3.1. Screening of WCO and PCO-Degrading Antarctic Bacterial Consortia
The bacterial consortia obtained by culturing from all 28 soil samples were tested for their ability to degrade WCO and PCO over a 5 days period. Most of the consortia were able to degrade >10% of the initial amount of WCO or PCO. The highest degradation of WCO was achieved by the consortium from sample BS23 (44.83%), followed by BS12, BS11 and BS14 (
Figure 1a). The highest degradation of PCO was achieved by the consortium from sample BS16 (51.24%), followed by BS14 and BS3 (
Figure 1b). Although BS23 achieved the greatest degradation of WCO, its ability to degrade PCO was lower than BS14. The consortium from sample BS14 (isolated from location 62°46′31″ S 81°18′48″ E), achieving degradation of 36.73% and 50.59% of WCO and PCO, respectively, was therefore selected for further optimisation using OFAT and RSM. Each consortium tested is likely to have different bacterial diversity and therefore different degradation capabilities. The consortium obtained from sample BS14 performed well compared to those from most other samples, clearly being able to produce the main enzymes involved in the process of breaking down oil and lipid [
44,
45].
The consortium from sample BS14 showed both high bacterial growth and a high degradation rate. Generally, the rate of degradation of contaminants is high when the bacterial growth of the samples increases [
46,
47]; nevertheless, some of the consortia examined here achieved oil degradation even with low bacterial growth rates (
Figure 1).
Normally, the group of identified Antarctic bacteria in biodegraded hydrocarbon comes from the same group of bacteria. Since cooking oil is classified as a hydrocarbon, the probability of Antarctic bacteria species occurring in this study is likely to be similar to the bacteria that have been identified as revealed in the past few decades.
Pseudomonas and
Rhodococcus genus are the best known bacteria with the capability of breaking down hydrocarbon; for instance,
Pseudomonas sp. strain ST41 isolated from Signy Island,
Pseudomonas sp. strain Ant 9 (Ross Island),
Pseudomonas sp. strain LCY 16 (King George Island),
Rhodococcus sp. strain AQ5-07 (King George Island),
Rhodococcus sp. strain DM1-21 (Marambio Island),
Rhodococcus sp. strain JG-3 (Ross Island) and
Rhodococcus sp. (South Shetland Island) (South Shetland Island) [
22,
27,
29,
48,
49,
50,
51,
52]. These Actinobacteria have been widely reported in most metagenomics studies, specifically in Antarctic Peninsula areas, including in the Korean Antarctic Research Station and Alexander Island [
53,
54,
55]. There are also other bacteria (less common) that have been listed as hydrocarbon-degrading bacterial species isolated from Antarctica and summarised by Wong et al. (2021):
Arthrobacter spp. strain AQ5-05 (King George Island),
Sphingomonas sp. strain Ant 17,
Sphingobium xenophagum strain D43FB (King George Island),
Planococcus sp. strain NJ41 and
Shewanella sp. strain NJ49 (Antarctic Ocean) [
56].
The degradation activity was observed to be different between WCO and PCO media for the same bacteria consortium, which might be caused by the different compositions of the substrates. The common fatty acid composition of canola oil (PCO) comprises 6% saturated fatty acid (palmitic and stearic) and 92% unsaturated fatty acid (oleic, linoleic and linolenic) [
2]. On the other hand, an increasing number of saturated fatty acids in waste cooking oil compared to the unused oil has been reported by Knothe and Steidley [
57], where the composition of steric and oleic compound increases along with the decreasing number of linoleic and linolenic fatty acids in waste oil. Lots of volatile organic compounds have also been found in the waste oil, which include acetaldehyde, methylamine, N,N,-dimethyl-octane, pyrazine, formamide, N,N-dimethylacetamide and furfural [
58]. These compounds might have been produced by chemical changes that occurred in waste cooking oil during the thermal heating processes (cooking), making the PCO more complex compared to the WCO.
3.2. Optimisation of Consortium BS14 Growth and Canola Oil Degradation Using One-Factor-at-a-Time
Overall, the optimum conditions identified using OFAT for WCO degradation were 0% (w/v) NaCl, pH 7.5, 1 g/L ammonium sulphate, 10 °C, 1.25 g/L yeast extract and 0.5–1% (v/v) initial substrate concentration. For PCO degradation, only the optimum nitrogen source concentration differed from that for WCO degradation, at 0.5 g/L (NH4)2SO4.
Sodium chloride (NaCl) was used to determine the influence of salt concentration on bacterial growth and degradation of WCO and PCO (
Figure 2a and (
Figure 3a). Both bacterial growth and oil degradation rates rapidly decreased as the concentration of NaCl increased above 0.25% (
w/
v). Degradation of both WCO and PCO was greatest at 0% NaCl, achieving 44.88% and 78.66%, respectively. Higher bacterial growth was also observed at 0% and 0.25% salt concentrations. These data are consistent with previous studies on the biodegradation of contaminants by Antarctic soil bacteria, which also documented greater growth and degradation at low salt concentrations. For instance, Zakaria et al. (2019) showed that an Antarctic isolate of
Rhodococcus boikonurensis was able to degrade an initial concentration of 0.2 g/L of phenol with up to 100% degradation within 48 h at a salt concentration of 0.01% (
w/
v) of NaCl [
59]. Similarly,
Arthrobacter sp. strain AQ5-05 showed the greatest hydrocarbon degradation rate at low salt concentration (0 to 3%
w/
v) through statistical optimisation [
60]. Salt concentrations in soils of the sampling area, despite its coastal location, can be categorised as low (0–0.3%) [
61]. Impacts of high salt concentrations include osmotic effects and reduced enzyme activity, soil microbial biomass and bacterial growth rate [
62,
63].
Bacteria are generally sensitive to pH variation in their environment [
64]. The data obtained here (
Figure 2b and (
Figure 3b) showed bacterial consortium growth and the degradation of both WCO and PCO were optimum at close to neutral pH and strongly inhibited at lower and higher pH. Both growth rate and WCO and PCO were most strongly inhibited by acidic conditions. At the optimum pH the degradation achieved was 46.06% for WCO and 81.67% for PCO. Ibrahim et al. (2020) reported that the Antarctic
Rhodococcus sp. strain AQ5-07 was most effective at degrading WCO at pH 7.5 [
22]. The same bacterial strain could also effectively degrade diesel at pH 7 [
29].
Nitrogen is a major element supporting function in microbial cells, being a key constituent of amino acids, nucleotides and all proteins [
65]. Inorganic nitrogen sources were trialled in this study, since the carbon source was solely provided from the WCO or PCO substrate. Use of (NH
4)
2SO
4 led to high degradation of both WCO and PCO, while use of NH
4Cl, NH
4NO
3 and (NH
4)
2HPO
4 also led to high degradation (>50%) of PCO (
Figure 2c and
Figure 3c). Bacterial growth was also high when supplied with (NH
4)
2SO
4. NH
4Cl stimulated the greatest bacterial growth, but had less influence on degradation rate compared to (NH
4)
2SO
4. It is possible that the different nitrogen sources may have stronger influences on cellular products and pathways other than lipolytic enzymes [
66]. Studies of Antarctic bacteria have previously reported that (NH
4)
2SO
4 is the most effective nitrogen source. For example, the cold-adapted Antarctic soil bacteria,
Arthrobacter sp. strain AQ5-05,
Arthrobacter sp. strain AQ5-06 and
Rhodococcus sp. strain AQ5-07, exhibited maximum degradation of phenol when provided with ammonium sulphate [
67].
Arthrobacter sp. strain AQ5-15 also achieved greater degradation of phenol when using ammonium sulphate as nitrogen source [
68]. Ammonium sulphate has the practical advantages of being low-cost, readily available and suitable for application on a large scale compared to other nitrogen sources [
69,
70] Ammonium bisulphate or ammonium sulphate have also been suggested to be the dominant forms present in Antarctic atmospheric studies, especially in coastal regions with high concentrations of marine vertebrates [
71,
72,
73].
Bacterial growth and oil degradation rates were high at concentrations of (NH
4)
2SO
4 of up to 1 g/L for WCO and 0.5 g/L for PCO (
Figure 2d and
Figure 3d). Maximum degradation was achieved at the lowest concentration trialled, decreasing by around 10% as the concentration was progressively increased. Previous studies have similarly reported that only low concentrations of ammonium sulphate were required to support bacterial growth and substrate degradation.
Arthrobacter sp. strains AQ5-05 AQ5-06 were able to degrade more than 40% of diesel when provided with 0.4 g/L ammonium sulphate [
30]. As shown in
Figure 2d (WCO medium), the BS14 bacterial consortium required a higher concentration of nitrogen source for optimum growth and degradation compared to that required in PCO medium (
Figure 3d). This might be due to the production of by-products from thermal degradation of the oil (from heat during cooking), including volatile and toxic compounds [
4]. Swiecilo and Zych-Wezyk (2013) stated that unfavourable environmental conditions lead to the activation of stress response mechanisms [
74]. Large energy expenditure is required for bacteria to activate the adaptive mechanisms to synthesise defence molecules. This may explain why the BS14 consortium in WCO medium required a greater N concentration.
Oil degradation and bacterial growth were tested at temperatures between 5 °C and 25 °C. Generally, degradation of WCO and PCO declined as temperature increased (
Figure 2e and
Figure 3e). Degradation of WCO was significantly greater at 10 °C than 5 °C and 15 °C, but there was no significant difference in the degradation of PCO between 10 °C and 15 °C. The temperature obtained during soil sample collection ranged from −0.1 °C to 8.5 °C using a thermocouple thermometer. However, the result showed that the bacteria consortia preferred the high temperature to the actual conditions in the Antarctic, which was at 10 °C. Numerous studies have also reported optimum activity of cold-adapted bacteria around 10–15 °C [
22,
30,
68,
75,
76]. While the degradation rate was lowest at 25 °C, bacterial growth was high (
Figure 2e and
Figure 3e). This may indicate that the subset of bacteria capable of surviving at this high temperature did not have the ability to degrade canola oil. Although the optimum temperature in degradation of WCO and PCO is relatively high from the actual value, the conditions of the Antarctic today also have an impact future application. According to the World Meteorological Organisation (2020), the new record temperature in the Northern Antarctic Peninsular was 18.4 °C during February 2020 due to global warming effects [
77]. Therefore, the highest activity obtained at 10 °C is acceptable for the application of this study in the Antarctic.
High concentrations of yeast extract increased both bacterial growth and WCO and PCO degradation (
Figure 2f and
Figure 3f). Yeast extract acts as a primary growth substrate for the bacteria to co-oxidise the oil [
78]. Previous studies have shown that the addition of yeast extract promoted the biodegradation of oil hydrocarbons and the growth of bacterial consortia [
79]. Yeast extract provides both macro- and micro-nutrients (such as metal ions) as well as vitamins and amino acids which are essential for bacterial growth and metabolism [
80,
81]. About 0.014 g/L yeast extract led to maximum diesel hydrocarbon degradation by
Acinetobacter beijerinckii strain ZRS [
80], and
Pseudomonas sp. sp48 required 5 g/L yeast extract to achieve maximum biodegradation of crude oil [
82]. The addition of yeast extract led to a more significant effect on the degradation of WCO compared to PCO. As noted above, the chemical changes accumulating in WCO as a result of repeated heating will include various chemical compounds from oxidation, hydrolysis and peroxidation processes, including acrylamide, fatty acids and aldehydes [
4,
83,
84].
High initial concentration of either WCO or PCO inhibited the bacterial consortium’s degradation ability, although bacterial growth was maintained (
Figure 2g and
Figure 3g). There was no significant difference in degradation at 0.5% and 1% initial concentration of either substrate. Only a single study has reported biodegradation of WCO using a culture of the Antarctic soil bacterium
Rhodococcus sp. strain AQ5-07 [
22]. The methodology differed from the current study, in particular using a much larger bacterial inoculum, which restricts comparison between the two studies. However, that study achieved greater degradation at an initial concentration of 3% WCO over 3 days, compared with around 40% over 7 days in the current study. The inhibition of degradation associated with high concentrations of substrate may indicate suppression of key enzymes [
85]. This is known to be the case especially in the biodegradation of hydrocarbons that are composed of aromatic elements [
86]. The ability of the bacteria to maintain growth when a high initial oil concentration is provided might result from the presence of yeast extract, providing the nutrient source required to fuel growth (rather than oil degradation).