Abstract/Summary

Iron and zinc deficiency present global issues affecting populations in both developed and developing countries. Efforts have been made to tackle these problems either through diet intervention, fortification or supplementation. Although these strategies had been beneficial to host nutrition, the impact of the unabsorbed nutrient on the gut microbiota has yet to be fully understood and this was investigated here using various in vitro approaches. The impact of iron on the gut microbiota was initially considered using Hungate tube and single vessel batch cultures approaches inoculated with human faecal slurries. Growth was monitored by measuring total bacterial number using Flow-FISH and the microbiota composition was analysed by Next Generation Sequencing (NGS). The total bacterial numbers and composition were similar between the control and iron regime in the Hungate tube system. This was considered to be due to the limited growth exhibited and the unregulated pH of the Hungate tube culture would obscure any influence of iron, although the presence of iron (haem/FeSO4) caused a substantial increase in Roseburia faecis. Inclusion of strong buffers in Hungate tubes was explored and 300 mM 2-(N-morpholino)ethanesulfonic acid was found to restrict pH fluctuation within a desirable range (pH range 5.5-6.7) and the bacterial counts were higher in the presence of buffer. Single vessel batch cultures containing gut model medium and 0.1% (w/v) faecal slurry from four different healthy donors were set up to investigate the impact of different forms of iron (haem and FeSO4) on the gut microbiota. The presence of haem caused a slower growth rate but all regimes showed similar total bacterial counts by 48 h. Haem increased the growth of Bacteroides, Parabacteroides, Clostridium, Lactobacillus and Rikenellaceae. Dorea formicigenerans, Ruminococcaceae and Veillonella dispar showed an increase in the presence of FeSO4 but not haem. Sutterella, Enterobacteriaceae, Bifidobacterium, Ruminococcus and Faecalibacterium prausnitzii showed a decrease in growth in the presence of haem with FeSO4. The effect of zinc on the gut microbiota was investigated using single vessel batch cultures and three-stage gut models containing ‘modified’ gut model medium supplemented with Zn at 77 (low), 192 (medium) and 770 µM (high) concentration. In the batch cultures, the presence of 77 or 192 µM zinc increased total bacterial counts while 770 µM zinc caused a lower growth. The presence of zinc increased the growth of Streptococcus and Sutterella but reduced the growth of Odoribacteria, Rikenellaceae, Roseburia faecis and Enterobacteriaceae. In the gut model, Faecalibacterium prausnitzii and Roseburia faecis showed a significant increase (p<0.05) in the presence of 770 µM Zn while Lachnospira displayed a significant decrease (p<0.05) in both the absence of zinc and presence of 77 µM Zn, indicating it favours a moderate zinc level. The ability of bacteria to degrade phytic acid, a major dietary inhibitor of zinc absorption, and use it as a phosphate and/or carbon source was investigated using an Escherichia coli triple mutant whereby all three known phytases were knocked out. The wild-type and mutant were grown in phosphate limited or carbon free M9 minimum medium, supplemented with 2.5, 5 and 10 mM phytic acid. Phytic acid was found to act as a good phosphate source but a poor carbon source for the growth of E. coli. However, the mutant showed comparable growth to the wild-type, indicating the phytases previously identified have no essential role in utilisation of phytate, suggesting a yet to be discovered phytate utilisation pathway. The impact of phytic acid on the gut microbiota was also investigated using single vessel batch cultures, containing phosphate-limited or carbon-free basal medium. Similar results were obtained whereby the gut microbiota were able to utilise phytic acid as a phosphate source but not a carbon source.