*3.3. Test Solutions*

Over the 8-week duration of the corrosion tests, the colours of the test solutions changed (see Figure 6), which is a potential indication of the metabolic and corrosion processes that took place. Of particular interest was the general blackening of solutions for tests T2 (orange tubercle inoculum), T5 (four isolates + *D. desulfuricans* aerobic) and T6 (*D. desulfuricans* anaerobic), and the black precipitation layer observed at the bottom of test condition T3 (*D. desulfuricans* aerobic). This blackening might be an indication of sulfate reduction and FeS production.

**Figure 6.** Changes in appearance of test bottles of six treatments during 8 weeks of immersion.

In general, the pH values of the solutions (Table 2) were reasonably constant for all of the test conditions over the 8-week incubation period, with pH values staying within ±0.5 of the initial value of 7.54. The greatest changes observed were for T4, which went slightly more basic to 7.9, and T6 which went slightly more acidic to just below 7.0.


**Table 2.** Bacterial counts and pH during 8-week incubation test of six treatments (Table 1). Initial pH of sterile media before inoculation was ~7.54. Total plate count in each treatment was the sum of both aerobic and anaerobic counts on marine agar (MA) and tryptic soy agar (TSA+) media, respectively.

#### *3.4. Analysis of Microbial Populations*

Total plate count results are provided in Table 2. While a reduction in counts for the *D. desulfuricans* inoculum tests (T3 and T6) was seen between the start and the end of the tests, in general the microbial populations were reasonably constant throughout incubation times. It is also worth noting that no colonies were observed in the aerobic plate tests for *D. desulfuricans* alone treatments (T3 and T6), suggesting that these solutions were free of any aerobic microbial contamination.

Figure 7 summarises the metabarcoding analysis of the mixed microbial consortium treatments (i.e., T2, T4 and T5) at week 2 (planktonic) and week 8 (planktonic and biofilm). The solution inoculated with the orange tubercle (T2) has, as expected, the most diverse community. While there were few bacterial taxa that reduced in relative abundance in the T2 solution during the test (e.g., *Exiguobacterium* spp. dropped from 13% to 3% in planktonic phase from 2 to 8 weeks), many bacteria maintained their presence in both planktonic and biofilm phases (e.g., Clostridiales and Rhizobiales). However, several bacteria were only detected after 8 weeks including *Desulfarculus baarsii*, *Desulfosporosinus* spp. and *Thioalkalivibrio* sp. Reasonable differences can also be seen between the planktonic and biofilm phases of the T2 solution at 8 weeks (e.g., Rhodobacteraceae spp. and *Clostridium thiosulfatireducens* more abundant in the biofilm phase).

The metabarcoding results for the combinations of isolates (T4 and T5) identified all of the bacteria inoculated in each test configuration in at least one of the time/phases tested. For both of these test configurations the proportion of *P. bellariivorans* tended to decrease from week 2 to week 8, and *H. korlensis* was only ever found in any reasonable level in the biofilm phase. A key difference between the two test configurations was for *B. aquimaris*, which had a strong presence in the T4 liquid (four isolates) but was only detected at low levels in the T5 liquid (four isolates + *D. desulfuricans*). *S. pontiacus* was detected at reasonably consistent levels for both test solution types throughout the tests and in both the planktonic and biofilm phases.

The metabarcoding data were further analysed for putative functions using METAGE-Nassist to interpret microbial metabolism and other traits, the results of which are presented in Table S2. Key aspects of the sulfur cycle, including sulfate reduction (detected at high levels in all samples at week 8) and sulfur oxidation (for the orange tubercle configuration at week 8) were detected.

**Figure 7.** Microbial communities identified by 16S rRNA gene metabarcoding of mixed consortium treatments (T2, T4 and T5), for planktonic communities (at weeks 2 and 8) and for the attached biofilms obtained by sonication (at week 8).
