Aeration, Agitation and Cell Immobilization on Corncobs and Oak Wood Chips Eﬀects on Balsamic-Styled Vinegar Production

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Article Aeration, Agitation and Cell Immobilization on Corncobs and Oak Wood Chips Eﬀects on Balsamic-Styled Vinegar Production

Ucrecia F. Hutchinson 1,2,3,* , Sivuyile Gqozo 1, Neil P. Jolly 1, Boredi S. Chidi 1,2, Heinrich W. Du Plessis 1 , Maxwell Mewa-Ngongang 1,2,3 and Seteno K. O. Ntwampe 2,3 1 Post-Harvest and Agro-Processing Technologies, ARC Infruitec-Nietvoorbij (The Fruit, Vine and Wine Institute of the Agricultural Research Council), Private Bag X5026, Stellenbosch 7599, South Africa 2 Bioresource Engineering Research Group (BioERG), Department of Biotechnology, Cape Peninsula University of Technology, P.O. Box 652, Cape Town 8000, South Africa 3 Department of Chemical Engineering, Cape Peninsula University of Technology, P.O. Box 652, Cape Town 8000, South Africa * Correspondence: [email protected]; Tel.: +27-21-809-3442

Received: 15 June 2019; Accepted: 22 July 2019; Published: 1 August 2019

Abstract: Optimum fermentor conditions are essential for desired microbial growth and activity in fermentations. In balsamic vinegar fermentation systems, the microorganisms used must endure several stressful conditions including high sugar concentration, low water activity, high osmotic pressure and high acetic acid concentration. Consequently, the present study was aimed at improving the performance of a microbial consortium of non-Saccharomyces yeast and acetic acid bacteria during balsamic-styled vinegar fermentation. Cell immobilization via adsorption on corncobs and oak wood chips in combination with aeration and agitation effects, have never been tested during balsamic-styled vinegar fermentation. Therefore, fermentations were initially conducted under static conditions 1 without aeration with successive fermentations also being subjected to low (0.15 vvm min− ) and 1 high (0.3 vvm min− ) aeration. The results showed improved acetification rates when cells were immobilized on corncobs under static conditions. Low aeration showed better acetification rates (1.45–1.56 g L day 1), while only free-floating cells were able to complete fermentations (1.2 g L day 1) · · − · · − under high aeration conditions. Overall, cells immobilized on corncobs showed higher acetification rates of 1.56 and 2.7 g L day 1 under low aeration and static fermentations, respectively. Oak wood · · − chips were determined to be less efficient adsorbents due to their relatively smooth surface, while the rough surface and porosity of corncobs led to improved adsorption and, therefore, enhanced acetification rates.

Keywords: aeration; adsorption; balsamic-styled vinegar; cell immobilization; corncobs; non-Saccharomyces yeast; oak wood chips

1. Introduction Similar to other vinegars, balsamic vinegar is a food ﬂavoring agent or condiment, which contains acetic acid as its main ingredient [1]. Balsamic vinegar is characterized by its sour and sweet taste [2] and can be used as a salad dressing, and for soothing sore throats among other uses [3]. Balsamic-styled vinegar (BSV) is a term adopted from Traditional Balsamic vinegar (TBV) of Modena and Reggio-Emilia Italy. BSV is a new type of Balsamic vinegar which has potential for being produced in South Africa using Chenin blanc wine grapes, since they are the most cultivated grape cultivar in South Africa [4]. Cell immobilization generally refers to a technique, which is performed to prevent cells from being freely suspended in the fermentation medium [5–7]. Cell immobilization improves biomass growth,

Foods 2019, 8, 303; doi:10.3390/foods8080303 www.mdpi.com/journal/foods Foods 2019, 8, 303 2 of 15 increases cell stability, protects cells from the toxic environment, and provides a reusability option [8]. Most fermentations generally employ the free-floating cell (FFC) method, which is effective in less stressful fermentation systems. However, during balsamic vinegar fermentation, there are multiple factors that have antagonistic effects on the microorganisms involved in the fermentation. For instance, the fermentation medium (cooked grape must) used for Balsamic-styled vinegar has a very high sugar content, low water activity and high osmotic pressure [9–11]. Furthermore, the high concentrations of acetic acid during fermentations can also have a negative impact on the microbial consortium used [12,13]. These aforementioned environmental stresses can cause the microbial consortia to enter the viable but non-culturable state, resulting in reduced microbial activity [14,15]. Consequently, to counteract these effects, cell immobilization is an ideal approach, which can minimize the impact of stressful conditions on the microorganisms used. Various methods are used to perform cell immobilization. These include entrapment in a gel matrix, immobilization on a solid surface and mechanical containment behind a barrier [16]; however, to date, numerous studies focus on cell immobilization via gel entrapment or immobilization on a solid surface (adsorption) [5]. In Balsamic-styled vinegar fermentation, cell immobilization has previously been studied using the gel entrapment technique [17], which is generally an expensive technique. However, cell immobilization by adsorption in combination with aeration and agitation effects has never been tested for balsamic vinegar production. Therefore, in this current study, cell immobilization on corncobs and oak wood chips was investigated using the adsorption technique, which is commonly classified as a simple and often cost-effective method depending on the support material used [8]. Corncobs are an inexpensive and abundant agricultural by-product obtained from the corn-milling process. They are currently used as animal feed, with 80% of their dry matter being comprised of cellulose and hemicellulose [18–22]. These aforementioned characteristics impart unique attributes that brand corncobs as an ideal material for cell immobilization. On the other hand, oak wood chips are abundantly used as an alternative to wooden barrel aging in the wine industry for economic reasons. They are known to improve the sensorial qualities of the final product due to the flavors they impart to wines [23–25]. For this reason, the current study selected oak wood chips, based on both their cell immobilization properties and potential impartation of organoleptic characteristics to the BSV. Nevertheless, oak wood chips are usually imported to South Africa and they cost more than corncobs. Consequently, this study investigated readily available inert materials which can be obtained at varying cost for cell immobilization, in order to allow producers to make an informed decision in achieving the desired BSV quality. Furthermore, the study explored the capabilities of the selected materials for cell affinity to the adsorbents selected and fermenter performance in both aerated and agitated cultures systems.

2. Material and Methods

2.1. Preparation of Fermentation Medium

The study commenced with the boiling of Chenin blanc grape must (22 ◦Brix) using a double jacketed steam pot (S.W.18, Aluminium Plant and Vessel Co. Ltd., London, UK) until a sugar concentration of 30 ◦Brix was obtained. The grape must was aliquoted into 3 L Erlenmeyer ﬂasks, covered with cotton wool stoppers, subsequent to autoclaving at 120 ◦C for 20 min. After autoclaving, the grape must was analyzed chemically for sugar ( B), pH, alcohol (% v/v) and total acidity (g L 1) ◦ · − using a density meter (Density meter DMA 35, Anton Paar, Graz, Austria), pH meter (Metrohm pH meter 632, Herisau, Switzerland), alcolyzer (Anton Paar, Graz, Austria) and minititrator (Hanna instruments minititrator HI 84502, Johannesburg, South Africa); respectively.

2.2. Inoculum Preparation Cryopreserved non-Saccharomyces yeast (n = 5) (Table1) and acetic acid bacteria ( n = 5) (Table2) cultures were used for this study. The yeast and bacteria cultures were individually grown in 400 mL Foods 2019, 8, 303 3 of 15

YPD (Merck, Modderfontein, South Africa) and in 400 mL GM broth (glucose 0.8%, mannitol 1.7%, peptone 0.3%, yeast extract 0.5%) (Merck, Modderfontein, South Africa), respectively. The yeast and bacteria inoculums were incubated at 28 ◦C for 48 and 72 h respectively, prior to inoculation.

Table 1. Non-Saccharomyces yeast used in the study.

Non-Saccharomyces Yeast Identity ARC Accession Numbers Origin Candida pulcherrima Y0839 Chardonnay grapes Candida zemplinina Y1020 Chardonnay grapes Hanseniaspora guilliermondii Y0558 Cabernet Sauvignon grapes Kloeckera apiculata C48V19 Chardonnay grapes Zygosaccharomyces bailii C45V69 Chardonnay grapes ARC: Agricultural Research Council of South Africa.

Table 2. Acetic acid bacteria used in the study.

Acetic Acid Bacteria Identity ARC Accession Numbers NCBI Accession Numbers Origin Acetobacter pasteurianus 171/19 CP 021922.1 Healthy grapes Acetobacter malorum 172/36 KU 686765.1 Shiraz wine Kozakia baliensis 179/48 CP 014681.1 Grape pomace Gluconobacter cerinus 126/34 KX 578017.1 Shiraz wine Gluconobacter oxydans 172/36 LN 884063.1 Grape pomace NCBI: National Center for Biotechnology Information.

The non-Saccharomyces yeasts were selected based on a previous screening investigation. The yeast used in this study were selected due to high acid formation on calcium carbonate agar plates, desired aroma of the ﬁnal fermented product, osmophilic characteristics and the ﬁnal concentration of the alcohol produced. Similarly, acetic acid bacteria were obtained from previous isolation procedures on various sources; namely, grape pomace, healthy grapes and Shiraz wine. The bacteria used for this study were selected based on their ethanol oxidation rate when inoculated in diluted grape juice or their sugar utilization abilities in autoclaved grape juice.

2.3. Sterilization of Corncobs and Oak Wood Chips Corncobs (CC) were dried in the oven prior to cutting into smaller pieces using a cutting tool (sizes are shown in Table3 and Figure1). French oak wood chips (OWC) were used for this study. The OWC did not require any drying. Subsequently, the CC and OWC were separately transferred into 4 3 L Erlenmeyer ﬂasks prior to autoclaving at 121 C for 20 min. Autoclaving was repeated × ◦ (n = 2) to maximize sterility.

Table 3. Size of corncobs and oak wood chips used in the study.

Surface Area of all Width/Diameter Circumference/ Surface Area of Adsorbents Length (cm) Adsorbents Used (cm) Perimeter (cm) One Piece (cm2) (cm2) Corncobs 6.00 1.05 4.00 0.66 12.57 100.53 402 (4 pieces) ± ± Oak woodchips 2.90 0.65 1.80 0.53 9.60 19.64 392 (20 chips) ± ± Length and width results are the average of repeats standard deviations. ± Foods 2019, 8, 303 4 of 15 Foods 2019, 8, x FOR PEER REVIEW 4 of 15

A B

Figure 1. Corncobs andand oakoak wood wood chips chips used used in thein the study, study, (A) corncobs(A) corncobs after after autoclaving autoclaving (B) oak (B wood) oak woodchips inchips cooked in cooked grape grape must. must.

Yeast and bacteria were immobilized separately due to their their difference difference in cell size. size. Furthermore, Furthermore, thethe differences differences in turbidity of the broth after yeast an andd bacteria cell growth wa wass assumed to be a a factor factor thatthat might affect affect cell adsorption between the yeasts and bacteria.

2.4. Cell Cell Immobilization Immobilization on on Corncobs Corncobs and Oak Wood Chips After yeastyeast and and bacteria bacteria were were fully-grown, fully-grown, the inoculums the inoculums were mixed were resultingmixed resulting in a 2 L consortium in a 2 L consortiumof yeast and of bacteria yeast and (Tables bacteria1 and (Tables2) (NB: 1 yeast and 2) and (NB: bacteria yeast and consortiums bacteria consortiums were initially were in separate initially inﬂasks). separate Subsequently, flasks). Subsequently, 1 L of the 1 yeastL of the consortium yeast consortium was transferred was transferred into 3 Linto Erlenmeyer 3 L Erlenmeyer ﬂasks, flasks,one containing one containing CC and CC another and another one containing one containing OWC. The OWC. same The procedure same procedure was performed was performed using the usingbacterial the consortium.bacterial consortium. The yeast The and yeast bacterial and cells bacterial were cells allowed were to allowed adsorb ontoto adsorb the surface onto the of CCsurface and ofOWC CC and (serving OWC as (serving a solid support as a solid surface support/bed) surface/bed) overnight atovernight 28 ◦C. at 28 °C.

2.5. Quantification Quantification of of Cells Cells Adsorbed Adsorbed on Corncobs and Oak Wood Chips Prio Prior-r- and Post-FermentationPost-Fermentation The number of cells adsorbed on the CC and OW OWCC for individual yeasts and bacteria were quantifiedquantified using the dry cell weight method method adapted adapted from from Stone Stone et et al. al. [26] [26] and and Nguyen Nguyen et et al. al. [27], [27], with minor modifications. modifications. Prior Prior to to this, this, the the yeast yeast an andd bacterial bacterial cell cell concentration (Table (Table 33)) inin liquidliquid suspension were individually quantified quantified following following the the procedure procedure described described in in Hutchinson Hutchinson et et al. al. [28]. [28]. Furthermore, thethe yeast yeast and and bacteria bacteria were were individually individually studied studied to assess to theassess variations the variations in cell adsorption in cell adsorptioncapabilities. capabilities. Yeast and bacteriaYeast and were bacteria grown were individually grown individually following the following procedure the described procedure in describedSection 2.2 in. Subsequently,Section 2.2. Subsequently, the yeast and the bacteria yeast and cells bacteria were individually cells were individually adsorbed onto adsorbed the CC onto and theOWC CC following and OWC the following procedure the described procedure in described Section 2.4 in. CC Section and OWC 2.4. CC were and removed OWC were from removed the broth from and thedried broth in an and oven dried set at in 40 an◦C. oven The set adsorbents at 40 °C. were The weighedadsorbents daily were until weighed a stationery daily weight until wasa stationery reached. weightTo was assess reached. the quantity of cells adsorbed post fermentation, the CC and OWC were transferred into grapeTo assess must the and quantity allowed of to cells ferment adsorbed for 20 post days. fermentation, Subsequently, the the CC adsorbents and OWC were were extracted transferred and intodried grape at 40 must◦C until and a allowed stationery to weightferment was for reached.20 days. Subsequently, To determine the the number adsorbents of cells were adsorbed extracted for and the driedprocedures, at 40 °C the until diff erencea stationery of the weight weight was of the reached. adsorbents To determine prior and the post number adsorption of cells was adsorbed computed. for the procedures, the difference of the weight of the adsorbents prior and post adsorption was 2.6. Inoculation computed. After the adsorption process, the OWC and CC were extracted from the broth and allowed to dry 2.6.for 4Inoculation h. A 3 L fermentation volume was used for this study. Four pieces of corncobs (2 pieces yeast, 2 pieces Acetic Acid Bacteria) (Table2) were used for inoculation in one flask. Twenty pieces of oak After the adsorption process, the OWC and CC were extracted from the broth and allowed to wood chips (Figure1B) were used for inoculation in another flask (10 pieces yeast, 10 pieces AAB) dry for 4 h. A 3 L fermentation volume was used for this study. Four pieces of corncobs (2 pieces (Table2). yeast, 2 pieces Acetic Acid Bacteria) (Table 2) were used for inoculation in one flask. Twenty pieces of oak wood chips (Figure 1B) were used for inoculation in another flask (10 pieces yeast, 10 pieces AAB) (Table 2).

Foods 2019, 8, 303 5 of 15

2.6.1. Phase 1: Static vs. Agitated Fermentations

Static and agitated fermentations were both incubated at 28 ◦C with agitated fermentations at 135 rpm. Agitated fermentations were conducted using an orbital shaker (FMH 200, FMH Instruments, Cape Town, South Africa). Under both static and agitated conditions, CC, OWC and FFC fermentations were conducted in triplicate.

2.6.2. Phase 2: Effect of Aeration Air pumps (Resun® AC 9906, Longgang, Shenzhen, China) were used to sparge air into the 1 Erlenmeyer flasks at different airflow rates, i.e., low aeration (LA = 0.15 vvm min− ) and high aeration 1 (HA = 0.30 vvm min− ). Erlenmeyer flasks were covered with loose cotton wool stoppers and fermentations were conducted in triplicate.

2.7. Data Handling Data was analyzed and computed using Microsoft Excel v2016 (Microsoft, Redmond, Washington, DC, USA). Substrate (S) (sugar) consumption rates and product (P) (acetic acid) formation rates were calculated using Equations (1) and (2) respectively, with an assumption that the initial reaction rate determines the overall fermentation rate. NB: change in time (t) was expressed as dt for both equations. Furthermore, relative differences as employed in other studies [28,29] were calculated using Equations (3) and (4) in order to show the significance of the differences observed under the different conditions studied. All results were the average of three biological repeats accounting for standard deviations which were calculated using Microsoft Excel v2016.

dS r = (1) s dt dP r = (2) p dt Absolute difference = Amount of increase Reference amount (3) − Absolute difference Relative difference = 100 (4) Reference amount ×

3. Results and Discussions

3.1. Phase 1: Non-Aerated Fermentations

3.1.1. Static vs. Agitated Fermentations TBV fermentation is normally carried-out under static conditions using batteria. This process is slow and inexpensive [1,9]; however, a rapid fermentation period for TBV is not a fundamental objective, since TBV is matured for a minimum of 12 years after fermentation [1], while BSV can be sold without ageing. Rapid vinegar production is a major goal for industrial processes. BSV can also be produced rapidly if the conditions are ideal for the microorganisms used. For the purposes of this study, it was important to evaluate the effects that agitation could have on BSV production in contrast to stationary fermentations. Agitation is also recommended for spirit industrial vinegar production to generally shorten the fermentation period [30]. In the current study, the sugar consumption rates for all treatments under agitated conditions ranged between 6.36 and 7.12 g L 1 day 1 whereas 2.68 and · − · − 6.10 g L 1 day 1 were observed under static conditions. The relative differences for sugar consumption · − · − between FFC and immobilized cells were calculated to be 14% (OWC and FFC) and 41% (CC and FFC) under static conditions, while the relative differences under agitated conditions were calculated to be 5% (OWC and FFC) and 9% (CC and FFC) under static conditions. The ethanol production step of the process was more effective under agitated conditions, as compared to static conditions Foods 2019, 8, 303 6 of 15 Foods 2019, 8, x FOR PEER REVIEW 6 of 15

2A,D).(Figure 2However,A,D). However, ethanol ethanol formatio formationn/consumption/consumption rates were rates werenot calculated, not calculated, since since alcohol alcohol is an is intermediatean intermediate product/substrate product/substrate which which is isproduc produceded and and consumed consumed simultaneously. simultaneously. Furthermore, Furthermore, acetic acid production was unsatisfactory for for all all treatments treatments under under agitated agitated conditions conditions (Figure (Figure 22F).F). 1 1 Agitation resulted in acetificationacetification ratesrates rangingranging fromfrom 0.110.11 toto 0.13 0.13 g g·LL−−1·dayday−−1,, with with the the highest highest acetic 1 · · acid concentration of 10.7 gg·LL−1 beingbeing achieved achieved at at day day 35. 35. However, However, for for a a successful successful BSV BSV fermentation, fermentation, · 1 an acetic acid concentration of at least 50 g L −1 is usually required [10]. Hutchinson et al. [17] made an acetic acid concentration of at least 50 g·L· − is usually required [10]. Hutchinson et al. [17] made similar observations when using using cell cell immobilization immobilization with with calcium calcium alginate alginate beads beads for for BSV BSV production. production. It is unclear unclear if if these these observations observations were species (ace (acetictic acid bacteria) dependent or due to the the agitation agitation settings used. In In light light of of these these observations, observations, other other vinegar vinegar studies studies have have successfully successfully applied agitation speeds between 100 [31] [31] and 200 rpm [[32],32], with industrialindustrial spirit vinegars successfully employing maximum agitation up to 900 rpm [30,33,34]. [30,33,34]. This suggested that the current agitation setting could not have negatively impacted the microbial activity in the fermentations. Furthermore, Furthermore, most vinegar production systems generally employ agitation and ae aerationration simultaneously [34, [34,35];35]; therefore, it is important to consider that agitation might only be beneficialbeneficial underunder such settings. Figure 22CC showsshows successfulsuccessful acetificationacetification/alcohol/alcohol oxidation profiles profiles for all treatments under static 1 1 fermentations. Total acid formation rates ranged from 1.46 and 2.70 g L −1 day −1 for all of the treatments fermentations. Total acid formation rates ranged from 1.46 and 2.70 g·L· − ··day− for all of the treatments studied. The The relative relative differences differences for total acid formationformation between agitated and static fermentations were 93%, 92% and 95% for FFC, OWC and CC, respec respectively.tively. Furthermore, Furthermore, th thee low low substrate substrate (sugar) (sugar) 1 1 consumption rates (2.68–6.10 g L −1 day−1 ) resulted in much lower alcohol formation rates (Figure2B) consumption rates (2.68–6.10 g·L· − ·day· −) resulted in much lower alcohol formation rates (Figure 2B) but highhigh totaltotal acid acid formation formation rates rates (Figure (Figure2C). These2C). These observations observations could mean could that mean the AABthat microbialthe AAB microbialactivity was activity ideal atwas low ideal alcohol at low concentrations, alcohol concentr suggestingations, thatsuggesting the agitation that the speed agitation should speed be optimized should bewith optimized the aim ofwith reducing the aim alcohol of reducing formation alcohol rates, formation in order rates, to benefit in order the AABto benefit used. the Furthermore, AAB used. Furthermore,if agitation is notif agitation suitable foris not BSV suitable fermentations, for BSV regardless fermentations, of the regardless speed, this of might the speed, benefit this producers, might benefitbecause producers, agitation isbecause energy agitation intensive is and energy the costintensive input and might the escalate, cost input not might only dueescalate, to the not energy only dueusage to butthe alsoenergy due usage to the but mechanical also due equipmentto the mechanical required equipment for agitation. required for agitation.

Static Fermentations Agitated Fermentations

350 350 A D 300 300 ) ) -1 -1 250 250

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Foods 2019, 8, x FOR PEER REVIEW 7 of 15

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-1 60 80 80 60 60 60 60 40 40 60 Alcohol (g.L 60 (g.L Alcohol 40 40 40 40 20 Alcohol (g.L Alcohol (g.L Alcohol (g.L Alcohol Alcohol (g.L Alcohol 20 40 40 20 20 Alcohol (g.L 20 20 (g.L Alcohol 0 20 0 20 0 0 02040 0 10203040 0 0 02040020400 Time (days) Time (days) 00 102030400 10203040 02040 TimeTime (days) (days) TimeTime (days) (days) 0 10203040 Time (days) Time (days) C

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Figure 2. Chemical developments under static (A–C) and agitated (D–F) conditions. Sugar (A,D), FigureFigure 2.Figure Chemical 2. Chemical 2. Chemical developments developments developments under under static under static (A– staticC(A) –andC) ( Aand –agitatedC) agitated and agitated(D– (FD) –conditions.F) ( Dconditions.–F) conditions. Sugar Sugar (A, Sugar D(A),, Dalcohol), (A ,D), (B,E) and total acid (C,F) developments during fermentation. Oak wood chips, alcoholalcohol (alcoholB, EFigure()B ,andE) ( Band ,2.totalE )Chemical andtotal acid total acid (C aciddevelopments, F()C developments, (FC), Fdevelopments) developments under during static during during fermen(A –fermenC fermentation.) tation.andtation. agitated (D Oak–FOak ) Oak conditions.wood wood wood chips, chips, chips,Sugar (A,D Corncobs,), Free-floating cells. Results are the average of three biological repeats Corncobs,Corncobs, alcoholCorncobs, (B ,E) and Free-floatingFree-floating totalFree-floating acid cells. cells.(C, Fcells. Results) Resultsdevelopments Results are ar thee arthe averagee during theaverage average of fermen three of threeof biologicaltation. three biological biological repeats repeatsOak accounting repeats wood accounting chips, for for standard deviation. accountingaccountingstandard for standardfor deviation. standardCorncobs, deviation. deviation. Free-floating cells. Results are the average of three biological repeats accounting for standard deviation. 3.1.2. Effect of the Adsorbents Used 3.1.2. Effect of the Adsorbents Used 3.1.2.3.1.2. Effect Effect of the of theAdsorbents Adsorbents Used Used The adsorption technique for cell immobilization has been studied using several inert materials 3.1.2.The Effect adsorption of the Adsorbents technique for Used cell immobilization has been studied using several inert materials TheThe adsorption adsorption technique technique for forcell cell immobilization immobilization has has been been studied studied using using several several inert inert materialsfor materials various vinegar production systems [31,32,36,37]. In the current study, CC were compared to for various vinegar production systems [31,32,36,37]. In the current study, CC were compared to OWC for forvarious various vinegar Thevinegar adsorption production production technique systems systems for[31,32,36, [31,32,36,cell immobilization37].37]. In theIn thecurrent hascurrent been study, study,studied CC CCwere using were compared several comparedOWC inert tofor materials tocell affinity and influence on BSV production. These materials differ in terms of their for cell affinity and influence on BSV production. These materials differ in terms of their biological OWCOWC for forforcell cell variousaffinity affinity vinegarand and influence productioninfluence on onBSV systems BSV prod prod uction.[31,32,36,uction. These37]. These Inmaterials the materials current differ differstudy, in termsin CC terms werebiologicalof theirof compared their composition to as they have different physical and structural attributes, as well as adsorption composition as they have different physical and structural attributes, as well as adsorption capacity biologicalbiological compositionOWC composition for cell as affinity they as they have and have different influence different physical onphysical BSV and prodand structural structuraluction. attributes, These attributes, materials as well as well asdiffer adsorption ascapacity adsorptionin terms and of surfacetheir chemistry. and surface chemistry. capacitycapacity andbiological and surface surface compositionchemistry. chemistry. as they have different physical and structural attributes, as well Sinceas adsorption agitated fermentations did not meet the required performance for BSV production, i.e., capacitySince agitatedand surface fermentations chemistry. did not meet the required performance for BSVachieving production, 60 i.e.,g·L−1 acetic acid, a more comprehensive discussion is provided in this section on the SinceSince agitated agitated fermentations fermentations1 did did not notmeet meet the therequired required performance performance for forBSV BSV production, production, i.e., i.e., achieving 60 g L− acetic acid, a more comprehensive discussion is provided in this section on the achievingachieving 60 g·L60Since −g·L1 acetic− 1agitated acetic· acid, acid, fermentationsa more a more comprehensive comprehensive did not meet discussion discussionthe required is provided is providedperformance in thisin this forsection sectionBSVfermentations onproduction, theon the thati.e., were only conducted under static conditions. Additional reasons attributed to the fermentations that were only conducted under static conditions. Additional reasons attributed to fermentationsfermentationsachieving that that were 60 were g·L only− only1 aceticconducted conducted acid, undera more under static comprehensive static conditions. conditions. Additionaldiscussion Additional reisasons providedreasons attributed attributed in thisunsuccessful to section the to the on agitated the fermentations, which were characterized by low acetification rates (0.11 to 0.13 thefermentations unsuccessful that agitated were fermentations, only conducted which under were static characterized conditions. Additional by low acetification reasonsg·L attributed− rates1·day (0.11−1) may to to the be due to the interference of cell adsorption by agitation conditions, which eventually unsuccessfulunsuccessful agitated agitated1 fermentations,1 fermentations, which which were were characterized characterized by lowby low acetification acetification rates rates (0.11 (0.11 to 0.13 to 0.13 0.13 g L− day− ) may be due to the interference of cell adsorption by agitation conditions, which g·L−g·L1·day−1·day−1)unsuccessful may−1)· may be· due be due toagitated the to theinterference fermentations,interference of cell of whichcell adsorption adsorption were bycharacterized agitationby agitation conditions, by conditions, low acetification which which eventuallyresulted eventuallyrates (0.11 in freely to 0.13 suspended cells. eventually resulted in freely suspended cells. resultedresulted ing·L freely in− 1freely·day suspended−1 )suspended may be cells.due cells. to the interference of cell adsorption by agitation conditions, whichModerately eventually higher sugar consumption rates of 8.14 and 5.14 g·L−1·day−1 for OWC and CC, Moderately higher sugar consumption rates of 8.14 and 5.14 g L 1 day 1 for OWC and CC, ModeratelyModeratelyresulted higher in higher freely sugar suspendedsugar consumpt consumpt cells.ion ion rates rates of 8.14of 8.14 and and 5.14 5.14 g·L −g·L1·day−1·day·−1− for−·1 forOWC− OWC andrespectively, and CC, CC, were observed at the initial stages (day 0–14) of the fermentation process (Figure 2A). respectively, were observed at the initial stages (day 0–14) of the fermentation process (Figure2A). respectively,respectively, wereModerately were observed observed higher at the at theinitialsugar initial stagesconsumpt stages (day (dion 0–14)ay rates 0–14) of ofthe of 8.14 thefermentation fermentationand 5.14 processg·L process−1·day (Figure−1 for(FigureAlcohol OWC 2A). 2A). formationand CC, rates were also moderately higher for CC and OWC fermentation than FFC Alcohol formation rates were also moderately higher for CC and OWC fermentation than FFC AlcoholAlcohol formationrespectively, formation rates rateswere were wereobserved also also moderately atmoderately the initial higher stageshigher for (d forCCay 0–14)CCand and OWCof theOWC fermentation fermentation processthanfermentations than FFC (Figure FFC 2A).(Figure 2B). While, total acid formation for cells immobilized on CC was consistently fermentations (Figure2B). While, total acid formation for cells immobilized on CC was consistently fermentationsfermentationsAlcohol (Figure (Figure formation 2B). 2B). While, ratesWhile, total were total acid alsoacid form moderatelyformationation for forcells higher cells immobilized immobilizedfor CC and on CConOWC CCwas fermentationwas consistently consistentlyhigher throughout than FFC the fermentation process, with total acid formation rates of 2.7 g·L−1·day−1 being higher throughout the fermentation process, with total acid formation rates of 2.7 g L 1 day 1 being higherhigher throughout fermentationsthroughout the thefermentation (Figure fermentation 2B). process,While, process, total with withacid total totalform acid ationacid formation formationfor cells rates immobilized rates of 2.7of 2.7g·L −ong·L1·day CC−1··dayachieved−1 − wasbeing·−1 beingconsistently− in the shortest fermentation period (20 days). Comparatively, FFC and OWC fermentations achieved in the shortest fermentation period (20 days). Comparatively, FFC and OWC fermentations achievedachieved inhigher the in theshortest throughout shortest fermentation fermentation the fermentation period period (2 0process, (2days).0 days). Comparatively, with Comparatively, total acid FFCformation FFC and and OWC rates OWC fermentations of fermentations 2.7resulted g·L−1·day in− 1lower being total acid formation rates of 1.64 and 1.46 g·L−1·day−1 with a longer fermentation resulted in lower total acid formation rates of 1.64 and 1.46 g L 1 day 1 with a longer fermentation resultedresulted inachieved lowerin lower total in total the acid shortest acid formation formation fermentation rates rates of 1.64periodof 1.64 and (2and 1.460 days). 1.46 g·L −g·LComparatively,1·day−1·day· −1− with·−1 with a− longer FFCa longer and fermentation OWCfermentationperiod fermentations of 33 and 37 days, respectively. Furthermore, the relative differences between FFC and period of 33 and 37 days, respectively. Furthermore, the relative differences between FFC and periodperiod of resulted33of and33 and 37in lowerdays,37 days, totalrespectively. respectively. acid formation Furthe Furthe rmore,ratesrmore, of the 1.64 therelative and relative 1.46 differences g·Ldifferences−1·day −1between with between a longerFFCimmobilized FFC and fermentation and cells were 11% (OWC and FFC) and 39% (CC and FFC). Therefore, it was deduced that immobilized cells were 11% (OWC and FFC) and 39% (CC and FFC). Therefore, it was deduced that immobilizedimmobilizedperiod cells cells ofwere 33were 11%and 11% (OWC37 (OWCdays, and andrespectively. FFC) FFC) and and 39% Furthe39% (CC (CC andrmore, and FFC). FFC).the Therefore, relative Therefore, differences it was it was deduced deduced between that that FFC and immobilized cells were 11% (OWC and FFC) and 39% (CC and FFC). Therefore, it was deduced that

Foods 2019, 8, 303 8 of 15 cells adsorbed on CC fermentations led to the highest microbial activity because of the adsorbents’ attributes, i.e., rough surface and porosity, which improved the adsorption of cells. As a result, the microbial consortium was able to form a highly effective community when adsorbed onto the CC. Contrarily, the OWC are relatively smoother than the porous CC, which consequently led to the poor adsorption of cells. Cell immobilization on CC was also studied for the production of tea vinegar, with acetification rates of 2.88 g L 1 day 1 being obtained [38]. Several studies often use wood shavings for cell · − · − immobilization instead of OWC. For example, Thiripurasundari and Usharani [39] and Kocher and Dhillon [40] reported acetification rates of 0.24 g L 1 day 1 and 5.76–22.8 g L 1 day 1 when using · − · − · − · − wood shavings for the production of cashew apple vinegar and sugar cane vinegar, respectively. It is not clear as to the reasons why the acetification rates for these two studies were different; however, other factors such as substrate availability, bacteria strain used and other fermentor conditions might have played a role. Furthermore, wood shavings and OWC may not be comparable materials due to their differentiated physical properties, and thus, differentiated adsorption capabilities. Table4 shows a comparison of other studies which investigated the e ffect of cell immobilization by adsorption in other vinegar production systems. The current work established a direct relationship between cell immobilization and higher acetification rates. Similarly, other studies [41] also reported a proportional relationship between cell immobilization and acetification rates, with the fibrous bed being reported to show a conspicuous increase in acetification compared to FFC (Table4). Furthermore, when a loofa sponge was used, it led to increased acetification rates [42] which were similar to the rates observed in the current study at static conditions.

Table 4. A comparison of the performance of immobilized cells in other studies.

Acetiﬁcation Rate Product Adsorbent Agitation Reference (g L 1 day 1) · − · − FFC: 0.11 Balsamic-styled Corncobs 135 rpm CC: 0.13 Current study vinegar Oak wood chips OWC: 0.11 FFC (lactose): 1.44 Acetic acid from FFC (milk permeate): 1.92 lactose and milk Fibrous-Bed/matrix 100 rpm [31] IC (Lactose): 12.96 permeate IC (milk permeate) 7.2 1 Hz reciprocating Rice wine vinegar Loofa sponge IC: 1.68–2.4 [42] shaking rate = 60 rpm IC: immobilized cells; CC: Corncobs; FFC: free-ﬂoating cell; OWC: oak wood chips.

Table4 also shows that agitation speeds of 100 rpm, including reciprocating shaking speed of 60 rpm were employed eﬀectively in other studies. These ﬁndings illustrate that agitation may also be dependent on several other factors, such as the microorganisms used, the type of vinegar being produced and other fermentor conditions.

3.2. Phase 2: Aerated Fermentations

3.2.1. Effect of Aeration Rates Aeration is generally a fundamental aspect in most vinegar fermentation systems [43,44]. Aeration systems vary between transferring pure oxygen or air, with pure oxygen systems being noted as expensive due to the cost of technical grade oxygen [45] and deleterious effects such as hyperoxia which leads to cellular death. For this study, aeration with air only was investigated as it was deemed cost effective. It was observed that both high aeration (HA) and low aeration (LA) led to similar alcohol formation/consumption profiles (Figure3B,E). The observations were surprising since alcoholic fermentation is often performed under anaerobic conditions or low dissolved oxygen conditions [46,47]. Consequently, yeast performance was expected to also be negatively affected by HA, albeit that Foods 2019, 8, x FOR PEER REVIEW 9 of 15

was deemed cost effective. It was observed that both high aeration (HA) and low aeration (LA) led toFoods similar2019, 8alcohol, 303 formation/consumption profiles (Figure 3B,E). The observations were surprising9 of 15 since alcoholic fermentation is often performed under anaerobic conditions or low dissolved oxygen conditions [46,47]. Consequently, yeast performance was expected to also be negatively affected by HA,yeasts albeit are generallythat yeasts characterized are generally as characterized facultative anaerobes as facultative and someanaerobes yeasts and can some still surviveyeasts can or growstill surviveunder aerobicor grow conditions under aerobic [48– 51conditions]. Since only [48–51]. air (withSince 21%only oxygen)air (with was 21% used, oxygen) it was was also used, plausible it was alsothat plausible diﬀerent that alcohol differ consumptionent alcohol consumption proﬁles could profiles have been could observed have been if technicalobserved gradeif technical oxygen grade was oxygentested—a was condition tested—a that condition can exacerbate that can hyperoxia.exacerbate hyperoxia.

Low Aeration High Aeration

350 350 A D 300 300 ) 250 ) 250 -1 -1 200 200 150 150 Sugar (g.L Sugar (g.L Sugar 100 100 50 50 0 0 0 1020304050 0 1020304050Foods 2019, 8, x FOR PEER REVIEW 7 of 15 FoodsFoods 2019 2019, 8, ,x 8 FOR, x FOR PEER PEER REVIEW REVIEW Time (days) Time7 (days)of7 15 of 15

120 B 120 E 120120 120120 60 B B B 60 E E 100 100 ) )

100100 E -1 100100 -1 80 80 ) ) ) ) ) ) -1 -1 -1 -1 -1 80-1 80 80 80 40 40 60 60 60 60 60 60 40 40 Alcohol (g.L 20 40 20 (g.L Alcohol 40 40 40 20 Alcohol (g.L Alcohol (g.L Alcohol (g.L Alcohol (g.L Alcohol Alcohol (g.L Alcohol Alcohol (g.L Alcohol 20 20 20 20 20 0 0 0 0 0 0 02040 0 10203040 0 0 0 102030405002040020400 1020304050 Time (days) Time (days) 00 10203040 10203040Time (days) Time (days) Time (days) TimeTime (days) (days) Time (days)

) 60 CC -1 60 F ) 60) 60 -1 -1 60 60 FF ) 60 40 C ) 60 -1 40

-1 F 40 40 40 40 40 40 20 20 20 20 20 20 acidity(g.L Total 20 0 acidity(g.L-1)Total

Total acidity(g.L Total 20 Total acidity(g.L Total 0 Total acidity(g.L-1)Total Total acidity(g.L-1)Total 0 10203040 02040 0 0 Total acidity (g.L

0 0 acidity(g.L Total 0 10203040 Time (days) 0 102030400 02040020400 Time (days) TimeTime (days) (days) 0 1020304050 TimeTime (days) (days)0 1020304050

Time (days) Time (days) Figure 2. Chemical developments under static (A–C) and agitated (D–F) conditions. Sugar (A,D),

FigureFigure 2. Chemical2. ChemicalFigure developments developments 3. Chemical under under developments static static (A –(AC under)– Cand) and lowagitated agitated (A–C )( D and –(DF) high– conditions.F) conditions. (D–F) aeration. Sugar Sugar ( SugarA ,(DA),,D (Aalcohol), ,D), alcohol (B,E) (Band,E) total acid (C,F) developments during fermentation. Oak wood chips, alcoholalcohol (B ,E(B) ,Eand) and totaland total totalacid acid acid(C ,(FC ()C ,Fdevelopments,F) )developments developments during during during fermen fermen fermentation.tation.tation. OakOak Oak wood wood wood chips, chips, chips, Corncobs,Corncobs, Free-floating cells. Results are the average of three biological repeats Free-ﬂoating cells. Results are the average of three biological repeats standardaccounting deviation. for standard deviation. Corncobs, Corncobs, Free-floating Free-floating cells. cells. Results Results ar ear thee the average average of ofthree three biological biological repeats ±repeats accountingaccounting for forstandard standard deviation. deviation. 3.1.2. Effect of the Adsorbents Used 3.1.2.3.1.2. Effect Effect of theof the Adsorbents Adsorbents Used Used The adsorption technique for cell immobilization has been studied using several inert materials TheThe adsorption adsorption technique technique for for cell cell immobilization immobilization has has been been studied studied using using several several inert inert materialsfor materials various vinegar production systems [31,32,36,37]. In the current study, CC were compared to forfor various various vinegar vinegar production production systems systems [31,32,36, [31,32,36,37].37]. In Inthe the current current study, study, CC CC were were compared comparedOWC tofor to cell affinity and influence on BSV production. These materials differ in terms of their OWCOWC for for cell cell affinity affinity and and influence influence on onBSV BSV prod production.uction. These These materials materials differ differ in interms terms biologicalof oftheir their composition as they have different physical and structural attributes, as well as adsorption biologicalbiological composition composition as theyas they have have different different physical physical and and structural structural attributes, attributes, as wellas well as adsorptionas adsorptioncapacity and surface chemistry. capacitycapacity and and surface surface chemistry. chemistry. Since agitated fermentations did not meet the required performance for BSV production, i.e., SinceSince agitated agitated fermentations fermentations did did not not meet meet the the required required performance performance for for BSV BSV production, production,achieving i.e., i.e., 60 g·L−1 acetic acid, a more comprehensive discussion is provided in this section on the achievingachieving 60 60g·L g·L−1 acetic−1 acetic acid, acid, a morea more comprehensive comprehensive discussion discussion is providedis provided in inthis this section sectionfermentations on onthe the that were only conducted under static conditions. Additional reasons attributed to the fermentationsfermentations that that were were only only conducted conducted under under static static conditions. conditions. Additional Additional reasons reasons attributed attributedunsuccessful to theto the agitated fermentations, which were characterized by low acetification rates (0.11 to 0.13 unsuccessfulunsuccessful agitated agitated fermentations, fermentations, which which were were characterized characterized by bylow low acetification acetification rates rates (0.11 (0.11g·L to− 10.13to·day 0.13 −1) may be due to the interference of cell adsorption by agitation conditions, which eventually g·Lg·L−1·day−1·day−1) may−1) may be bedue due to theto the interference interference of cellof cell adsorption adsorption by byagitation agitation conditions, conditions, which which eventually resultedeventually in freely suspended cells. resultedresulted in freelyin freely suspended suspended cells. cells. Moderately higher sugar consumption rates of 8.14 and 5.14 g·L−1·day−1 for OWC and CC, ModeratelyModerately higher higher sugar sugar consumpt consumptionion rates rates of of8.14 8.14 and and 5.14 5.14 g·L g·L−1·day−1·day−1 for−1 for OWC OWC andrespectively, and CC, CC, were observed at the initial stages (day 0–14) of the fermentation process (Figure 2A). respectively,respectively, were were observed observed at theat the initial initial stages stages (d ay(d ay0–14) 0–14) of ofthe the fermentation fermentation process process (Figure (FigureAlcohol 2A). 2A). formation rates were also moderately higher for CC and OWC fermentation than FFC AlcoholAlcohol formation formation rates rates were were also also moderately moderately higher higher for for CC CC and and OWC OWC fermentation fermentation thanfermentations than FFC FFC (Figure 2B). While, total acid formation for cells immobilized on CC was consistently fermentationsfermentations (Figure (Figure 2B). 2B). While, While, total total acid acid form formationation for for cells cells immobilized immobilized on onCC CC was was consistently consistentlyhigher throughout the fermentation process, with total acid formation rates of 2.7 g·L−1·day−1 being higherhigher throughout throughout the the fermentation fermentation process, process, with with total total acid acid formation formation rates rates of of2.7 2.7 g·L g·L−1·day−1·dayachieved−1 being−1 being in the shortest fermentation period (20 days). Comparatively, FFC and OWC fermentations achievedachieved in thein the shortest shortest fermentation fermentation period period (20 (2 days).0 days). Comparatively, Comparatively, FFC FFC and and OWC OWC fermentations fermentationsresulted in lower total acid formation rates of 1.64 and 1.46 g·L−1·day−1 with a longer fermentation resultedresulted in inlower lower total total acid acid formation formation rates rates of of1.64 1.64 and and 1.46 1.46 g·L g·L−1·day−1·day−1 with−1 with a longera longer fermentation fermentationperiod of 33 and 37 days, respectively. Furthermore, the relative differences between FFC and periodperiod of of33 33and and 37 37days, days, respectively. respectively. Furthe Furthermore,rmore, the the relative relative differences differences between between FFCimmobilized FFC and and cells were 11% (OWC and FFC) and 39% (CC and FFC). Therefore, it was deduced that immobilizedimmobilized cells cells were were 11% 11% (OWC (OWC and and FFC) FFC) and and 39% 39% (CC (CC and and FFC). FFC). Therefore, Therefore, it was it was deduced deduced that that

Foods 2019, 8, 303 10 of 15

Nevertheless, total acid formation, which is a key step in BSV production was higher (1.42–1.56 g L 1 day 1) at LA (Figure3C) as compared to HA (0.14–1.2 g L 1 day 1) (Figure3F) for cell immobilized · − · − · − · − treatments. However, FFC completed the fermentations under both aeration settings with acetification rates of 1.27 and 1.46 g L 1 day 1 for HA and LA, respectively. Additionally, over oxidation has been · − · − reported as a common challenge in most vinegar production systems [10]. It seems unlikely that the excessive/increased oxidation rates may have affected the fermentations in this study; since the alcohol profiles under HA and LA conditions (Figure3B,E) were similar to some degree. Other studies reported higher acetification rates compared to the current study when aeration was employed. Rubio-Fernández et al. [44] reported acetification rates of 17.24 and 32.4 g L 1 day 1 for wine vinegar production when air · − · − and oxygen rich air (0.06 vvm) were used respectively. Additionally, Qi et al. [52] reported acetification rates of 43.44 g L 1 day 1 for industrial vinegar production when oxygen (0.13 vvm m 1) was used. · − · − − It is important to mention that the observations between the current study and other studies are influenced by several factors, which include media composition and the AAB strains used. The most obvious influential factor is that BSV fermentation involves high sugar concentration/high osmotic pressures with the simultaneous involvement of non-Saccharomyces yeasts and AAB. Furthermore, spirit vinegar production is generally faster than the production of other vinegars. It is unfortunate that there are no similar BSV studies with which to compare the current data, due to the traditional techniques used for Balsamic vinegar production.

3.2.2. Performance of Adsorbents Used Under Aerated Conditions When cells adsorbed on the different materials were evaluated at the initial stages of fermentation, sugar consumption showed similar profiles with sugar consumption rates between 20.15 to 27.2 g L 1.day 1 under both aeration (HA and LA) settings for all treatments (Figure3A,D). After 7 days, · − − sugar consumption at LA was higher for OWC fermentations with rates between 3.32 g L 1 day 1 · − · − followed by FFC and CC with a sugar consumption rate of 1.80 and 0.11 g L 1 day 1 respectively · − · − (Figure3A). On the other hand, HA, CC and OWC fermentations led to relatively similar results, as well as the highest sugar consumption rates (2.70–3.46 g L 1 day 1) compared to FFC (1.28 g L 1 day 1) · − · − · − · − (Figure3D). The lowest sugar consumption rates were observed in FFC fermentations under HA with trends that we cannot compare with any previously available literature. However, it is possible that the activity of freely suspended yeast cells under HA was reduced by AAB FFC that eventually led to a higher production of acetic acid. Overall, sugar consumption rates were 4.14, 5.78 and 7.4 g L 1 day 1 · − · − under LA and 7.62, 4.82 and 7.23 under HA for CC, FFC and OWC, respectively. The relative differences for sugar consumption between LA and HA fermentations were 2%, 17% and 46% for OWC, FFC and CC; respectively. Furthermore, alcoholic fermentation was successful on all immobilized and FFC fermentations for both aeration settings. Alcohol formation/consumption profiles were moderately similar for all treatments studied under both aeration settings. Total acid formation provided an insight on the productivity of the process evaluated. It was evident, that CC fermentations had the highest total acid formation at LA, followed by FFC and then OWC fermentations. The differences observed were minor, since the total formation rates were 1.56, 1.46 and 1.42 g L 1 day 1 with a fermentation period of 31, 33 and 34 days for CC, FFC and OWC · − · − fermentations, respectively. Under HA, only FFC fermentations completed the fermentation with an acetification rate of 1.2 g L day 1 in 38 days, while CC and OWC fermentations initiated by showing an · · − increase in total acid subsequent to its decrease from day 14 to 42. It was not well understood as to how the FFC fermentations were able to reach the required levels of total acid concentration. A credible assumption could be that HA may have disrupted the adsorbed cells, thus causing and maintaining a low microbial activity. Overall, based on the results obtained, it was evident that agitation and HA did not favor the production of BSV, where cell immobilization by adsorption was used. Foods 2019, 8, 303 11 of 15

3.3. Variations in Cell Adsorption Capabilities among the Yeast/Bacterial Species Used

3.3.1. Individual Yeast Adsorption on Corncobs and Oak Wood Chips The phenomenon of adsorption efficiency is critical to the current study. The adsorption by different microorganisms varied due to cell affinity differences to the adsorbents surface used. However, it is possible that microorganisms employed will adsorb differently when tested as a consortium. With regards to yeast adsorption on CC, it was observed that C. zemplinina had the lowest number of cells adsorbed prior and post fermentation. Z. bailii initially exhibited low cell adsorption before fermentation; however, a relatively higher cell adsorption (92.93%) after fermentation was observed. Yeasts species such as C. pulcherrima, H. guilliermondii and K. apiculata showed a noticeably high cell adsorption prior and post fermentation (Table5).

Table 5. Evaluation of yeast cells adsorbed on corncobs and oak wood chips.

Yeast Cells Adsorbed on Corncobs Yeast Cells Adsorbed on Oak Wood Chips Cell Before After Relative Before After Relative Concentration Identity Fermentation Fermentation Diﬀerence Fermentation Fermentation Diﬀerence YPD Broth (g g 1) (g g 1) (%) (g g 1) (g g 1) (%) (Cells mL 1) · − · − · − · − · − Candida pulcherrima 2.34 105 0.0371 1.1580 96.80 0.0214 0.1751 87.78 × Candida zemplinina 7.40 105 0.0006 0.0290 97.93 0.0903 0.0566 37.32 − Hanseniaspora × 7.70 105 0.0110 0.7906 98.61 0.1001 0.1032 3.00 guilliermondii × Kloeckera apiculata 1.95 106 0.0283 1.6955 98.33 0.0926 0.1804 48.67 Zygosaccharomyces × 3.97 105 0.0400 0.5661 92.93 0.1785 0.1393 21.96 bailii × −

Furthermore, yeast cell adsorption was different for both OWC and CC. Some yeast species such as C. zemplinina (59.54%) and Z. bailii (28%) showed a relative decrease in cell adsorption post fermentation (Table5). The trends showed that C. zemplinina and Z. bailii might have low cell affinity to the smooth ‘easy-to wash-off’ OWC surface during the fermentation compared to CC surfaces. On the other hand, C. pulcherrima (87.78%), H. guilliermondii (3.00%) and K. apiculata (48.67%) showed a relative increase in cell adsorption capacity during the fermentation, with H. guilliermondii showing the lowest adsorption. Overall, the cells adsorbed (g g 1) on CC and OWC were not comparable since they have · − different densities, including surface chemistry properties.

3.3.2. Individual Bacteria Adsorption on Corncobs and Oak Wood Chips The adsorption of bacteria onto CC and OWC was also evaluated (Table6). A. pasteurianus and A. malorum appeared to have the highest (0.1461 and 0.095 g g 1, respectively) cell adsorption · − efficiency on CC before fermentation (Table6). K. baliensis, G. cerinus and G. oxydans had relatively similar adsorption efficiency on CC before fermentation, with relatively low variations (97.76%, 99.26%, 96.12%, respectively) observed post fermentation. Similar decreases in adsorption efficiency patterns on OWC were observed between A. malorum and K. baliensis (Table6). A. pasteurianus had the lowest initial cell adsorption (0.0020 g g 1) before fermentation; however, it had the highest percentage cell · − increase (98.87%) after fermentation. G. cerinus and G. oxydans showed a similar pattern before and after fermentation, with 52.40% and 56.33% in cell adsorption after fermentation. Other studies have investigated the cell adsorption on CC and wood shavings; however, these studies did not report the number of cells adsorbed on the adsorbents used, which leads to the lack of comparative data. Foods 2019, 8, 303 12 of 15

Table 6. Evaluation of bacterial cells adsorbed on corncobs and oak wood chips.

Bacteria Cells Adsorbed on Corncobs Bacteria Cells Adsorbed on Oak Wood Chips Cell Before After Relative Before After Relative Concentration Identity Fermentation Fermentation Diﬀerence Fermentation Fermentation Diﬀerence GM Broth (g g 1) (g g 1) (%) (g g 1) (g g 1) (%) (Cells mL 1) · − · − · − · − · − Acetobacter 3.97 105 0.1461 1.1968 87.79 0.0020 0.1772 98.87 pasteurianus × Acetobacter malorum 1.25 106 0.0948 1.0882 91.29 0.1851 0.1545 16.53 × − Kozakia baliensis 3.13 105 0.0260 1.1589 97.76 0.1224 0.1122 8.33 × − Gluconobacter cerinus 2.22 106 0.0116 1.5618 99.26 0.1498 0.3147 52.40 Gluconobacter × 6.38 105 0.0426 1.0977 96.12 0.1008 0.2308 56.33 oxydans ×

Overall, these results showed a successful adsorption on CC and OWC for both yeasts and bacteria, but with varying adsorption eﬃciency. However, the data also displayed an unstable cell adsorption proﬁle on OWC, since a decrease in cell adsorption was observed for some yeast and bacterial species. The data further suggested a more sustainable approach on the reusability of immobilized cells on CC and OWC treatments.

4. Conclusions According to all the results obtained, it is evident that cell immobilization improves acetification rates during BSV fermentation. However, the highest acetification rates were obtained under static and non-aerated conditions. Corncobs were observed to be the most suitable material for cell immobilization presumably due to their physical structure. Consequently, the shortest fermentation period was 20 days when cells were immobilized on corncobs under static fermentation conditions. A study to investigate the concurrent optimization of agitation and aeration as well as the effect of corncobs and oak wood chips on organoleptic properties is recommended. The current study therefore, serves as a foundation for cell immobilization by adsorption on materials during balsamic-styled vinegar production.

Author Contributions: Conceptualization, U.F.H. and N.P.J.; methodology, U.F.H., S.G., B.S.C., N.P.J., M.M.-N. and H.W.D.P.; formal analysis, U.F.H., M.M.-N., N.P.J., B.S.C., H.W.D.P., and S.K.O.N.; investigation, U.F.H., S.G. and M.M.-N.; resources, N.P.J., H.W.D.P. and S.K.O.N.; data curation, U.F.H., S.G., B.S.C., H.W.D.P. and S.K.O.N.; writing—original draft preparation, U.F.H., review and editing, U.F.H., S.G., N.P.J., B.S.C., H.W.D.P., M.M.-N., and S.K.O.N.; Supervision, N.P.J., H.W.D.P. and S.K.O.N.; funding acquisition, N.P.J. and S.K.O.N. Funding: Financial support was provided by the Agricultural Research Council (ARC) and the Cape Peninsula University of Technology (South Africa). Acknowledgments: The authors would acknowledge the Bioresource Engineering Research Group (BioERG), Department of Biotechnology, Cape Peninsula University of Technology (RK 16) and the Agricultural Research Council (ARC). Conﬂicts of Interest: The authors hereby declare that they have no conﬂict of interest.

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