microbial production of 1,3-propanediol. Appl. Microbiol. Biotechnol. 1999, 52, 289–297. (6) 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. (7) Manning, J. H.; Hutten, I. M. Synthetic fiber paper having a permanent crepe. U.S. Patent 5,094,717, 1992. (8) New World Encyclopedia. Acetic acid. http://www.newworldencyclopedia.org/entry/Acetic_acid (accessed on February 4, 2017).

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endemic bacteria augmented with Lactobacillus panis PM1B. J. Agric. Food Chem. 2016, 64, 7940–7948.

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(14) Ratanapariyanuch, K. Recovery of protein and organic compounds from secondary-fermented

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thin stillage. Ph.D. Thesis. University of Saskatchewan, Saskatoon, SK, Canada, 2016.

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CG11 and partial structure analysis of the polymer. Appl. Environ. Microbiol. 1994, 60, 3914–3919.


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(23) Hammes, W.; Gänzle, M. Sourdough breads and related products. In Microbiology of fermented

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foods, 2nd ed.; Wood, B. J. B., Ed.; Blackie Academic and Professional: London, UK, 1998; pp 199–216.

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Food Microbiol. 2007, 24, 120–127. (25) 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. (26) 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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sequencing-based diversity studies. Nucleic Acids Res. 2012, 41, 1–11.

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(28) Bradford, M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 1976, 72, 248–254.

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(29) Mustafa, A. F.; McKinnon, J. J.; Christensen, D. A. Chemical characterization and in vitro crude

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protein degradability of thin stillage derived from barley- and wheat-based ethanol production. Anim.

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Feed Sci. Technol. 1999, 80, 247–256.

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(30) Mustafa, A. F.; McKinnon, J. J.; Ingledew, M. W.; Christensen, D. A. The nutritive value for

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ruminants of thin stillage and distillers' grains derived from wheat, rye, triticale and barley. J. Sci. Food

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Agric. 2000, 80, 607–613.

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of environmental and genetic factors on biofilm formation by the probiotic strain Lactobacillus rhamnosus

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GG. Appl. Environ. Microbiol. 2007, 73, 6768–6775.

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(33) Yildiz, F. H.; Schoolnik, G. K. Vibrio cholerae O1 El Tor: Identification of a gene cluster required

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for the rugose colony type, exopolysaccharide production, chlorine resistance, and biofilm formation.

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Proc. Natl. Acad. Sci. U. S. A. 1999, 96, 4028–4033.

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(34) Badel, S.; Bernardi, T.; Michaud, P. New perspectives for lactobacilli exopolysaccharides. Biotechnol. Adv. 2011, 29, 54–66. (35) Arendt, E. K.; Ryan, L. A. M.; Dal Bello, F. Impact of sourdough on the texture of bread. Food Microbiol. 2007, 24, 165–174. (36) Corsetti, A.; Settanni, L. Lactobacilli in sourdough fermentation. Food Res. Int. 2007, 40, 539– 558.

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(37) Plessas, S.; Fisher, A.; Koureta, K.; Psarianos, C.; Nigam, P.; Koutinas, A. A. Application of

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Kluyveromyces marxianus, Lactobacillus delbrueckii ssp. bulgaricus and L. helveticus for sourdough

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bread making. Food Chem. 2008, 106, 985–990.

459 460

(38) Valence, F.; Deutsch, S. M.; Richoux, R.; Gagnaire, V.; Lortal, S. Autolysis and related proteolysis in Swiss cheese for two Lactobacillus helveticus strains. J. Dairy Res. 2000, 67, 261–271.

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(39) Pedersen, C.; Jonsson, H.; Lindberg, J.; Roos, S. Microbiological characterization of wet wheat

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distillers' grain, with focus on isolation of lactobacilli with potential as probiotics. Appl. Environ.

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Microbiol. 2004, 70, 1522–1527.

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(40) Wine, E.; Gareau, M. G.; Johnson-Henry, K.; Sherman, P. M. Strain-specific probiotic

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(Lactobacillus helveticus) inhibition of Campylobacter jejuni invasion of human intestinal epithelial cells.

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FEMS Microbiol. Lett. 2009, 300, 146–152.

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(41) Giraffa, G.; Chanishvili, N.; Widyastuti, Y. Importance of lactobacilli in food and feed biotechnology. Res. Microbiol. 2010, 161, 480–487. 20 ACS Paragon Plus Environment

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(42) Fujiwara, S.; Seto, Y.; Kimura, A.; Hashiba, H. Establishment of orally-administered

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Lactobacillus gasseri SBT2055SR in the gastrointestinal tract of humans and its influence on intestinal

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microflora and metabolism. J. Appl. Microbiol. 2001, 90, 343–352.

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(43) Roy, D.; Ward, P.; Vincent, D.; Mondou, F. Molecular identification of potentially probiotic lactobacilli. Curr. Microbiol. 2000, 40, 40–46. (44) Saito, T. Selection of useful probiotic lactic acid bacteria from the Lactobacillus acidophilus group and their applications to functional foods. Anim. Sci. J. 2004, 75, 1–13.

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(45) 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. Appl.

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

479 480 481 482 483 484

(46) Waldherr, F. W.; Meissner, D.; Vogel, R. F. Genetic and functional characterization of Lactobacillus panis levansucrase. Arch. Microbiol. 2008, 190, 497–505. (47) Porter, M. C. Concentration polarization with membrane ultrafiltration. Ind. Eng. Chem. Prod. Res. Dev. 1972, 11, 234–248. (48) Lucarini, A. C.; Kilikian, B. V. Comparative study of lowry and bradford methods: interfering substances. Biotechnol. Tech. 1999, 13, 149–154.

485



Page 22 of 41

486

FIGURE CAPTIONS

487

Figure 1. (A) Liquid I and (B) slurry I from fermentation.

488

Figure 2. Concentration of (A) glycerol, (B) 1,3-PD, (C) lactic acid, and (D) acetic acid from small-scale

489

TSF determined by DPFGSE-NMR analysis.

490

Figure 3. Graphical representation of main bacteria group present in slurry I in small-TSF. Other

491

microorganisms indicated Acetobacteraceae, Bifidobacteriaceae, and unidentified bacteria. Percentage

492

was not indicated if less than 3%.

493

Figure 4. Concentration of (A) glycerol, (B) 1,3-PD, (C) lactic acid, (D) acetic acid, and (E) 3-HPA when

494

fermented at 25 and 37 °C in duplicate 25 L fermentation determined by DPFGSE-NMR analysis. The

495

concentration of 3-HPA was under detection limit when fermenting W-TS at 37 °C .

496

Figure 5. Graphical representation of bacterial species present in slurry I from 25 °C fermentation. Other

497

microorganisms indicated Acetobacteraceae, Acinetobacter, Alicyclobacillus acidocaldarius, Bacillus, B.

498

coagulans, Bifidobacteriaceae, Chryseobacterium, Clostridiacae, Clostridium, Comamodaceae, Delftia,

499

Enterococcus,

500

Pediococcus, P. acidilactici, Pedobacter, Prevotella, Rhodocyclaceae, Thermoanaerobacterium

501

saccharolyticum, Xanthomonadaceae, and unidentified bacteria. Percentage was not indicated if less than

502

3%.

503

Figure 6. Graphical representation of bacteria species present in slurry I from 37 °C fermentation. Other

504

microorganism identified included members of Acetobacteraceae, Acinetobacter, Alicyclobacillus

505

acidocaldarius, Bacillus, B. coagulans, Bifidobacteriaceae, Chryseobacterium, Clostridiacae,

506

Clostridium, Comamodaceae, Delftia, Enterococcus, Flectobacillus, Gluconobacter, Janthinobacterium

507

lividum, Mogibacteriaceae, Pediococcus, P. acidilactici, Pedobacter, Prevotella, Rhodocyclaceae,

508

Thermoanaerobacterium saccharolyticum, Xanthomonadaceae, and unidentified bacteria. Percentage

509

was not indicated if less than 3%.

510

Figure 7. Fermentation stages in (A) 23 h, (B) 93 h, and (C) 172 h in small-scale TSF.

Flectobacillus,

Gluconobacter,

Janthinobacterium

lividum,

Mogibacteriaceae,


Page 23 of 41


511

Figure 8. Moisture and protein contents of TSF product from fermentation at 25 °C of small-scale

512

fermentation where (A) moisture content of TSF products fermenter 1, (B) protein content of TSF

513

products fermenter 1, (C) moisture content of TSF products fermenter 2, and (D) protein content of TSF

514

products fermenter 2. Each value is presented as the mean ± SD (n = 2). Total nitrogen was determined

515

by the Kjeldahl method. Nitrogen in sample contributed by GPC and betaine was determined by DPFGSE-

516

NMR. The nitrogen in sample contributed by these materials to total nitrogen was subtracted prior to

517

calculation of protein content. Corrected protein was calculated using conversion factor 5.7 as expressed

518

as crude protein. There was no sampling at 141 h of fermentation as the solution was settling at this time.

519

Therefore, the fermentation medium was left for 24 h to let the slurry I precipitate.

520

Figure 9. Diagram of AGF process.

521

Figure 10. Transmembrane (10 kDa MWCO) flux of liquid I from (A) fermenter 1 and (B) fermenter 2.

522

Filtered volume of liquid I passed through a 10 kDa MWCO membrane of liquid I from (C) fermenter 1,

523

and (D) fermenter 2. The time in the legend presents the fermentation time.

524

Figure 11. Moisture and protein contents of TSF products from fermentation at 25 °C where (A) moisture

525

content of TSF products fermenter 1, (B) protein content of TSF products fermenter 1, (C) moisture

526

content of TSF products fermenter 2, and (D) protein content of TSF products fermenter 2. Each value is

527

presented as the mean ± SD (n = 2). Total nitrogen was determined by the Kjeldahl method. Nitrogen in

528

sample contributed by GPC and betaine was determined by DPFGSE-NMR. The nitrogen in sample

529

contributed by these materials to total nitrogen was subtracted prior to calculation of protein content.

530

Corrected protein was calculated using conversion factor 5.7 as expressed as crude protein.

531

Figure 12. Moisture and protein contents of TSF products from fermentation at 37 °C where (A) moisture

532

content of TSF products fermenter 1, (B) protein content of TSF products fermenter 1, (C) moisture

533

content of TSF products fermenter 2, and (D) protein content of TSF products fermenter 2. Each value is

534

presented as the mean ± SD (n = 2). Total nitrogen was determined by the Kjeldahl method. Nitrogen in

535

sample contributed by GPC and betaine was determined by DPFGSE-NMR. The nitrogen in sample 23 ACS Paragon Plus Environment


Page 24 of 41

536

contributed by these materials to total nitrogen was subtracted prior to calculation of protein content.

537

Corrected protein was calculated using conversion factor 5.7 as expressed as crude protein.

538

Figure 13. Moisture and protein contents of replications of small-scale TSF of twelve fermenters where

539

(A) moisture content of TSF products fermenter 1, (B) protein content of TSF products fermenter 1, (C)

540

moisture content of TSF products fermenter 2, and (D) protein content of TSF products fermenter 2. Each

541

value is presented as the mean ± SD (n = 2). Total nitrogen was determined by the Kjeldahl method.

542

Nitrogen in sample contributed by GPC and betaine was determined by DPFGSE-NMR. The nitrogen in

543

sample contributed by these materials to total nitrogen was subtracted prior to calculation of protein

544

content. Corrected protein was calculated using conversion factor 5.7 as expressed as crude protein.


Page 25 of 41


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

W-TS2

W-TS3

W-TS4

W-TS5

0.58 ± 0.02

0.68 ± 0.00

0.66 ± 0.00

0.46 ± 0.01

0.56 ± 0.01

betaine nitrogenc

0.011 ± 0.001 0.009 ± 0.000 0.013 ± 0.000 0.009 ± 0.000 0.010 ± 0.000

GPC nitrogenc

0.006 ± 0.000 0.006 ± 0.000 0.008 ± 0.000 0.005 ± 0.000 0.006 ± 0.000

moisture

91.74 ± 0.02

91.05 ± 0.00

91.15 ± 0.00

93.64 ± 0.01

92.01 ± 0.00

3.18 ± 0.10

3.77 ± 0.01

3.65 ± 0.06

2.54 ± 0.11

3.10 ± 0.04

38.57 ± 1.28

42.10 ± 0.19

41.28 ± 0.66

40.03 ± 1.71

38.86 ± 0.31

corrected proteind (wb) protein (db) a

W-TS1

b

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

the Kjeldahl method. cNitrogen contributed by GPC and betaine was determined by DPFGSE-NMR. 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.



Page 26 of 41

Table 2. Concentration (g/L) of Organic Solutes of W-TS Samples a component

W-TS1

W-TS2

W-TS3

W-TS4

W-TS5

1,3-PD

0.63 ± 0.01

2.91 ± 0.01

0.61 ± 0.01

0.31 ± 0.01

0.80 ± 0.01

acetic acid

1.85 ± 0.01

3.92 ± 0.06

1.99 ± 0.04

0.89 ± 0.02

2.34 ± 0.06

betaine

0.94 ± 0.03

0.77 ± 0.05

1.12 ± 0.02

0.74 ± 0.01

0.87 ± 0.05

0.35 ± 0.00

0.40 ± 0.01

0.47 ± 0.01

0.12 ± 0.01

0.29 ± 0.02

10.56 ± 0.08

7.61 ± 0.13

11.23 ± 0.20

9.18 ± 0.18

8.34 ± 0.22

GPC

1.06 ± 0.01

1.16 ± 0.01

1.40 ± 0.07

0.89 ± 0.04

1.05 ± 0.04

isopropanol

0.34 ± 0.00

0.38 ± 0.00

0.40 ± 0.01

0.32 ± 0.01

0.42 ± 0.02

lactic acid

6.04 ± 0.07

6.76 ± 0.02

5.96 ± 0.15

3.28 ± 0.02

5.78 ± 0.30

ethanol b

glycerol

a

phenethyl alcohol 0.38 ± 0.00 0.43 ± 0.03 0.47 ± 0.01 0.34 ± 0.01 0.34 ± 0.02 b Each value is presented as the mean ± SD (n = 2). The concentration of glycerol in W-TS samples may

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


Page 27 of 41


Table 3. Protein Contenta (g/L) of Filtrate from Ultrafiltration of Liquid I from Fermentation at 25 °C of Small-scale Fermentation at Different Fermentation Time container

0h

23 h

45 h

69 h

93 h

117 h

0.39 ± 0.01

0.21 ± 0.00

0.20 ± 0.02

0.22 ± 0.00

0.19 ± 0.00

0.19 ± 0.00

fermenter 2 0.41 ± 0.01 0.21 ± 0.00 Each value is presented as the mean ± SD (n = 2).

0.20 ± 0.01

0.21 ± 0.00

0.19 ± 0.01

0.19 ± 0.00

fermenter 1 a



Page 28 of 41

A

B

Figure 1.



Concentration (g/L)

Concentration (g/L)

Page 29 of 41

A

12 10 8 6 4 2 0

6 4 2 0 0

9.0000 8.0000 7.0000 6.0000 5.0000 4.0000 3.0000 2.0000 1.0000 0.0000

50

100

150

0

200

8

C8

6

6

4

4

2

2

0 0

0

B

8

10

50

20

100

30

150

40

200

50

100

150

200

D

0 50

0

50

60

100

70

150

80

200

90

100

Fermentation time (h) Liquid I fermenter 1

Liquid I fermenter 2

Slurry I fermenter 1

Slurry I fermenter 2

Figure 2.



Page 30 of 41

Figure 3.


Page 31 of 41


A

10 8

4

6

3

4

2

2

1

0

0

Concentration (g/L)

0

20

40

60

80

100 120

0

C

6

B

5

5

20

40

60

80

100

120

D

8 6

4 3

4

2

2

1 0

0 0

20

40

60

80

100

120

0

20

40

60

80

100

120

E

0.8 0.6 0.4 0.2 0.0 0

20

40

60

80

100 120

Fermentation time (h) 37 C slurry I fermenter 1 37 C slurry I fermenter 2 25 C slurry I fermenter 1 25 C slurry I fermenter 2

37 C liquid I fermenter 1 37 C liquid I fermenter 2 25 C liquid I fermenter 1 25 C liquid I fermenter 2

Figure 4. 31 ACS Paragon Plus Environment


Page 32 of 41

Figure 5.


Page 33 of 41


Figure 6.



Page 34 of 41

Figure 7.


Page 35 of 41


A

50

96

40

93

30

90

20

87 84

C

99 96 93 90 87 84

99 96 93 90 87 84

Protein content (%, w/w, db)

Moisture content (%, w/w, wb)

99

B

10 0

50

D

40 30 20 10 0

Fermentation time (h) Slurry I

Liquid I

Figure 8.



Particle in W-TS

Page 36 of 41

Anoxic gas bubble

Figure 9.


Page 37 of 41


A

B

35

30

30

25

25

40

20

Flux (L/M2/h

Flux (L/M2/h)

35

15 10 5

20

30

15

20

10

10

5

0

0

0

0

0.2

0.5

0.4

1

0 0.8

0.6

1.5

2

1

1.2

0

0.5

1.4

1.6

1

1.5

2

Volume concentrate factor (log scale)

Volume pass through membrane (mL)

W-TS

23 h

45 h

69 h

C

12 10

93 h

117 h

D

12 10

8

8

12 10 8 6 4 2 0

6 4 2 0

6 4 2 0

0

0.5

0.5

1

1.5

1

2

1.5

2.5

3

0

2

0

0.5

2.5

1

1.5

3

2

2.5

3

Filtration time (h) W-TS

23 h

45 h

69 h

93 h

117 h

Figure 10.



A

99

50

90 87 84 W-TS

24

47

72

101

121

C

99 96

87 84

99 99 96 96 93 93 90 90 87 W-TS 24 87 84 84



40

93

90

B

60

96

93

Page 38 of 41

30 20 10 0 W-TS

24

47

72

101

121

D

60 50 40 30 20 10 0

47

72

101

121

W-TS

24

47

72

101

121

Fermentation time (h)

Fermentation time (h) Slurry I Liquid I Slurry I Liquid I

Figure 11.


Page 39 of 41


A

96

50

93

40

90 87 84 W-TS

24

47

72

101

121

C

99 96 93 90 87 84

99 96 93 90 W-TS 87 24 84

B

60



99

30 20 10 W-TS

24

47

72

101

121

D

60 50 40 30 20 10

47

72

101

121

W-TS

24

47

72

101

121


Liquid I

Figure 12.



A

96

40

93

30

90 87 84 W-TS

22

46

94

C

99 96 93 90 87 84

99 96 93 90 87 W-TS 84

B

50



99

Page 40 of 41

20 10 0 W-TS

22

46

94

D

50 40 30 20 10 0

22

46

94

W-TS

22

46

94


Liquid I

Figure 13.


Page 41 of 41


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ACS Paragon Plus Environment

Production of Protein Concentrate and 1,3 ... - ACS Publications

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