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Process Biochemistry 42 (2007) 1561–1565 www.elsevier.com/locate/procbio

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A new pilot plant scale acetifier designed for vinegar production in Sub-Saharan Africa Bassirou Ndoye b,c,*, Stephane Lebecque a, Jacqueline Destain b, Amadou Tidiane Guiro c , Philippe Thonart a,b a

Centre Wallon de Biologie Industrielle, Universite ´ de Lie`ge, B-40, Sart-Tilman, B-4000 Lie `ge, Belgium b Faculte´ Universitaire des Sciences Agronomiques de Gembloux, Unite ´ de Bio-industries, 2 Passage des De ´porte´s, B-5030 Gembloux, Belgium c Institut de Technologie Alimentaire de Dakar, Route des Pe `res Maristes, BP 2765, Dakar, Senegal Received 21 November 2006; received in revised form 2 July 2007; accepted 21 August 2007

Abstract A novel thermotolerant strain Acetobacter senegalensis sp. nov. (CWBI-B418T) isolated in Senegal from mango fruit, previously freeze-dried and conserved at 4 8C under vacuum packaging was successfully rehydrated into an acetifying medium. It was used as an inoculum culture and then applied into a new pilot plant scale acetifier (300 L) for vinegar production. This latter was specifically designed to produce a high volume and quality of vinegar in Sub-Saharan Africa at fermentation temperature of 35 8C. Several semi-continuous cycles of acetic acid fermentations were carried out. The behaviour of substrate and product concentrations, population of bacteria into the reactor was analysed as well as the evolution of acidity, acetification rates and stoichiometric yields. Operation with this novel bioreactor allowed achieving 8% (v/v) of acetic acid concentration at 35 8C. # 2007 Elsevier Ltd. All rights reserved. Keywords: Acetobacter senegalensis sp. nov.; Thermotolerant acetic acids bacteria; Freeze-dried starter culture; Vinegar cultivation; Acetifier bioreactor

1. Introduction Vinegar is widely used as food condiment in Sub-Saharan Africa. The main biotechnological process involved in vinegar making is acetic acid fermentation. It consists of a biological oxidation (strictly aerobic and thermodynamically favoured), in which a substrate with a low content of alcohol is partially oxidised by means of acetic acids bacteria to produce acetic acid and water [1]. The stoichiometry for the conversion of substrate into product is 1:1 (v/v) [2]. In a technological processing viewpoint, two methods of vinegar production were carried out: (i) The Orleans method by the stationary surface culture, particularly adapted for vinegar production from cereal or

´ Universitaire des Sciences Agronomiques * Corresponding author at: Faculte de Gembloux, Unite´ de Bio-industries, 2 Passage des De´porte´s, B-5030 Gembloux, Belgium. Tel.: +32 81 62 2305; fax: +32 81 61 4222. E-mail address: [email protected] (B. Ndoye). 1359-5113/$ – see front matter # 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2007.08.002

fruit juice. Although, the equipment used by this method authorizes only low yields and volumes of production. (ii) The continuous submerged culture is a modern mass production process, by aeration, into an acetator. The latter process gives higher fermentation rate and yield of acetic acid; however, it requires precise control of fermentation for the efficient vinegar production [3]. The advantages of submerged fermentation over the traditional methods are: (i) the submerged fermentation permits 30 times faster oxidation of alcohol; (ii) greater efficiency is achieved; (iii) a smaller reactor is needed; (iv) yields are 5–8% higher and more than 90% of the theoretical yield is obtained; (v) the process can be highly automated; (vi) The ratio of productivity to capital investment is much higher [4]. However, a great quantity of heat is generated due to the oxidation of ethanol in the submerged acetic acid cultivation. A large scale cooling system becomes necessary to maintain the optimum temperature [3]. Furthermore, the optimum temperature for bacterial growth in most industrial vinegar production is 30 8C [5]. Since these bacteria are mesophilic, slight

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temperature elevation by 2 or 3 8C causes marked decrease in both the rate and yield [6]. If the vinegar cultivation can be conducted at about 35 8C by using a thermophilic strain, more than 50% of the cost for cooling water will be reduced in Sub-Saharan Africa. Previous studies have characterized two thermotolerant acetic acids bacteria (TAAB) useful for vinegar manufactures in Sub-Saharan Africa [7]. These TAAB were prepared as freeze-dried starters and their characteristics of preservation during storage were optimised [8]. The aim of this paper was to apply a freeze-dried thermotolerant strain for a sustainable development of vinegar production into a new pilot plant scale acetifier via a semicontinuous process. In this respect, the abilities of acetate production of this strain were optimised to produce wine vinegar at temperature of 35 8C. 2. Materials and methods

oxygenation into the reactor, heat exchange and charge–discharge operations during the acetification cycles. The aeration is enhanced by a system of venturi, which favours the air compression. Two sensors (OMRON, Japan, E2KC25MY1) control the filled and refilled volume during the acetification cycles. Many other valves and gauges complete the described system graphically shown in Fig. 1.

2.2. Acetification phases during the fermentation cycles The most commonly used operation in industrial acetifiers for vinegar production is the semi-continuous one. Once the reactor is completely filled, the semi-continuous cycles begin [9–11]. The initial substrate is constituted by 200 L of acetifying medium (AM) with initial ethanol and acetic acid concentration of 5% (v/v) and 2.5% (v/v), respectively. In such conditions, in a few hours, the acidity of the medium is shortly increased by fermentation with consumption of the present ethanol. When the process reached the optimum acidity, the system is considered completely finished. The starting of a new cycle begins with the discharge of 30% of the total volume (200 L). At this point, the biomass population has been diluted and new environmental conditions are established. In a few hours, a new cultivation occurs in similar conditions to the previous one. So then, in every cycle, a production of 70 L of vinegar (70%) is obtained with a desired or an optimum acidity. Such protocol is schemed in Fig. 2.

2.1. Bioreactor description 2.3. Micro-organism and culture media The pilot plant acetification equipment, depicted in Fig. 1, consisted basically of a plastic (PVC) cylinder reactor (Mertens Plastic, Liege, Belgium) inspired to the pilot plant model of Chansard fermentation (Lyon, France) with 1.75 m height and 0.6 m internal diameter (working volume of 200 L and total volume of the cylindrical reactor 300 L). The air inlet is equipped of a gyrometric flowmeter (Georg Fischer, Type: SK51, No. 198-801-881). A polarographic dissolved oxygen sensor measures the partial dissolved oxygen pressure into the reactor and allows the oxygen consumption by cells in their metabolic and growth functions. The temperature of operation is settled by an external heat exchanger (S.A.G. Charlier Heat Exchangers, Belgium) connected to the reactor. The heat exchanger consists of fine tubes and a double envelope where circulates of the cold water which dissipates generated heat. It fills three roles: (i) the cooling fresh wine; (ii) the air–liquid exchange step in which the oxygenation is carried out at the time of the circulating fresh wine and (iii) the recycling fresh wine and its constant mixture. A single centrifugal pump (ALFA LAVAL) in combination with the heat exchanger ensures aeration for

The strain used in this study is a novel thermotolerant acetic acid bacterium Acetobacter senegalensis sp. nov. (CWBI-B418T) isolated from mango fruit (Mangifera indica) in Senegal [12]. This strain is freeze-dried and conserved under vacuum packaging at 4 8C without oxygen, moisture and free of light. The freeze-dried A. senegalensis sp. nov strain (0.5 g) conserved at 4 8C about 6 months under vacuum packaging was revitalized in the acetifying medium (AM) containing: yeast extract (Organotechnie, France) 1 g/L, glucose 2 g/L, MgSO4 1 g/L, (NH4) 2HPO 4 1 g/L, KH2PO4 1 g/L, citrate Na 31 g/L with a pH medium of 4. Ethanol 2.5% (v/v) and acetic acid 0.5% (v/v) were added after sterilization at 121 8C for 20 min. The double medium contained into embossed flasks (3 L) was inoculated and incubated at 30 8C for 1 h on a rotary shaker with 130 rpm. The starting inoculum consisted of the revitalized CWBI-B418 strain (100 mL) as a seed culture inoculated into an YGM (yeast, glucose and mannitol) medium previously described [7]. This subculture was incubated at 30 8C on a rotary shaker with 130 rpm for 24 h.

Fig. 1. Industrial acetification equipment used in the experimental work. (1) Reactor; (2) electrical equipment box; (3) oxygen sensor; (4) temperature sensor; (5) ventilation tube; (6) wine supplying tube; (7) recycling pump; (8) heat exchanger; (9) venturi air; (10) venturi wine; (11) vinegar outlet valve; (12) feed inlet valve; (13) wine wood; (14) vinegar wood.

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Fig. 3. Experimental data on acetic acid concentration, ethanol concentration and biomass during the start-up process. Fig. 2. Protocol for the semi-continuous operation procedure.

The cultivation medium used for all the experiments was the acetifying medium (AM) described above with an initial ethanol and acetic acid concentration of about 5% (v/v) and 2.5% (v/v), respectively. This medium is introduced into the bioreactor at the beginning of every single discontinuous cycle. In the first cycle of every series, the fresh medium and a given percentage of the final product are mixed with non-extreme concentrations of ethanol.

2.4. Analytical methods The total biomass evolution was followed by using the turbidimetrical method (the optical density (OD) measured by spectrophotometer, Pharmacia) at 540 nm. The ethanol concentration (%, v/v) of wine vinegar was determined using the enzymatic UV-method kit (Boehringer Mannheim, R-Biopharm, REF 61270). Total acidity (%, v/v) of the wine vinegar was measured by titration with 0.1N NaOH using phenolphthalein as pH indicator.

3. Results 3.1. Start-up process: growth and acetification phases The freeze-dried thermotolerant strain was applied to the semi-continuous operation procedure into the described bioreactor (Fig. 1) for several stages as described by De Ory et al. [9,10]:  The fresh medium (70 L) is inoculated into the bioreactor and adequately mixed with the starting inoculum which is composed of a huge population of acetic acid bacteria in their exponential growth phase; when the desired concentration of ethanol is reached, a partial filling-up with a fresh medium (30 L) is added into the reactor to arrive at a final volume of 100 L.  The second step is monitored by adding a new volume of fresh medium (100 L) into the reactor to arrive at a working volume of 200 L. The process continues in successive stages and finishes when the desired working volume is reached.

Consequently, the number of total stages will depend on the percentage of starting inoculum and the reactor working volume. Fig. 3 shows the various stages from the start (inoculation) to the end of the whole operation. This is the start-up procedure. At the starting operation (inoculum at 70 L), no significant acetic acid production was observed even though with the exponential phase of growth from 5 to 20 h of culture. At this moment, micro-organism used the main proportion of its energy resources to synthesize the required enzymes for substrate degradation [13]. After mixing the inoculum and fresh medium to arrive at a step volume of 100 L, the strain grew without lag phase. It still grew at the working volume of 200 L and a stationary phase is observed from 40 h until the end of the culture. Concomitantly, there is a high acidic production and a significant decrease of ethanol concentration until 50 h, namely the working volume of 200 L is achieved. At the end of the culture, the low acidic production phase did not favour a bacterial growth due to its toxic effects, while a decrease of the ethanol concentration is noticed. This decrease might be caused by the ethanol volatilization at a higher cultivation temperature at 35 8C [5,14]. Then, step-by-step during the start-up process, the microorganism grew optimally without any appreciable lag phase and increased its acetic acid production. These results were obtained recently during previous studies of physiological characteristics to determine the thermotolerant properties of this new acetic acid bacterium [7]. 3.2. Production scheme All the developed experiments are summarised in Table 1 with experimental results. The evolution of the concentrations of substrate (ethanol), product (wine vinegar) and the bacterial population in every cycle was depicted in Fig. 4. The results indicate that it is possible to operate in semicontinuous regime in the proposed bioreactor and to obtain

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Table 1 Production scheme: experimental data for the study of semi-continuous cycles in the bioreactor Experiment

70 L 100 L 200 L Cycle Cycle Cycle Cycle Cycle Cycle Cycle Cycle Cycle Cycle Cycle Cycle

1 2 3 4 5 6 7 8 9 10 11 12

Time (days)

Total acidity (%) Initial

Final

1 1 2 4 3 2 1 1 3 3 3 3 3 3 4

0.3 0.5 0.7 1.5 3.1 3.7 4.1 4.8 5.5 6.1 6.5 6.4 6.5 6.3 6.5

0.64 1.7 3.2 5.4 6.6 7.02 7.9 8.3 8.6 9.2 9.4 9.7 9.9 9.9 9.9

Acidity produced (%)

Acetification rate (% day1)

Stoichiometric yield (%)

0.34  0.1 1.2  0.1 2.5  0.2 3.9  0.5 5.1  0.1 5.5  0.5 6.4  0.1 6.8  0.3 7.1  0.4 7.7  0.1 7.9  0.2 8.2  0.3 8.4  0.2 8.4  0.2 8.4  0.1

0.34 1.2 1.25 0.97 1.7 2.76 6.4 6.8 2.36 2.56 2.63 2.72 2.8 2.8 2.1

68 100 60 100 100 100 100 99 97 93 90 100 100 100 100

Acidity produced was obtained by calculating the difference between total acidity and initial acidity. Acetification rate represents the rapport between acidity produced and the time. The stoichiometric yields were evaluated by calculating the moles of acetic acid produced per mole of ethanol consumed in the liquid medium.

Fig. 4. Biomass, ethanol and acetic acid concentrations vs. time during the cultivation cycles. (C) Cycle.

wine vinegar (70 L) with a high acidity at the end of the last cycle (8–9% of total acidity). 4. Discussion The behaviour of ethanol and acetic acid concentrations versus process time shows the typical trends for the microorganism. After a completed working volume of the bioreactor (200 L) corresponding to the starting acetic acid fermentation (Fig. 4), the micro-organism grew and begun immediately to produce acetic acid by consuming the ethanol. Such behaviour was not observed from other strains like those used in European industrial vinegar. It is usually demonstrated that these strains presented a lag phase in the first hours and after replacement with fresh wine [9,10]. This is due to the initial concentrations of substrate and product, previous history of the culture, etc. A stationary and a death phase of growth were observed when the high product concentrations are obtained (8%, v/v). Viable cells from this stage are those that had been adaptated in the culture medium. In general terms, this could be resumed as a

phase in which bacteria are spending their major energy on the metabolic adaptation to the new environmental conditions of the culture [15]. This phase of production demonstrates that the use of this thermotolerant strain overcame the lag phase that was nonproductive from the fermentative point of view. The average acetification rate for every cycle, shown in Table 1, expresses the ability of the strain to produce acetic acid during fermentation process. The progressive increasing of the rate could be explained by the synthesis of new enzymes from metabolic pathways useful for the fermentation ability. The present biomass progressively acclimatizes to the reactor. This phase is achieved by probably the dissolved oxygen used as a critical factor for maintaining cellular viability [5,14,16]. De Ory et al. [10] explained the increasing of the rates in terms of cellular adaptation of the fermentation culture conditions. These latter were an initial mixed culture submerged in the medium in which the best-adapted strains were going to be selected. The novel strain cultivated at 35 8C increases its ability for the fermentation, and then the acetification rates until an optimum observed in cycles 4 and 5. From this stage, the rate decreases due to the toxicity of the product. It is state of the art that bacteria react with particular sensitivity to changes of the medium conditions [17]. A direct answer by bacteria to inhibiting these changes is an increasing production of certain protective or stress substances. Among these, the haponoids are well known to be able to slow down the diffusion of acetic acid through the cell membranes [18]. These conditions favour the pH-controlling enzyme cascade and the cell is enabled to keep the internal pH value on a tolerated level in spite of increasing acetic acid concentrations [17]. The stoichiometric yields shown in Table 1 have been calculated as the moles of acetic acid produced per mole of ethanol consumed during the fermentation time. It represents consequently the efficiency of the closed system designed. As indicate in Table 1, when the yield is 100%, any evaporation on

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substrate has been registered during the process time and all of it has been stoichiometrically converted. However, the elevated fermentation temperature could produce a volatilisation of the ethanol during the process. Ohmori et al. [2] have reported that a temperature of 30 8C was established as the most suitable for industrial vinegar production (11–12% acetic acid). This is the temperature currently employed by the industry [1]. The temperatures above of 30 8C tend to increase damage to bacteria due to the concentration of acetic acid in the cultivation medium [6,19]. However, Saeki et al. [14] have isolated a thermotolerant acetic acid bacterium, which reached a final acetic acid concentration less than 70 g/L with very low process productivity. 5. Conclusion Results have shown that the new thermotolerant strain [12], used as functional freeze-dried starter culture, grew immediately and produced acetic acid as well during the start-up as during the acetification processes at fermentation temperature of 35 8C. In other hand, the proposed pilot acetifier is designed to maximise productivity and to be technically viable. Then, the designed acetifier bioreactor could be appropriate for vinegar production in Sub-Saharan Africa due to the reduction of cooling water expenses, the relatively high quality and volume of vinegar produced, etc. Acknowledgements This research was supported by the partnership between Walloon Region (Belgium) and Senegal (DRI Contract No. 27/ 11/2003-134-S). The authors gratefully acknowledge the International Foundation for Science (IFS), Sweden, for financial support to B. Ndoye via a Grant Program (Grant No. 3595-1) awarded to him. References [1] Adams MR. Microbiology of fermented foods. Blackie Academic & Professional; 1998. p. 1–44. [2] Czuba J. Growth of Acetoacter biomass and product formation during acetic fermentation. Acta Alimentaria Polonica Vol. XVII/XLI/No. 3 1991 pp. 205–211.

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[3] Ohmori S, Masai H, Arima K, Beppu T. Isolation and identification of acetic acid bacteria for submerged acetic acid fermentation at high temperature. Agric Biol Chem 1980;44...