A new pilot plant scale acetifier designed for vinegar production in sub saharan africa

Short communication
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-B418
T
) 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 aceti c 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
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 acet ic acid cultivation. A
large scale cooling system becomes necessary to maintain the
optimum temperature [3]. Furthermore, the optimum tempera-
ture for bacterial growth in most industrial vinegar production
is 30 8C [5]. Since these bacteria are mesophilic, slight
www.elsevier.com/locate/procbio
Process Biochemistry 42 (2007) 1561–1565
* Corresponding author at: Faculte
´
Universitaire des Sciences Agronomiques
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: (B. Ndoye).
1359-5113/$ – see front matter # 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.procbio.2007.08.002
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 8Cby
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 semi-
continuous 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
2.1. Bioreactor description
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
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, E2K-
C25MY1) 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 con-
centration 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.
2.3. Micro-organism and culture media
The strain used in this study is a novel thermotolerant acetic acid bacterium
Acetobacter senegalensis sp. nov. (CWBI-B418
T
) 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 8Cabout
6 months under vacuum packaging wasrevitalizedintheacetifying medium(AM)
containing: yeast extract (Organotechnie, France) 1 g/L, glucose 2 g/L, MgSO
4
1 g/L, (NH
4
)2HPO
4
1g/L,KH
2
PO
4
1 g/L, citrate Na
3
1 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.
B. Ndoye et al. / Process Biochemistry 42 (2007) 1561–15651562
The cultivation medium used for all the experiments was the acetifying
medium (AM) described above with an initial ethanol and acetic acid con-
centration 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]:
 Th e 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.
 Th e second step is monitored by adding a new volume of
fresh med ium (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 highe r cultivation temperature
at 35 8 C [5,14].
Then, step-by-step during the start-up process, the micro-
organism 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 semi-
continuous regime in the proposed bioreactor and to obtain
Fig. 2. Protocol for the semi-continuous operation procedure.
Fig. 3. Experimental data on acetic acid concentration, ethanol concentration
and biomass during the start-up process.
B. Ndoye et al. / Process Biochemistry 42 (2007) 1561–1565 1563
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 micro-
organism. 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 stationar y 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 non-
productive 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 latt er were an initial mixed culture submerged in the
medium in which the best-adapte d strains were going to be
selected. The novel strain cultivated at 35 8C increases its
ability for the fermentation, and then the acetificat ion 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, whe n the yield is 100%, any evaporation on
Table 1
Production scheme: experimental data for the study of semi-continuous cycles in the bioreactor
Experiment Time
(days)
Total acidity (%) Acidity
produced (%)
Acetification
rate (% day
À1
)
Stoichiometric
yield (%)
Initial Final
70 L 1 0.3 0.64 0.34 Æ 0.1 0.34 68
100 L 1 0.5 1.7 1.2 Æ 0.1 1.2 100
200 L 2 0.7 3.2 2.5 Æ 0.2 1.25 60
Cycle 1 4 1.5 5.4 3.9 Æ 0.5 0.97 100

Cycle 2 3 3.1 6.6 5.1 Æ 0.1 1.7 100
Cycle 3 2 3.7 7.02 5.5 Æ 0.5 2.76 100
Cycle 4 1 4.1 7.9 6.4 Æ 0.1 6.4 100
Cycle 5 1 4.8 8.3 6.8 Æ 0.3 6.8 99
Cycle 6 3 5.5 8.6 7.1 Æ 0.4 2.36 97
Cycle 7 3 6.1 9.2 7.7 Æ 0.1 2.56 93
Cycle 8 3 6.5 9.4 7.9 Æ 0.2 2.63 90
Cycle 9 3 6.4 9.7 8.2 Æ 0.3 2.72 100
Cycle 10 3 6.5 9.9 8.4 Æ 0.2 2.8 100
Cycle 11 3 6.3 9.9 8.4 Æ 0.2 2.8 100
Cycle 12 4 6.5 9.9 8.4 Æ 0.1 2.1 100
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.
B. Ndoye et al. / Process Biochemistry 42 (2007) 1561–15651564
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 8Ctendtoincreasedamagetobacteriadueto
the concentration of acetic acid in the cultivation medium
[6,19]. However, S aek i et al. [14] have isolated a thermo-
tolerant acetic acid bacteriu m, 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 immedi-
ately 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.
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