Table 3.

Summary of factors contributing to E. coli blooms and methods to mitigate such blooms.

Factor contributing to E. coli blooms	Recommended methods to mitigate E. coli blooms
Prolonged storage of FSI at −80 °C	Obtain ethical approval for collecting faecal samples from volunteers on multiple occasions during the course of the project/study.
	Perform all micro-Matrix experiments within 8 weeks of preparing aliquots of FSI if possible and practical.
	Perform Quality Control (QC) checks at regular intervals to assess microbial biodiversity in FSI aliquots.
Prolonged thawing of FSI	Thaw for minimum amount of time as possible (10–15 min) in anaerobic chamber.
Presence of high concentrations of simple low molecular weight sugars/free amino acids following INFOGEST	Perform dialysis for 24–30 hrs with regular replacement of water, using 500 Da dialysis membranes post-INFOGEST.
	After dialysis, perform lyophilisation to negate dilution of digested substrates post-INFOGEST.
	After lyophilisation, reconstitute dialysed digested substrates in original starting volume with water to negate any dilution.
High starting DO concentrations in substrates/FFM mix	Pre-equilibrate FFM, water, reagents, universal sterilins, falcon tubes, pipette tips and all other plastic-ware overnight under anaerobic conditions before commencing experiments.
High starting levels of E. coli in FSI	Perform QC checks from T0 samples and/or sequence aliquots of FSI at regular intervals to assess microbial biodiversity and E. coli relative abundance.
	Recruit volunteers who are in good health and have not taken antibiotics, proton pump inhibitors and/or other medication possibly leading to loss of microbial diversity/loss of strict anaerobes/dysbiosis, as a confounding factor.
	Perform regular QC checks by using FSI aliquots from other projects within the department (within the remit of ethical approval documentation) for sequencing QC checks and detection of E. coli blooms.
pH either too high or too low	Maintain pH equilibrium within the 6.2–7.2 range by using 20 % v/v liquid ammonia and CO2 to increase and decrease pH respectively, throughout the experiment.
Preparation of FFM	Prepare FFM as close as possible to the day of the micro-Matrix experiment.
	Store at 4 °C for a maximum of 7 days prior to commencing the micro-Matrix experiment.