Investigations Concerning Bacterial Life in Strong Brines

In recent years the biocoenonis of salt lakes has been particularly studied by Prof. Baas Becking and collaborators. In these investigations little attention has been paid to the bacterial flora. Subject of this study therefore was to investigate which bacterial groups may be present and physiologically active in natural brines. As it was impossible for the author to carry out such an investigation on the spot, a number of samples of salt mud and crude salt were submitted to a bacteriological examination. Bacterium trapanicum Fetter and two Bacillus species were isolated from a sample of mud from the salt-garden of Grissee (Java) by plating on salt containing media. Bacterium trapanicum produces a red pigment and is obligate halophilic. The two Bacillus species which developed on common peptone agar (containing 0,5 % salt) could not further be classified. A colourless, motile, obligate halophilic species, the characteristics of which were not further studied, was isolated from a sample of mud from Pamekasan (Madura). From a sample of crude salt from Searles Lake (California), which was about three years old no bacteria could be isolated. Enrichment cultures with the above mentioned samples as inoculum for halophilic representatives of lactic acid bacteria, bacteria belonging to the colon group, bacteria causing methane fermentation of fatty acids, urea bacteria and denitrifying bacteria were unsuccessful. Sulfate reducing bacteria were present. The development of this group could be observed in salt concentrations up to 30%. Colourless sulfur bacteria (thionic acid bacteria) developed also in media containing up to 30% salt. Although a spontaneous development of green sulfur bacteria was observed in a sample of mud from the salt garden of Pamekasan (Madura) special enrichment cultures with various salt samples as inoculum gave no result. Enrichment cultures of bacteria which attack pectin were positive up to the medium with 18% NaCI under aerobic as well as under anaerobic conditions. Although the result of this part of the investigation is rather scanty, chiefly caused by the relatively long time elapsed after collecting of the samples used, it may be concluded that the samples contain both typical halophilic forms and non halophilic forms. It also appears that long (3 — 5 year) storage of salt seems to destroy various bacterial forms originally present. Since in the sample mentioned above the representatives of only few bacterial groups could be detected experiments were carried out to determine the maximal salt concentration which is tolerated by ’’normal” soil bacteria. Bacteria which would develop in these experiments in culture media containing high percentages of salt may certainly be present in natural brines and thrive therein as soon as suitable nutritive conditions are fulfilled. First several pure cultures (present in the collection of the ’’Laboratorium voor Microbiologie” at Delft) were tested on their ability to develop in media containing increasing amounts of salt. It appeared, however, that the maximal concentrations tolerated were rather low. Most of the species tested tolerated 6 % NaCl at most. In a few cases f.i. with Azotobacter chroöcoccum – growth was already inhibited at a concentration of 3%. T aking into account that bacteria as occurring in nature may adapt themselves better to a life under abnormal conditions than pure cultures (Kluyver and Baars (47) ) enrichment cultures were made for various bacterial groups in media with increasing percentages of salt and with garden soil as inoculum. In this way cultures were obtained of lactic acid bacteria in media containing up to 6 % NaCl, bacteria belonging to the colon group up to G% NaCl, butyric acid bacteria up to 24% NaCl, urea bacteria up to 24% NaCl, denitrifying bacteria up to 30% NaCl, Azotobactcr chroöcoccum up to 3 % NaCl, sulfate reducing bacteria up to 6% NaCl, colourless sulfur bacteria up to 12% NaCl, bacteria which attack proteins up to 24% NaCl and bacteria which attack pectin under aerobic conditions up to 18% NaCl. Comparison of the maximal concentrations tolerated by pure cultures with those tolerated in the corresponding enrichment cul- lures shows that in general, the latter are much higher. Pure cultures seem therefore to, possess a smaller potentional environment than the corresponding forms which occur under natural conditions. The possibility of an independent cycle of life in natural brines was discussed on the basis of the results obtained and of those found in the literature. In a salt lake containing brine of about the most important bacterial processes may take place. Since it appeared that many bacteria occurring in garden soil tolerated rather high percentages of salt the question arose whether the bacteria occurring in natural brines might be adaptation forms of ’’normal” soil bacteria. Forms isolated from natural brines as a rule possess a high preference for salt. Many of them do not develop in media with a small amount of salt. If the supposition that they are adaptation forms of ’’normal” bacteria is true, some ’’normal” soil bacteria should be able to adapt themselves in such a way that they lose the property to develop in media without salt. To investigate if it is possible to isolate such adaptation forms from salt free material several bacterial strains were isolated from the enrichment cultures in salt containing media with garden soil as inoculum. The media for isolation and propagation contained the same percentage of salt as the enrichment cultures. The isolated forms were tested on their ability to maintain development on media without salt. The possibility to give adaptation forms was investigated for Bacterium coli. Strains of this form were Isolated from enrichment cultures with well water as inoculum and with o, 3 and 6% NaCl. The three strains, however, did not show any difference as totheir behaviour in salt containing media. So the existence of an adaptation form of Bacterium coli could not be demonstrated. Urea bacteria were isolated from enrichment cultures in media containing z% Ca citrate, z% urea and o, 3 and 6% NaCl and inoculated with garden soil. Urobacterium O and III (the Roman numerals indicate the percentage of salt present in the enrichment culture and in the media for isolation and propagation of the form) developed in media with o, 3 and 6 % NaCl, in a medium with 12% no growth was observed. Urobacterium VI developed in media with o up to 24% NaCl. So the latter form tolerated much higher salt concentrations than the forms O and III. From enrichment cultures in peptone water with 5 % urea and o, 3, 6, 12 and 24% NaCl and with garden soil as inoculum once more urea bacteria were isolated. The forms O, III, VI and XII developed on peptone agar with 1% urea and 0,5% NaCl, but Urobacterium XXIV did not give development on this medium. Consequently the latter form must be considered as a typical adaptation form. Quantitative data of urea decomposed in media with increasing percentages of salt were obtained for Urobacterium VI from the citrate medium and for Urobacillus XII and Urobacterium XXIV from the peptone medium. The diagram of these data (p. 140) shows that each of the strains used represents a special type. Urohcict. VI is a halophilic type, Urobacillus XII is highly halotolerant and Urobacterium XXIV is obligate halophilic. Denitrifying bacteria were isolated from enrichment cultures with o, 3,6, ia, 18, 24 and 30% salt. It appeared that the forms HI, VI, XII and XVIIIa developed on common peptone agar (with o,j NaCl). The forms XVIIIb, XXIV and XXX, however, did not give development on this medium. So the latter are adaptation forms and may be considered to be ’’artefacts” originated from ’’normal” soil bacteria during the enrichment and isolation. No adaptation forms could be isolated from Betacoccus dextranicus. Enrichment cultures of butyric acid bacteria were obtained in media with o, 3, 6 and 24% salt. A pure culture could not be isolated form the medium with 24% salt because sporulation did not occur. Nevertheless it was made probable that in this culture an adaptation form had developed for transfer in a medium without salt gave no development and in a medium with 24% salt again fermentation appeared. From a culture in peptone water with 30% salt inoculated with garden soil an obligate halophilic form was isolated. A number of bacterial strains were isolated from different salt containing agar plates inoculated with salt free material as garden soil, canal water and sewer mud. Only one of the strains isolated appeared to be an adaptation form for it was the only one that did not develop on common peptone agar. Prof. C. B. van Niel was so kind to send ten strains of bacteria isolated from a soil sample from Ames (Iowa) by enrichment cultures in yeast extract with 20% NaCl. It appeared that all strains were halotolcrant for they developed better on peptone agar with 0,5% salt thans on peptone agar with 18% salt. . Prom the results obtained it may be concluded that in fact it is possible to isolate from salt free material forms which have adapted themselves in such a way to a life in a salt environment that they do not develop subsequently in media without salt. There- fore it is very probable that many if not all forms occurring in natural brines are adaptation forms from ’’normal” soil bacteria. Finally some observations were made on the bacterial flora of a few vegetable salt preserves viz. sauerkraut, gherkins, cucumbers and beans. In order to investigate for which purpose salt is added in the preparation of sauerkraut, krauts were prepared with 2,5 % and without salt. The result showed that salt chiefly is added to obtain sufficient juice. An important influence of the addition of salt on the bacterial flora and the acid formation was not observed in the experiment made. In about the same manner as sauerkraut gherkins and cucumbers are preserved. In this case the percentage of salt added is as a rule much higher. Fabian, Bryan and Etchhll (27) mention in their publication on cucumber fermentation that in America cucumbers are fermented in relatively high salt concentrations (8 % salt in the Low salt curing and 10% in the Fligh salt curing). During the fermentation the strength of the brine is still increased. As they do not mention by which kind of lactic acid bacteria the fermentation was caused an effort was made to obtain a spontaneous lactic adid fermentation in a 10% brine. Therefore gherkins were placed in brine of 10% and of 6% NaCl. A lactic acid fermentation, however, appeared only in the brine of 6% NaCl. From this brine a species of the genus Betabacterium Orla Jensen was isolated which was able to develop in a medium with 9 % NaCl. In a medium with 12% NaCl this form gave no development. In our country the amount of salt added in the preservation of cucumbers and gherkins is much lower. A sample of brine of fermented cucumbers from Amsterdam contained 3,6% NaCl. From this sample a species of Betabacterium was isolated. A sample of brine from Roelofarendsveen contained 5,6% NaCl. From this sample a Streptobacterium species was isolated. In both samples yeasts were present. During the preservation of salted beans no lactic acid fermentation appears. The percentage of salt added is as a rule much higher than in the preservation of gherkins and cucumbers. Several samples of salted beans were investigated. The percentage of salt varied from 6—29%. To my surprise in all samples numerous bacterial and yeast cells were present. Occasionally salted beans become purple. A sample of such beans received from Mr. E. M. v. d. Zijl at Warffum (Groningen) was submitted to a furher investigation. It was found that a Pseudomonas species to which the name Pseudomonas Beijerinckii was given is responsible for the formation of the purple colour. This bacterium produces the purple pigment only when it grows in bean extract or extracts of some other vegetables. In peptone water or yeast water no pigment is produced. The same bacterium was found in a number of samples of salted beans, which did not possess a purple colour. It appeared that the pigment was only produced when the bacterium grows under certain conditions. The formation of the pigment is chiefly dependent on the pH of the medium and of the oxygen tension. The purple pigment could not be identified. It is insoluble in water as well as in carbon disulfide as in all organic solvents tested. When shaking with air the purple pigment turns to brown. By addition of hydrosulfite the colour again turns to purple. By addition of acids f.i. diluted acetic acid a yellow solution is obtained. Evaporation of this solution in vacuo at 6o° C. gives a purple amorphous residue. Even in this form the pigment does not dissolve in any solvent. In the brine of salted beans yeasts always occurred. They were identified as Debaryomyces membranaefaciens and Debaryomyces Guilliermondi. On old salted beans we often encountered Torula epizoa a brown mould.