## 2 Materials and Methods  
### Isolation, Identification, and Biodefoamer Production  
Four pure strains, namely, _Bacillus subtilis_ (GCA_000009045.1), _Aerononas veronii_ (GCA_000204115.1), _Klebsiella grimontii_ (UGJQ01000001.1), and _Comomonas testosteronei_ (GCA_900461225.1), were isolated as previously reported elsewhere [21], to constitute a consortium for biodefoamer production. These isolates were obtained from the spout of a commercial poultry slaughterhouse (Cape Town, South Africa). Their identification was made by initially extracting the DNA using the Quick-DNA Fungal/Bacterial Miniprep kit (Zymo Research, Irvine, CA, USA, Catalogue No. D6005), following the kit manufacturer's instructions without modification. Subsequently, the purified DNA fragments were analyzed using an Applied Biosystems(tm 3500 xL Genetic Analyzer (Applied Biosystems, Waltham, MA, USA) for each strain with the instrument producing ab1 files, which were used to identify the individual strains using Basic Local Alignment Search Tool (BLAST) search provided by the National Centre for Biotechnology Information (NCBI, [[http://www.ncbi.nlm.nih.gov](http://www.ncbi.nlm.nih.gov)]([http://www.ncbi.nlm.nih.gov](http://www.ncbi.nlm.nih.gov)) (accessed on 4 December 2022)). To ascertain the efficacy of the isolates' ability to produce biodefoamers individually, a biodefoamer production protocol, as listed in the subsequent paragraph, was followed. Biofoam collapse (decay) and destabilization rates were used to quantify the efficacy of the biodefoamer produced by the individual isolates [21]. The foam generated was generated using an apparatus described in [PERSON] et al. [21], with the formed biofoam being visualized.  
To produce the biodefoamer, a seed culture was first developed by inoculating a volume (50 mL) of sterile nutrient broth in conical flasks (250 mL) with a loop full of the individual pure strains grown overnight (24 h, 37 \({}^{\circ}\)C) in agar plates. The inoculated nutrient broth was put into an orbital shaker (120 rpm, Labwit ZVYR-240 shaking incubator, Labwit Scientific, Burwood East, VIC, Australia) for a further 24 h at 37 \({}^{\circ}\)C. To assess the seed culture's viability, a volume (1 mL) of the consortium was plated for 24 h at 37 \({}^{\circ}\)C. Subsequently, the seed culture (1 mL), i.e., after 48 h of incubation at 37 \({}^{\circ}\)C in an orbital shaker, was inoculated in 99 mL of nutrient broth in conical flasks to constitute a 100 mL fermentation broth. The fermentation broth (1.5 mL) was sampled from each flask used daily for 120 h and centrifuged (Hermle-Z233M-2 centrifuge, Labortechnik GmbH, Wasserburg, Germany) at 15,000 rpm for 30 min to recover a biomass-free supernatant. Supernatant samples (from \(n=3\) flasks) were pooled such that a sufficient volume of the crude biodefoamer was obtained. To characterize the crude biodefoamer produced, cold (4 \({}^{\circ}\)C) ethanol was mixed with cell-free biodefoamers using a 2:1 ratio (cold ethanol:supernatant) followed by shaking at 4 \({}^{\circ}\)C overnight to produce a precipitate, which was collected by initially centrifuging the mixture (15,000 rpm, 30 min) subsequent to pooling the collected precipitates and washing using sterile distilled water. After that, the washed precipitate was vacuum-dried in a desicctor for 24 h, with Fourier transform infrared (FTIR) (Spectrum Two, PerkinElmer, Waltham MA, USA) and \({}^{1}\)H nuclear magnetic resonance (NMR, Bruker 400, Bruker Nano GmbH, Berlin, Germany) spectroscopy analyses being conducted [21].