lakes was at least partly due to the combined action of two groups of micro- organisms- 

 (1) sulphate - reducing bacteria, which reduced the sulphate in the lake water to sul- 

 phide, and (2) photosynthetic sulphide- oxidising bacteria which oxidised the sulphide 

 produced in (1) to elemental sulphur. 



Experiments with pure cultures. Experiments with crude cultures cannot be ac- 

 cepted as proof that specific organisms are responsible for what occurs. For final 

 confirmation of the hypothesis, experiments were carried out with pure cultures of sul- 

 phate reducers and of Chromatium and Chlorobium. All cultures originated from the 

 lakes. 



Mixed pure cultures of D. desulphuricans + Chromatium and of D. de sulphuric ans 

 + Chlorobium were prepared in various media, based on combinations of the media 

 used for the separate growth of the organisms and the composition of the lake water. 

 The cultures were incubated anaerobically at 32°C in an illuminated cabinet. No 

 source of sulphur other than sulphate was used. Growth of both pairs of bacteria oc- 

 curred in nearly all cultures. Those containing Chlorobium and sulphate reducers de- 

 posited a yellow layer of sulphur (Fig. 2). No such layer appeared in the Chromatium 

 cultures, but under the microscope the Chromatium cells were seen to be almost com- 

 pletely filled with sulphur globules. The best yield of sulphur (judged by inspection) 

 was obtained from D. desulphuricans + Chlorobium grown in the medium in Table 3. 



These experiments show that elemental sulphur can be produced from sulphate by 

 the combined action of pure cultures of sulphate reducers and the photosynthetic green 

 and red sulphide oxidisers in a common medium. They provide evidence that some of 

 the sulphur in the Cyrenaican lakes was produced by a similar combination of sulphate 

 reducers and Chlorobium. 



Reducing agent for sulphate reduction. The reduction of sulphate by D. desul- 

 phuricans requires a reducing agent, either hydrogen or an organic compound such as 

 lactic acid. The source of reducing agent for sulphate reduction in Ain-ez-Zauia was 

 not clear since the organic content of the water was low (see Table 1), though the pos- 

 sibility exists that the continuous supply of this organic material by the spring was 

 used for reduction. It was also possible that the coloured sulphide -oxidising bac- 

 teria, which can satisfy their carbon requirements by photosynthesis from COj, pro- 

 vided suitable organic matter for the sulphate reducers. To test this, mixed pure cul- 

 tures of D. desulphuricans + Chromatium and of D. desulphuricans + Chlorobium were 

 prepared with no carbon source other than NaHCOs, and incubated in light at 30°C. 

 In order to avoid false results due to carry-over of organic material in the inocula, 

 the mixed populations were sub- cultured at least three times. At each stage they 

 were inspected for sulphate reducers microscopically. In both cases the coloured sul- 

 phide oxidisers grew readily, and at each stage sulphate reducers were detected mi- 

 croscopically. The sulphate reducers were more plentiful in symbiosis with Chroma- 

 tium, and if thiosulphate was used in place of sulphide as a sulphur source their 

 presence could be detected chemically by blackening (FeS) after addition of a ferrous 

 salt. 



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