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

This latter was specifically designed to produce a high volume and quality of vinegar in Sub-Saharan Africa at fermentation temperature of 35 8C.. nov.; Thermotolerant acetic acids bacte

Short communication

A new pilot plant scale acetifier designed for vinegar

production in Sub-Saharan Africa Bassirou Ndoyeb,c,* , Stephane Lebecquea, Jacqueline Destainb,

Amadou Tidiane Guiroc, Philippe Thonarta,b

aCentre Wallon de Biologie Industrielle, Universite´ de Lie`ge, B-40, Sart-Tilman, B-4000 Lie`ge, Belgium

bFaculte´ Universitaire des Sciences Agronomiques de Gembloux, Unite´ de Bio-industries,

2 Passage des De´porte´s, B-5030 Gembloux, Belgium

cInstitut 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

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 acetic acid cultivation A large scale cooling system becomes necessary to maintain the

tempera-ture for bacterial growth in most industrial vinegar production

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:ndoye.b@fsagx.ac.be(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 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

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 inFig 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 inFig 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-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) 2HPO41 g/L, KH2PO41 g/L, citrate Na31 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) 1561–1565 1562

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] :

 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 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

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 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 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 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

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

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]

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

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 1)

Stoichiometric yield (%) Initial Final

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) 1561–1565 1564

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

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

thermo-tolerant 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

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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