**1. Introduction**

The Mediterranean region is the main producer of olive oil, concentrating more than 95% of the world's olive trees. Within this area, Spain represents around half of total world manufacturing, considering this industry as one of the most important agri-food sectors for this country [1,2].

Presently, the main industrial process to obtain olive oil is continuous extraction by two-phase or three-phase systems. Depending on the oil extraction system used, considerable amounts of solid and liquid waste as final products are generated. Although the three-phase extraction process mainly generates alpechin and pomace as final residues, the main waste stream from two-phase extraction system is the alperujo (AL) [3]. The pomace is a solid waste composed of the pulp and pits of the olive, commonly employed as fertilizer, biofuel production, or animals feed. The alpechin corresponds with the liquid effluent, composed of water and minerals and characterized by a high organic matter load. This waste stream is considered, together with the AL, a highly polluting residue, for which reuse is not an easy task so its revalorization is still being investigated [4,5]. Due to the global increase of olive oil demand, the excessive amount of waste streams generated throughout the olive oil industry is a growing problem that poses an environmental challenge [6].

In Spain, the two-phase olive oil extraction system is used in approximately 90% of olive mills [4]. The application of this extraction system generates about 800 kg of AL per ton of processed olive, which represents an annual production of around four million tons for the Spanish oil industry [3]. The AL obtained is a semi-solid waste stream composed of vegetable water and olive pomace with high moisture content (60%) that still contains a certain amount of oil [7]. The AL is subjected to a second centrifugation, to obtain a pomace oil. The resulting residue is usually dried in rotary heat dryers at high temperatures and the by-product is subjected to an extraction with hexane to recover more oil, which

**Citation:** Díaz, A.I.; Ibañez, M.; Laca, A.; Díaz, M. Biodegradation of Olive Mill Effluent by White-Rot Fungi. *Appl. Sci.* **2021**, *11*, 9930. https:// doi.org/10.3390/app11219930

Academic Editor: Filomena De Leo

Received: 30 September 2021 Accepted: 20 October 2021 Published: 24 October 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

requires a large amount of energy and incurs high costs [8,9]. Dried or wet AL can also be used in composting processes. However, due to its low porosity, the addition of bulking agents such as bark chips or cotton gin is necessary [9,10]. In addition, its high content of lignocellulosic compounds and polyphenols, which are toxic to animal cells, plants, insects, and microorganisms, is an important drawback [11]. Discharging these wastes without treatment would cause serious damage to aquatic systems, such as the reduction of soluble oxygen. Furthermore, its strong odour would also cause serious problems for the population living near the discharge area [12,13]. Therefore, the removal of the pollutant compounds of the AL, and therefore favouring of the subsequent biological/physical treatment, is one of the main problems that the olive oil industry must confront.

The literature has been mainly focused on the treatment of olive mill wastes coming from the three-phase extraction system, with the aim of removing the organic and phenolic compounds and improving the biodegradability of the effluent. Regarding biological methods, anaerobic digestion or aerobic activated sludge processes have mainly been applied for the treatment of olive mill wastewater (OMW). However, these methods are not usually applied directly to the effluent due to the presence of recalcitrant molecules, polyphenols, the low nutrient load, and the acidic pH of the waste, which make treatment difficult [14]. Previous studies have reported an improvement in the efficiency of these processes reducing the acidity, adding nutrients such as cobalt, or extracting polyphenols before biodegradation [15]. Due to the high level of antimicrobial compounds present in OMW, the acclimatization of biomass or the use of physical CaCO3 supports has also been required to improve the biodegradation and methanization process [16]. Therefore, the traditional biological methods are not as effective as would be desirable. Physical–chemical methods such as nanofiltration, ultrafiltration, ultrasound, hydrothermal carbonization, and different advances oxidation processes have been reported for the treatment of OMW reducing its chemical oxygen demand (COD) and phenol content [17,18]. However, these techniques have several drawbacks, such as the addition of chemicals, fouling of the membrane, and high pressure and temperature conditions [5]. Therefore, the search for alternative methods that allow the treatment of OMW and AL in an economic and ecofriendly way is crucial.

The use of fungi has been described as a promising alternative over the use of bacteria for OMW treatment due to its ability to grow under adverse conditions and to produce a great variety of extracellular enzymes that make possible the degradation of recalcitrant compounds [19]. In this way, fungi can break down the complex recalcitrant compounds making them more assimilable to be used by themselves or by the bacteria in a subsequent treatment [20,21]. The OMW treatment by fungi has mainly focused of removing COD, phenolic content, and colour, as well as obtaining by-products with biotechnological interest, such as fungal enzymes [22,23]. White-rot fungi can degrade the lignin present in lignocellulosic wastes due to the release of enzymes, mainly lignin peroxidase and manganese peroxidase [24]. These fungi have been investigated for the treatment of recalcitrant compounds and colour degradation of OMW, obtaining good results. For example, Ntougias et al. [25], who studied the capacity of several strains of *Pleurotus* and *Ganoderma* fungi to treat OMW, reported significant removals of COD, TOC, and phenolic compounds, as well as a reduction of the toxicity of the effluent. COD degradations around 50% have been achieved by the fungus *Phanerochaete chrysosporium* immobilized on loofah [26]. Great removals of colour, phenolic compounds and COD have also been obtained when OMW was treated with fungi from genus *Aspergillus* [22,27].

As far as we know, the bioremediation with fungi has been mainly applied to treat OMW or pomace coming from the three-extraction system. However, its application to AL waste, obtained from the two-phase extraction system, has been hardly studied. Therefore, the main objective of this study was to investigate the capability of the whiterot fungus *Phanerochaete chrysosporium* to treat AL waste, to reduce its COD, colour, and phenolic compounds.
