TRATAMIENTOS QUÍMICOS APLICADOS A LAS FIBRAS

3.9.1 Overview

The previous section presented additional groundwater quality monitoring data and analysis of geochemical processes, and argued that SO4 reduction was a key biogeochemical process controlling the transport and reactivity of SO4 in HHF groundwaters. At the typical groundwater temperatures of the HHF (15 to 20 0C), SO4 reduction can only occur through the action of specialised sulfate reducing bacteria (SRB), as discussed in Section 3.6.2. The presence of SRB has not been investigated at Loy Yang or in the Latrobe Valley previously. It was necessary, therefore, to obtain samples from the HHF to test for SRB.

The sampling and identification of bacteria or microorganisms within groundwater is a complex and challenging task (Chapelle, 1993; Bedient et al., 1994). The organisms may be free-floating within groundwater, or they may be preferrentially attached to colloidal particles or aquifer sediments (Chapelle, 1993). Further complications arise with SRB due to their general intolerance of oxygen (Postgate, 1984). Ideally, it would be preferrable to obtain both samples of groundwater and aquifer sediments for analysis. At the time of preparing this work in May 1997, no new groundwater bores were being planned from which sediment samples could be obtained. A series of groundwater samples were subsequently obtained for further analysis. The primary objective was to detect SRB in HHF groundwaters, and not a systematic study of microbial ecology. Thus samples of groundwater were considered sufficient for this objective.

3.9.2 Methodology

In July 1997, a series of groundwater samples were obtained by gently and carefully using a bailer. The bores 2105U, 2124U, 3135U, the RWP access shaft and the surface outflow pipe from the RWP were sampled on July 18, and bores 2175U, 2176U, 2178, 3282L and 3282U on July 24, 1997. The samples were stored in sterilised bottles within a chilled esky. They were tested according to APHA (1992) methods for culturing and identification of SRB by EML Consulting Services Pty Ltd. The following methodology and results are based on EML (1997a & 1997b).

The samples were analysed by membrane filtration through sterile 0.45 µm pore-size membranes and incubating on specialised SRB-prepared agar plates for up to 21 days. The bores 2105U and 3135U had higher turbidity and only small volumes of less than 1 mL were filtered. Anaerobic conditions were used in a controlled environment at 30 0C with hydrogen-based atmosphere. The cultures were examined on an adjacent open bench for no more than 10-15 minutes. The specialised SO4-reducing agar medium used was prepared according to the required standards in APHA (1992).

3.9.3 Results of SRB Analysis

There was considerable growth during incubation, although there did not appear to be any change observed to the bacterial colonies after about 6 days. The majority of samples produced discrete olive or khaki coloured bacterial colonies, with a typical example shown in Figure 3.14. Some agar plates appeared to be too moist and the bacterial colonies became overgrown and ran into each other (see Figure 3.14). The samples from both the RWP access shaft and bore 2124U, however, were an exception and produced a distinct dark brown colony, suggesting some pyrite formation (black) and thus SO4 reduction. The colonies produced by bacteria from the RWP sample are shown in Figure 3.15. There was no detection in any sample of Desulfovibrio sp. colony forming units (CFU) per 100 mL. Further work was undertaken by EML to attempt to identify the bacteria that were being cultured from the groundwater samples.

Figure 3.14 - Bacterial colonies from bore 2173U after incubation (10 mL and 1 mL), typical of most colonies cultured from groundwater samples (EML, 1997b)

Figure 3.15 - Bacterial colonies from the RWP after incubation (50 mL), similar to the colonies cultured from bore 2124U (EML, 1997a)

The cultured samples from the RWP and bore 2124U were restreaked and recultured on a new agar plate. This failed to produce further dark colonies, instead producing the olive / khaki coloured colonies typical of most groundwater samples tested (see Figure 3.14). Bacterial smears were then prepared from bores 2176U, 2178 and 3282L, heat- fixed, stained by the Grams method and examined under the microscope. Due to the extended age of the samples, the bacteria were staining Gram-negatively and sporulation was occurring. The microscopic examination revealed that some of the bacterial isolates appeared to be SO4-reducing Clostridia sp. or possibly Desulfotomaculum sp., a more commonly known SO4-reducing bacteria. The Clostridia sp. examined under the microscope are shown in Figures 3.16 and 3.17. No samples exhibited the microscopic morphology typical of Desulfovibrio sp.

3.9.4 Summary of SRB Analysis

The sampling and analysis of HHF groundwaters for SRB has proven to be a challenging task. The samples from bore 2124U and the RWP suggested some degree of SO4 reduction and hence activity of SRB, while all remaining samples produced olive / khaki coloured bacterial colonies and no direct indication of SRB. All samples appear to have active bacterial populations but no indication of typical Desulfovibrio sp. Further investigation by EML suggested that the cultured bacteria may be either Clostridia or Desulfotomaculum sp. Given the indications of darkening in the cultures from 2124U and the RWP, and the possible presence of Desulfotomaculum sp. it would therefore appear reasonable to assume that SRB are active within HHF groundwaters. Further research is required to more accurately determine the bacteria present, preferrably both from groundwater and aquifer sediment samples, as well as determining bacterial population dynamics.

Figure 3.16 - Clostridial spores, separated from vegetative cells. Visualised under oil immersion (x1,000) (EML, 1997b)

Figure 3.17 - Clostridial cells, rod-shaped, containing terminal endospores. Old cultures now staining Gram-positively. Visualised under oil immersion (x1,000) (EML, 1997b)