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Ganou-Parfait B., Valadon M., Parfait A., 1991. Contribution à la bactériologie des eaux de fabrication de distilleries de la Guadeloupe. AFCAS : 1re Rencontre internationale en langue française sur la canne à sucre, 296–302.
Contribution to the bacteriology of manufacturing waters of Guadeloupe distilleries.
GANOU-PARFAIT B., VALLADON M., PARFAITA.
INRA, LAPRA, CRITT-BAC, Pointe à Pitre, Guadeloupe
Water resources have two main origins: rivers and groundwater. The inventory of the bacterial microflora is oriented towards the search for germs that may have consequences in manufacturing.
Several factors influence the quantitative and qualitative composition of the bacterial population: thermal processes, mineral content and the use of antiseptics. Results are provided for some industrial sites.
Keywords: distillery, rum, bacteria, water, mineralization, contamination, antiseptic.
The distilleries of the Guadeloupe archipelago are located in three islands: Basse-Terre, Grande-Terre and Marie-Galante. The last two are characterized by a dry climate and calcareous soil. The water table and small rivers constitute the own water resource. Basse-Terre is mountainous and humid; surface waters are more abundant.
Manufacturing waters are not treated, they bring with the raw materials, most of the germs of contamination. To protect fermentative media from bacterial growth and activity, acidification is caused by sulfuric acid and certain fluoride-based antiseptics are used.
The bacterial flora also participates in the formation of the aroma.
Materials and methods
Water samples are taken at the feed of the industrial site. The operation continued for two seasons, 1989 and 1990 for a period from February to May. The treatment scheme is that of Figure 1. The different groups of bacteria are isolated from selective media (Table I). The counts of certain anaerobic bacteria, including sulphate-reducing bacteria are made according to the most favorable number method with a 95% confidence coefficient (ALEXANDER, 1982) and for this purpose on three tubes by dilution.
Other bacteria are counted by the Sartorius membrane filter method. The water is diluted and filtered on a filtration ramp. The filtration membranes for counting cellulose nitrate have a porosity of 0.2 μ and are then applied to the agar medium agar plate. The incubation takes place at 30°C for aerobic germs and gaspak jar at 30°C for anaerobic germs.
The determination of the minerals of the water samples was carried out by HPLC; these are first filtered under vacuum to facilitate degassing. After dilution, the samples are clarified by passage over Sep-pak columns. The anions are separated by passing through a Mitsubishi SCA03 column at room temperature with potassium phthalate eluting at pH greater than 8. They are analyzed on the universal UV detector JASCO 875 uv. The cations are separated by passing through a Mitsubishi SCK 01 column at room temperature. The eluent for alkalis is nitric acid and for alkaline earths is tartaric acid supplemented with ethylene diamine. They are detected by a WESCAM 215 conductivity meter. Figure 2 shows examples of separation. The different ions are quantified and recorded thanks to the integrator SIC calculator: Chromato corder 12.
From a bacteriological point of view, there are aerobic germs (lactic acid bacteria, corynebacteria, Bacillus) more present in cane juice-based media and thermophilic and anaerobic germs (clostridia, Bacillus, sulphate-reducing bacteria) more abundant in molasses media (Table II). Antiseptics are used in the distillery to limit the activity of bacteria. The most commonly used is sulfuric acid (41/100 hl); it makes it possible to lower the pH of the meal to 4.5, but increases the concentration of sulphate ions in the fermentation medium.
It is possible that the metabolism of sulfur in yeast and in bacteria (BSR) has consequences on the aromatic fraction of rum and the quality of vinasses, a source of pollution on the environment. Other antiseptics have been used in Guadeloupe.
Quite often, besides the necessity of adapting yeasts to these antiseptics, resistant bacteria develop.
Table III: The waters of the rivers. Variation of mineral contents.
Laboratory tests have shown that bacteria in fermentative fermentation media can grow in the presence of high concentrations of sodium fluoride (0.28 g/l), sodium acid, sodium chloride and ethanol. This indicates an adaptation of bacterial germs to sodic antiseptics, to ethanol (up to 8 °GL in general) We have studied the variation of the manufacturing water contents in ions sodium, calcium, magnesium, chloride, nitrate and sulfate and the variation of the corresponding bacterial flora (Tables III, IV, V and VI).
Table IV : Variation of the bacterial flora in the water.
At the beginning of the febriermars campaign, their number is of the order of 7.10^6 bacteria / ml of water then it regresses and stabilizes until the end of the campaign at 6.10^3 bacteria / ml
At the beginning of the campaign it was possible to detect 1.10^3 aerobes / ml of water. This number remains stable during the campaigns except in April when it increases slightly to 1.10^4 bacteria / ml
In March, we detected 1.10^3 aerobic bacteria in the river water. In April they are of the order of 15.10^4 on average. Storage in concrete tanks shows an increase in aerobic flora: 11.10^6 bacteria / ml of water in April
The rains of April seem to favor the number of anaerobic bacteria, generally stable during the campaign. The population goes from 1 to 6.10^3 to 1 to 5.10^5 bacteria / ml
The water contains few anaerobic bacteria at the beginning of the campaign: 1.10^2 bacteria / ml in 1989, 2.10^4 bacterias / ml in 1990. We detected periods of strongest contamination as in March 1989 (Easter): 2.10^4 bacteria. At the end of the 1990 campaign, the number of anaerobes reached 5.10^4 bacteria / ml
We observed an increase of the anaerobic flora in time during the campaigns, it went from 10^2 to 10^4 bacteria / ml of water. However, the number has increased in the deep steel tank as it has decreased in the reservoirs, very wide and shallow concrete ponds: 50 to 100 bacteria / ml
The number of sporulated bacteria declines from beginning to end of the season. It goes from 10^3 to 10^1 bacteria for anaerobes and 6.10^2 for aerobes
Their population decreases during the campaigns. The sporulated anaerobes remain few: 4 to 7.10^1 and are not found in the countryside. Spore-forming aerobes are rarer. We observe a maximum in April of the order of 10^1 bacteria / ml
Spore-forming anaerobic bacteria are rare in water. Their number is around 6 to 10 in the tanks. Aerobic spore-forming bacteria increased in April from 10 bacteria / ml to 1.10^6 bacteria / ml
The first months of the industrial campaign made it possible to count 1 to 2.10^2 bacteria / ml. This number of bacteria evolves by regressing the following months until being canceled at the end of the campaign
Bacteria are generally in small quantities except at periods of a kind of contamination (March 1989 for example): 10^1 to 2.10^3 bacteria / ml. At the end of the campaign, we do not detect any more.
Their population is generally non-existent except in April: 10.10 bacteria / ml. At this same time their number is, in the steel tank, 2.10^2 bacteria / ml
Depending on the geographical location and the type of water, we note quite large differences between the diluting waters of the distillery raw materials.
The groundwater of Grande-Terre and Marie-Galante are in contact with seawater at certain times of the year. This probably explains the high chloride, sodium and sulphate contents of these waters. Well water from Marie-Galante has larger amounts of sodium, calcium, magnesium, chloride and sulphate than well water from Grande-Terre. It contains in addition nitrate.
Table V: Variation in Mineral Content in Well Waters (There was no well on the lowlands.
Table VI: Variation of the bacterial flora of well water.
Surface water is less mineralized than well water. The reserve water of Grande-Terre is equivalent to the mineral of Basse-Terre; it is in fact from this that the irrigation water of Grande-Terre is conveyed.
The bacterial flora of Basse-Terre, Grande-Terre and Marie-Galante surface waters are similar; however, we note that Grande-Terre’s irrigation water is a little richer in anaerobic bacteria and sulphate-reducing bacteria. The river water of Marie-Galante sees its flora vary quantitatively when it is stored. Masonry ponds seem to favor an increase in bacterial flora especially if they are poorly maintained and deep.
The well waters of Marie-Galante are more contaminated than the well water of Grande-terre; only the population of sporulated bacteria appears more numerous in the water of Grande-Terre. All these results show that there is a certain relationship between the mineralization of manufacturing waters and their bacterial population. Well waters that contain high concentrations of minerals are more contaminated by bacteria than surface water. It is difficult to say that this or that ion is responsible for the variation of the population of this or that bacterial group for the moment. We have simply found, for example, that in the brackish waters of the groundwater there are groups of BSRs, one of which has a halophilic tendency and that in some waters the decrease in time of concentration of sulphate ions corresponds to an increase in the number of SSBs.
ALEXANDER. M., 1982. Most probable number method for microbial population. Methods of soil analysis part 2. Chemical and Microgiological poperties. Agronomy monograph n’ 9 (2nd édition), p. 815-820
ANON, 1985. Standards methods for the examination of water and wastewater 16th edu. Washington D.C. American Public Health Association.