Simultaneous production of biosurfactant and extracellular unspecific

Crude oil plays a significant role in supporting global economic development and human welfare. However, natural geophysical processes and human activities associated with the extraction, transportation, and refining of crude oil have led to progressively severe crude oil spills, causing water and soil pollution, as well as substantial ecological disasters [10]. Crude oil consists of a complex mixture of compounds, including n-alkanes, cycloalkanes, polycyclic aromatic hydrocarbons (PAHs), resins and asphaltenes, many of which exhibit carcinogenicity and teratogenicity [70]. These hazardous pollutants are persistent organic compounds and enter organisms through the food chain, ultimately causing extensive and permanent damage to human health and ecosystems [63], [69]. Currently, a variety of remediation methods are available to tackle crude oil pollution, including physical, chemical, and biological approaches. However, physical and chemical methods have limitations, such as low efficiency, high cost, potential secondary pollution and changing natural ecosystems [12], [70]. In contrast, microorganisms, which play a crucial role in maintaining ecosystem balance and fostering sustainable environment [64], have emerged as promising living cell reactors to provide a cost-effective, efficient, and sustainable approach for crude oil bioremediation [11], [19], [8].

The ability of microorganisms to degrade crude oil primarily relies on their hydrocarbon oxidases, which facilitate biodegradation by introducing oxygen to substrates [65], [70]. Currently, they are predominantly found to be intracellular enzymes, including soluble/particulate methane monooxygenases, AlkB related alkane hydroxylases, eukaryotic cytochrome P450 (subfamily CYP52), bacterial cytochrome P450 (subfamily CYP153) and dioxygenases [1], [65]. One well-characterized crude oil degrading enzyme is cytochrome P450, which catalyzes the oxidation of terminal C-H groups of alkanes to form corresponding alcohols [1], [14]. For instance, Yarrowia lipolytica harbors twelve ALK genes encoding cytochrome P450, enabling it to hydroxylate n-alkanes of varying carbon chain lengths in crude oils [30]. However, the catalytic activity of P450 requires nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) as a reducing agent and flavin reducing protein to facilitate electron transfer [26], [58]. Such complexity inevitably leads to the electron-transport uncoupling, resulting in the wastage of reducing equivalents and reducing the catalytic turnover [26]. In addition, it is noteworthy that long-chain and multiple aromatic ring hydrocarbons are difficult to be transported into cells for degradation due to the intracellular location of P450 [27], [72], thereby limiting the ability to degrade the numerous complex hazardous pollutions in crude oil. In addition to P450s, unspecific peroxygenases (UPOs), a kind of extracellular heme-thiolate peroxidases produced by fungi, exhibits the ability to efficiently catalyze a broad range of selective oxyfunctionalization of non-activated C–H- and CC-bonds as well as C–C-bond cleavage [33]. Structural analysis of UPO from Hypoxylon sp. EC38 revealed that the active site of the UPO is accessible to broad molecules of varying bulkiness [53]. Hence, UPOs exhibit significant potential in the oxidation of a diverse array of compounds present in crude oil. Moreover, unlike P450s, UPOs depend solely on H₂O₂ as an electron acceptor and oxygen donor, making them self-sufficient in catalytic activity [17], [53]. These advantages make the fungi with UPOs have significant potential for crude oil degradation. However, there is relatively limited research focused on this area.

During the remediation of crude oil, the limited water solubility significantly impedes the degradation process [28]. Surfactants have proven to be effective in reducing the interfacial tension of organic pollutants and facilitating crude oil degradation [32], [43]. However, numerous identified crude oil-degrading microorganisms, such as bacteria Geobacillus and Anoxybacillus and fungi Penicillium, Fusarium, Yarrowia and Candida, do not inherently produce biosurfactants [11], [44], [5], [75]. Therefore, in certain instances, exogenous chemical surfactants are supplemented to enhance microbial crude oil degradation [29], [44], [71]. However, these approaches come with additional costs and the potential for secondary pollution. Notably, some microorganisms display the capacity to synergistically degrade crude oil and generate biosurfactants, primarily limited to rhamnolipids-producing bacteria such as Pseudomonas aeruginosa MF069166, Ochrobactrum intermedium R2, and Alcaligenes faecalis R8, and sophorolipids-producing fungi such as Meyerozyma sp. MF138126 and…