**1. Introduction**

Cochlear implantation (CI) is the surgical implantation of an electrical device that can directly stimulate the auditory nerve through bypassing a nonfunctional inner ear. Through this device, speech and other sounds can be heard by severe to profound deaf people [1].

The first "true" CI was performed by William House and John Doyle on January 9, 1961; the surgery was performed through postauricular incision using mastoidectomy posterior tympanotomy approach (MPTA) to the middle ear. The electrode array was inserted then, after exposure of the round window (RW) membrane, into the scala tympani [2].

Interestingly, the surgical approach used and described by William house in 1961 became the classic or the standard approach for CI; for more than half a century, there was no major advancement or change in the surgical approach. However, there were different alternative approaches and some technical advancements, each of them having relative advantages and disadvantages.


*Review* **Impact of Co-Culture on the Metabolism of Marine Microorganisms**

**Flore Caudal 1, Nathalie Tapissier-Bontemps 2,3 and Ru Angelie Edrada-Ebel 4,\***

**Abstract:** Natural products from plants have been listed for hundreds of years as a source of biologically active molecules. In recent years, the marine environment has demonstrated its ability to provide new structural entities. More than 70% of our planet's surface is covered by oceans, and with the technical advances in diving and remotely operated vehicles, it is becoming easier to collect samples. Although the risk of rediscovery is significant, the discovery of silent gene clusters and innovative analytical techniques has renewed interest in natural product research. Different strategies have been proposed to activate these silent genes, including co-culture, or mixed fermentation, a cultivation-based approach. This review highlights the potential of co-culture of marine microorganisms to induce the production of new metabolites as well as to increase the yields of respective target metabolites with pharmacological potential, and moreover to indirectly improve the biological activity of a crude extract.

**Keywords:** co-culture; natural products; marine bacteria; marine fungi

#### **1. Introduction**

Secondary metabolites are molecules found in nature that are produced by organisms from one or several primary metabolites. Secondary metabolites are not required for basic viability but are essential for the organism's survival strategies. These natural products are synthesised by many organisms, such as bacteria, fungi, plants, and sponges, and are found in all environments, both terrestrial and marine.

Natural products from terrestrial plants and microorganisms are a common source of modern drug molecules. Historically, they have been used since ancient times and in traditional medicine for the treatment of many diseases and illnesses.  The earliest records dating from around 2600 BC documented the use of about 1000 substances derived from plants in Mesopotamia, such as oils of *Cupressus sempevirens* (cypress) and *Glycyrrhiza glabra* (liquorice). One of the best-known documents is the "*Ebers Papyrus*", dating from 1500 BC, recording more than 700 medicines, most of them of plant origin [1–3].

Recently, Newmann and Cragg (2012) reported that between 1981 and 2010, 48.6% of the 175 small drug molecules used in cancer therapy were either natural products or derived therefrom. In other areas, such as anti-infectives, the influence of natural products as lead structures has been quite remarkable [4,5].

Regarding microorganisms, it is estimated that the number of existing microbial species would be around 105 to 106, but only several thousand have been isolated into

**Citation:** Caudal, F.; Tapissier-Bontemps, N.; Edrada-Ebel, R.A. Impact of Co-Culture on the Metabolism of Marine Microorganisms. *Mar. Drugs* **2022**, *20*, 153. https://doi.org/10.3390/ md20020153

Academic Editor: Sang-Jip Nam

Received: 11 January 2022 Accepted: 19 February 2022 Published: 21 February 2022

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

**Copyright:** © 2022 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/).

pure cultures [6]. It is still possible to find biologically active molecules with novel structures or unprecedented carbon skeletons and unique modes of action from uncultivable and undiscovered species. Additionally, unlike macro-organisms, microorganisms have significant advantages in terms of their resilience and feasibility in terms of the cost of an industrial-scale fermentation. For the preservation of their environment of origin, the possibility to collect samples sustainably without having an impact on the biodiversity of the natural environment is of great importance. Furthermore, microorganisms are a prolific source of structurally diverse bioactive metabolites and have yielded some of the most important antibiotics in the pharmaceutical industry—one best known example is the discovery of penicillin by Fleming in 1928 [2].

Plants have also been proven to be very good sources of bioactive natural products, but in recent years, the marine environment has demonstrated its ability to provide new structural entities as well. More than 70% of our planet's surface is covered by oceans, and in certain marine ecosystems, such as coral reefs, experts estimate that the biological diversity is higher than in a tropical rainforest [7]. Exploration of the marine environment has for a very long time been limited to coasts and ocean surfaces, but with technical advances in diving and remotely operated vehicles (ROVs), it is becoming easier to take samples at deeper ocean depths. As for terrestrial microorganisms, marine bacteria and fungi quickly proved to be prolific sources of novel bioactive compounds [8].

Therapeutic molecules from the marine environment are now available in the market. In 2007, trabectedin (**1**) (Yondelis®) was the first marine-derived anti-neoplastic drug to be approved in the European Union. Trabectedin (Figure 1) was initially isolated from the Caribbean tunicate *Ecteinascida turbinata* [9]. Currently, PharmaMar commercially prepares Yondelis® by chemical synthesis [10,11]. Another example is salinosporamide A (**2**) (Marizomib®), which was isolated from a marine-derived microorganism. In 2002, Feling et al. of the Fenical and Jensen group initially isolated salinosporamide A (**2**) (Figure 1) from a sediment obligate marine actinomycete, *Salinospora tropica* [12]. Salinosporamide A (**2**) is a 20S proteasome inhibitor that was granted an orphan drug designation by the European Medicines Agency (EMA) for the treatment of multiple myeloma [13].

**Figure 1.** Chemical structures of trabectedin (**1**), salinosporamide A (**2**), isopropylchaetominine (**3**), isoterrelumamide A (**4**), and 5- -epi-averufanin (**5**).

Natural products therefore represent a non-negligible share of molecules on the market, but currently, the risk of rediscovery is always high. They have been laid aside for several years by the pharmaceutical industry in favour of synthetic molecules. However, with the discovery of silent gene clusters and the significant improvement of screening, analytical, and molecular biological techniques, a renewed interest in natural products research has been increasingly flourishing.

Whole-genome sequencing of different microorganisms has disclosed inconsistencies between the number of groups of genes identified using informatic approaches and the number of metabolites produced by the organisms [14]. The genes that are present but not expressed under laboratory conditions are called silent gene clusters. A hypothesis was then proposed for activating these silent gene clusters to potentially access new secondary metabolites that could be of pharmacological interest. Different strategies for activating these silent gene clusters are described below.

Various strategies have been put together to influence the production of secondary metabolites by microorganisms when grown in the laboratory. As described in a 2014 review paper by Bertrand et al., it is possible to explore organisms at different levels, from their genome to their metabolome via their transcriptome and proteome [15]. At the genome level, metabolic engineering was among the first techniques to be exploited, and was defined by Khosla and Keasling as "the process of rerouting metabolic pathways by genetic manipulation" [16]. In recent years, metabolic engineering has enabled the development of several techniques of genetic manipulation, the most common of which involves the biosynthesis of secondary metabolites in a heterologous host. When the natural producer of certain targeted secondary metabolites do not grow well under laboratory or industrial conditions, the biosynthetic gene cluster of interest is inserted into a different but more robust microorganism, which is selected on the basis of the involved biosynthetic pathway of targeted metabolites [17]. Mutasynthesis is another technique in which a mutant microorganism that is deficient in a key part of the biosynthetic pathway is employed and to which analogous precursors are supplemented to augment the production of new metabolites of interest [18]. These methodologies applied on the microbial genome have also been utilised along with combinatorial biosynthesis. With combinatorial biosynthesis, genes and modules of closely related synthetic pathways of secondary metabolites would be rearranged to create new congeners [17]. The success rate of combinatorial synthesis depends on the position of the introduction of the modification. Modifications at the later stages of the synthetic pathway would afford higher success rates. Indeed, there will potentially be less enzyme downstream of the point of alteration, due to having to tolerate these modifications of the substrate and complicating the process [17]. For this kind of method, it is essential that the biosynthetic gene clusters of the microorganisms are sequenced and the functions of the genes of interest are also assigned [15].

Epigenetic modification is another commonly used strategy utilised on the transcriptome and the proteome, where microorganisms were treated with epigenetic modifiers such as DNA methyl transferase or histone deacetylase inhibitors to modulate the DNA histone remodelling the chromatin for initiating the transcription of silent genes [19]. 5-Azacytidine is among the most commonly used DNA methyl transferase inhibiting molecules. Akone et al. (2016) of the Proksch group in Düsseldorf, Germany, used 5-azacytidine on fungal endophytic cultures of *Chaetomium* sp. to generate five additional polyketide congeners along with enhancing the production of targeted anticancer metabolites produced in their coculture experiments [20]. Alternatively, the Oshima group utilised suberoyl bis-hydroxamic acid, an example of a histone deacetylase inhibitor that afforded the production of a novel series of three new prenylated tryptophan analogues known as luteorides from cultures of the entomopathogenic fungus, *Torrubiella luteorostrata* [21]. Epigenetic modification has demonstrated the accumulation of new natural products of interest.

Microbial cultures grown under laboratory conditions may afford a different metabolomic profile when found in their original environment, and therefore it is important to take into account the chemical–ecological relationships that occur in their communities [15]. Moreover, genomic data are not available for all microorganisms. Therefore, non-genomedependent techniques have also been developed. One of the strategies often related to chemical ecology is a cultivation-based approach, namely, the "one strain many compounds" (OSMAC) technique, which underlines how a single strain can produce different molecules when grown under different inoculation and incubation conditions [14]. Different parameters can be changed, such as the salinity of the medium, its composition, the addition of different salts, elicitors, differences in temperature or agitation conditions, and many more. One of the other parameters to be taken into account is also the observed differences in metabolomic profiles between cultures grown in solid and liquid media, as illustrated in a review paper by Romano et al. (2018) of the Dobson group in Cork, Ireland [14]. An example to illustrate the OSMAC approach is a comparative study on the changes in the metabolomic profile of a sponge-associated fungus, *Aspergillus carneus* [22]. In this study, Özkaya et al. (2018) of the Proksch group isolated three new natural products (Figure 1)—isopropylchaetominine (**3**), isoterrelumamide A (**4**), and 5- -epi-averufanin (**5**)—after the fungus was inoculated on three different media. Isoterrelumamide A (**4**) was present only after fermentation on modified Czapek medium, while 5- -epi-averufanin was only afforded when incubated on solid rice medium with or without sea salt. It can thus be seen that depending on the composition of the culture medium in which the microorganism was inoculated, the detected secondary metabolites were not always the same [22].

Finally, the third option is by co-culture, also called mixed-fermentation, which is another cultivation-based approach. In this technique, two or more different microorganisms are inoculated together to mimic the natural habitat in which symbiotic or competing interactions between microorganisms are simulated and/or enhanced. Natural habitats could be characterised by limited access to resources and nutrients, as well as by an exchange of metabolites between micro- and/or macroscopic organisms. Competition between microbes is deliberately provoked by inducing stress factors, which stimulates the activation of silent gene clusters that were not expressed under classical culture conditions [8].

In this review, we focus on secondary metabolites from marine microorganisms and how co-culture techniques can induce their production. The different techniques implemented for mixed fermentations are discussed. Finally, the review is concluded through a critical enquiry on the potential for producing new metabolites by employing different novel co-culture techniques. This review, however, will not differentiate marine fungi from marine-derived fungi. Marine-derived fungi are species that do not belong to the well-documented lineages of obligate marine fungi, or when the source macro-organism cannot be conclusively identified as marine [23]. Nevertheless, these microorganisms could be isolated from oceans; ocean-dwelling animals; marine algae; and from marine–terrestrial transitional habitats, e.g., mangroves and other halophytes.

#### **2. Co-Cultivation between Marine Microorganisms**

The different results obtained from various representative examples of co-culture conditions presented in publications from 2010 to 2020 were surveyed in this review paper. The next three sections will tackle, and present publications found under the search terms: marine culture "coculture" OR "mixed fermentation" OR "co-culture" AND "fungi" OR "bacteria". Articles were then classified into the three sections according to the type of coinoculated microorganisms: (1) bacteria–fungi, (2) between bacteria, and (3) between fungi.

#### *2.1. Co-Cultures between Fungi and Bacteria*

In this section, only the three most relevant sample articles were found to correspond to the mixed fermentation of marine fungi and bacteria. One example of the association between marine-derived fungi and a terrestrial bacterium is also presented.

Yu, Ding, and Ma (2016) co-cultured the fungus *Aspergillus flavipes* with the actinomycete *Streptomyces* sp., which were both isolated from marine coastal sediment from the Nanji Islands in China. The mixed fermentation increased the production yield of new cytochalasin analogues from the corresponding fungal monoculture [24]. This co-culture

induced the production of six cytochalasans shown in Figure 2, namely, rosellichalasin (**6**), aspochalasin E (**7**), aspochalasin P (**8**), aspochalasin H (**9**), aspochalasin M (**10**), and 19,20-dihydro-aspochalasin D (**11**). These secondary metabolites were produced in monocultures of *Aspergillus flavipes* but at very low concentrations that were below the limit of quantification through HPLC-TOF-MS. Yu, Ding, and Ma (2016) detected the respective *m*/*z* ion peaks, demonstrating the enhancement of cytochalasan production that required physical contact between non-inactivated co-cultivated microbes. All six cytochalasans exhibited strong activity against *Streptomyces* sp. with an inhibition rate of 50–80% at concentrations between 2 and 16 μg/mL but had no effect on the fungus *A. flavipes* at the same concentration. This result indicated that the cytochalasans assisted *A. flavipes* to compete with *Streptomyces*.

**Figure 2.** Chemical structures of rosellichalasin (**6**), aspochalasin E (**7**), aspochalasin P (**8**), aspochalasin H (**9**), aspochalasin M (**10**), and 19,20-dihydro-aspochalasin D (**11**).

From the same perspective, Wakefield et al. (2017) of the Jasper group in Aberdeen, United Kingdom, described co-cultures of a marine-derived fungus, *Aspergillus fumigatus* MR2012, with two hyper-arid desert bacterial isolates *Streptomyces leeuwenhoekii* strain C34 and strain C58 [25]. The co-culture with strain C34 afforded a new luteoride derivative, luteoride D (**12**); a new pseurotin congener, pseurotin G (**13**); and two known compounds, terezine D (**14**) and 11-*O*-methylpseurotin A (**15**), which were not found in the axenic fungal culture (Figure 3). Interestingly, some of the major fungal metabolites produced by the respective axenic cultures were suppressed. The microbial co-culture with strain C58 dramatically increased the production of chaxapeptin (**16**) that was already initially found in the axenic culture of the bacteria while inducing the production of a known pentalenic acid (**17**). It is interesting to note that the co-culture process could induce the production of new compounds and increase the yield of existing metabolites from both axenic microbial cultures and not in only one of the two microorganisms involved in the co-culture. However, co-culturing could also result in a loss of production of some major families of fungal metabolites present in its axenic culture, while none of the bacterial metabolites were proven to have antifungal effects.

**Figure 3.** Chemical structures of luteoride D (**12**), pseurotin G (**13**), terezine D (**14**), 11-*O*methylpseurotin A (**15**), chaxapeptin (**16**), and pentalenic acid (**17**).

To observe the induction of antimicrobial compounds as well as the production of biosurfactants and quorum-sensing inhibitors, Dusane et al. (2011), from Pune University in India, worked with four marine epibiotic bacteria (*Bacillus* sp. S3, *Bacillus pumilus* S8, *Bacillus licheniformis* D1, and *Serratia marcescens* V1) that exhibited bioactivity against pathogenic or biofouling fungi [26]. Dusane et al. demonstrated the induction and/or enhancement of antimicrobial activity by co-culturing marine epibiotic bacteria with *Candida albicans* and *Yarrowia lipolytica*. Except for S8, monocultures of S3, D1, and V1 displayed antifungal activity against *C. albicans*. On the other hand, none of the axenic cultures of epibiotic bacteria showed any antifungal activity against *Y. lipolytica*. However, cell-free supernatants of mixed fermentation cultures of marine *Bacillus* sp. S3 and *C. albicans* induced antifungal activity against *Y. lipolytica*, while co-cultures of D1 and *C. albicans* enhanced biosurfactant production. Dusane et al. illustrated the induction and enhancement of the production of certain metabolites of interest with specific biological activity by co-culturing microorganisms with pathogenic microbial strains.

#### *2.2. Co-Cultures between Two Bacterial Strains*

In recent years, several reports on co-cultures between bacteria have been published. Based on the search terms mentioned above, we chose the 11 most appropriate publications reported on co-cultures between marine bacteria for further evaluation, including one of the papers presented at the end of the previous section. Bacteria in the marine environment are found in numerous habitats, not only in sediments, but also in association with algae and other macro-organisms. These microbes can have a free mode of life in water or sessile in the form of a biofilm in which bacteria can communicate by quorum sensing [27].

In the last paper presented in the previous section, Dusane et al. also co-cultured four marine epibiotic bacteria with other bacteria, such as *Pseudomonas aeruginosa* PA and *Bacillus pumilus* BP [26]. The respective cell-free supernatants of co-cultures of marine *Bacillus* sp. S3 and S8 with *Ps. aeruginosa* and *B. pumilus* allowed for the induction of antifungal activity. Again, antifungal activity was evident when marine isolates were co-cultivated with either pathogenic or biofouling bacterial cultures. However, the enhancement of the antibacterial activity was less apparent when compared with the distinctive induction of antifungal activity by the cell-free supernatants of the co-cultures. Exceptionally, the antibacterial activity of *B. licheniformis* D1 against *Ps. aeruginosa* was significantly increased

when co-cultured with the latter pathogen. Similarly, a significant enhancement of biosurfactant production was observed when *S. marcescens* V1 was co-cultivated with the biofouling bacteria *B. pumilus*, which was not observed when inoculated with other nonbiofouling marine bacterial isolates. On the other hand, cell-free supernatants of co-cultures of *Ps. aeruginosa* and *Bacillus* sp., as well as of their respective axenic cultures, were also observed to exhibit quorum sensing inhibitory activity. Co-cultivation of marine epibiotic bacteria with pathogenic and fouling bacteria were able to either induce or enhance all the indicated bioactivity in this research paper, which signposted the production of secondary metabolites with antibiotics, anti-quorum sensing, and biosurfactant activities.

In another study, Haque et al. (2016) focused on the antifungal and anticancer activity of an ethyl acetate crude extract obtained from a co-culture of two marine *Streptomyces* sp., ANAM-5 and AIAH-10, isolated from mangrove forest soil samples of Sundarbans, Bangladesh [28]. Only crude extracts of the co-cultures were implemented for the bioassays, while no isolated purified compounds could be utilised. The crude extracts exhibited antifungal activities against both *Saccharomyces cerevisiae* and *Aspergillus niger* with an MIC value of 64 μg/mL as well as against *Candida albicans* with an MIC value of 32 μg/mL. Haque et al. evaluated the antineoplastic activity of the co-culture extracts by measuring cell growth inhibition and the enhancement of the life span of Ehrlich ascites carcinoma (EAC) cell-bearing mice. The efficiency of the activity of the crude extract was compared with the standard anticancer drug bleomycin. Co-cultured bacterial crude extracts effectively inhibited cell growth rate at 75.75% at a of dose 100 mg/kg (i.p.), which is quite comparable to that of bleomycin at 0.3 mg/kg. Crude extracts of the co-cultured bacteria also effectively increased the life span of tumour-bearing mice by 71.79% at a dose of 100 mg/kg (i.p.). In conclusion, the extract obtained from the co-culture displayed interesting antimicrobial and anti-neoplastic activity. Reported biological activity was enhanced by 70% with the crude extracts of the co-culture, but there is no record whether new compounds were generated or merely enhanced concentration of biologically active components.

Ravi et al. (2017), from VIT University in India, briefly described co-culturing marine and terrestrial actinomycetes to enhance anticancer bioactivity [29]. The co-cultured bacterial extract gave an IC50 value of 20 μg/mL, while the extract from the monoculture afforded a weaker IC50 of 40 μg/mL. It was interesting to perceive that the co-culture of two actinomycetes species from different ecological origin could augment the production of potential anticancer metabolites. Ravi et al. also disclosed that under co-culture conditions, albeit with the occurrence of normal and healthy actinomycete filaments, spore production was greatly suppressed.

Yu et al. (2015) of the Ma group from Zhejiang University, Hangzhou, developed a relatively simple fermentation method to increase the production of algicidal tryptamine derivatives [30]. Tryptamine derivatives, as shown in Figure 4, were initially introduced as very promising molecules for the control of harmful algal blooms, but their application and further study was halted by the low production rate of the bioactive metabolites in axenic cultures of *Bacillus mycoides*. Bacterial co-cultures were established using the OSMAC technique to optimise the production of potent algicidal tryptamine congeners. Yu et al. subjected *Streptomyces* sp. CGMCC4.7185 and *Bacillus mycoides* to mixed fermentation, and various experiments were carried out to find the best culture medium and pH to enhance the production of the desired tryptamine analogues. The mean production yields of the respective tryptamine derivatives from a 68 L fermentation culture under optimised conditions were 14.9 mg/L for *N*-acetyltryptamine (**18**), 2.8 mg/L for *N*-propanoyltryptamine (**19**), 3.0 mg/L for bacillamide A(**20**), 13.7 mg/L for bacillamide B (**21**), and 9.6 mg/L for bacillamide C (**22**). The total tryptamine yield represented more than 50% of the crude ethyl acetate extract (3.0 g in the 5.5 g total extract). Under normal axenic culture conditions, the occurrence of various tryptamine analogues was undetectable by HPLC equipped with a diode array detector at a UV range between 200 and 400 nm but perceivable by TLC when visualised with iodine vapour.

**Figure 4.** Chemical structures of the co-culture-induced compounds *N*-acetyltryptamine (**18**), *N*propanoyltryptamine (**19**), bacillamide A (**20**), bacillamide B (**21**), and bacillamide C (**22**).

Low biomass yields with bacterial cultures are a frequent challenge. Under a coculture environment, a higher production yield of the targeted secondary metabolites was facilitated. With higher yields, the various analogues can be further evaluated for their other potential activities and cytotoxicity studies. However, subsequently, for a large (industrial) scale-up, as the fermentation parameters can change and are not directly reproducible, then it may be necessary to employ other techniques or in combination with co-cultivation such as hemisynthesis or one of the metabolic engineering methods described above in Section 1.

Cho and Kim (2012), from Soon Chun Hyang University, Korea, increased the production of a target antifouling compound by co-culturing a bacterium producing the metabolite of interest with a competitor bacterium isolated from the same host macroorganism [31]. The active antifouling diterpene lobocompactol (**23**) (Figure 5) was initially isolated from the marine *Streptomyces cinnabarinus* PK209. To increase lobocompactol production, Cho and Kim selected *Alteromonas* sp. KNS-16 as the lobocompactol-resistant bacterium for a co-culture competitor. Both bacterial strains, PK209 and KNS-16, were isolated from the surface of a seaweed rhizosphere. The co-culture of these two bacterial strains resulted in a 10.4-fold increase in the production of lobocompactol compared to the PK209 monoculture (2.7 mg/L instead of 0.25 mg/L). Lobocompactol (**23**) exhibited significant antifouling activity with an EC50 of 0.18 μg/mL against the macroalga *Ulva pertusa* and 0.43 μg/mL against the diatom *Navicula annexa*. The extract also demonstrated its activity against the fouling bacteria. These studies from Yu et al. [30] as well as Cho and Kim [31] presented the potential of co-cultures in increasing the production of a particular target metabolite.

Co-cultures have also led to the discovery of new bioactive molecules. Shin et al. (2018) from Seoul National University elucidated a new piperazic acid-bearing cyclic peptide, dentigerumycin E (**24**) (Figure 5), afforded by the co-culture of the marine *Streptomyces* sp. JB5 and *Bacillus* sp. GN1, isolated from an intertidal mudflat in Wando, Republic of Korea [32]. *Streptomyces* sp. JB5 was co-cultivated with seven different bacterial strains including *Bacillus* sp. HR1, *Paenibacillus* sp. CC2, *Brevibacillus* sp. PTH23, *Streptomyces* sp. SD53, *Streptomyces* sp. UTZ13, *Hafnia* sp. CF1, and *Mycobacterium* sp. Myc06. However, only the co-culture with *Bacillus* sp. HR1, which is phylogenetically close to *Bacillus* sp. GN1, afforded dentigerumycin. Although the mechanism triggering the biosynthesis of dentigerumycin E (**24**) by *Bacillus* strains remains unclear, the results of these co-culture experiments imply that *Bacillus* strains, which are most closely related to *B. cereus*, may share the ability to induce production of dentigerumycin E (**24**) in *Streptomyces* sp. JB5. Full genome sequencing of *Streptomyces* sp. JB5 allowed for the identification of the BGC for dentigerumycin E (**24**), and thus it was confirmed that it was indeed produced by the *Streptomyces* strain. Different experiments have shown that dentigerumycin E had antimetastatic potential against breast cancer cells.

**Figure 5.** Chemical structures of lobocompactol (**23**), dentigerumycin E (**24**), and janthinopolyenemycins A (**25**) and B (**26**).

Anjum et al. (2018) of the Lian group from Zhejiang University, Hangzhou, isolated janthinopolyenemycins A (**25**) and B (**26**), two rare polyketides (Figure 5), from a co-culture of two *Janthinobacterium* spp. strains ZZ145 and ZZ148 obtained from a marine soil sample, and the strains were co-inoculated in different media including rice solid medium [33]. Both polyketides exhibited antifungal activity against *C. albicans* with MIC and MBC values of 15.6 and 31.25 μg/mL, respectively, although their bioactivities were less potent than the control (amphotericin B). The janthinopolyenemycin congeners were also found to be active against methicillin-resistant *Staphylococcus aureus* (MRSA) and *E. coli*. The co-culture of ZZ145 and ZZ148 induced the generation of the new janthinopolyenemycin congeners.

Many of the marine bacteria used in co-cultures were derived from soil sediment. However, the literature has also shown co-cultures of sponge-derived actinomycetes [34]. Dashti et al. (2014) of the Quinn group in Griffith University co-cultured *Nocardiopsis* sp. RV163, derived from the Mediterranean sponge *Dysidea* along with *Actinokineospora* sp. EG49 from the Red Sea sponge *Spheciospongia vagabunda* to induce the biosynthesis of three compounds (Figure 6), namely, *N*-(2-hydroxyphenyl)-acetamide (**27**), 1,6-dihydroxyphenazine (**28**), and 5a,6,11a,12-tetrahydro-5a,11a-dimethyl[1,4]benzoxazino[3,2-*b*][1,4]benzoxazine (**29**), of which compound **28** was produced at a very high yield at 12% of the crude extract weight. Dashti et al. monitored the induced production of new metabolites in the co-culture by 1H NMR analysis of the respective culture extracts. The 1H NMR spectral data also indicated the suppression of the production of some of the metabolites present in the axenic cultures, which was similarly observed by Wakefield et al. in 2017 [25]. All three afforded compounds (**27** to **29**) were tested for bioactivity against *Bacillus* sp. P25, *Escherichia coli* and *Fusarium* sp. P21, and human parasites *Leishmania major* and *Trypanosoma brucei*, as well as *Nocardiopsis* sp. RV163 and *Actinokineospora* sp. EG49 cultures. Only 1,6-dihydroxyphenazine (**28**) exhibited bioactivity against *Bacillus* sp.; *Trypanosoma brucei*; and, interestingly, against *Actinokineospora* sp. EG49, which may indicate that compound **28** was biosynthesised by *Nocardiopsis* sp. RV163. These last three studies demonstrated the potential of a co-culture to induce the production of new metabolites.

**Figure 6.** Chemical structures of *N*-(2-hydroxyphenyl)-acetamide (**27**), 1,6-dihydroxyphenazine (**28**), 5a,6,11a,12-tetrahydro-5a,11a-dimethyl[1,4]benzoxazino [3,2-b][1,4]benzoxazine (**29**), and keyicin (**30**).

The last two studies in this section are articles by the Bugni group from the University of Wisconsin. Unlike all the previous articles, these latter studies used microscale fermentation [35]. Microscale cultivation was carried out in 96-well plates, in volumes of 500 μL. Adnani et al. (2015) selected *Mycobacterium* sp. (WMMA-183) and *Rhodococcus* sp. (WMMA-185) as co-culture strains for various species from within the multiple genera of Micromonosporaceae. From the Micromonosporaceae family, 65 species were chosen for analysis of their monoculture and co-culture metabolomic profiles. While some culture samples showed antibiotic induction, some significant limitations also occurred, as this method is likely to miss antibiotics produced in the co-culture due to the initial activity observed in axenic cultures from an unrelated antibiotic. To resolve such methodological limitations, a complementary approach by LC–MS-based metabolomics was employed to analyse the types of compounds produced during the interaction and exhibition of antibiotic activity. For the 130 co-culture combinations, a total of 12 Micromonosporaceae demonstrated discernible diversity, with six producing a unique chemistry but shared similar chemical profiles when co-cultured with *Mycobacterium* sp. or *Rhodococcus* sp., while the other six produced different metabolomic profiles that were dependent on the bacterium with which it was co-cultured. Co-cultures producing similar chemical profiles were clustered, while secondary metabolites exclusively produced in co-culture were further evaluated for novelty. As an example, co-culture of *Verrucosispora* sp. and *Rhodococcus* sp. exclusively produced a total of 29 identified compounds. Of the 29 compounds, 27 were determined to be novel, and 24 could be reproduced in the scale-up. This latter study demonstrated the ability to screen and identify the generation of new metabolites from a microscale fermentation approach.

In their article, Adnani et al. (2017) described the production of keyicin (**30**), a new *bis*-nitroglycosylated anthracycline (Figure 6), by a co-culture of a *Rhodococcus* sp. and a *Micromonospora* sp. [36]. Keyicin (**30**) was produced exclusively via mixed fermentation, but physical contact between the two bacteria was not necessary. Monocultures of the

two bacterial strains were incubated in distinct chambers but were connected through a tunnel separated by a diffusible membrane allowing the exchange of metabolites without any cellular transfer between chambers. Through genome sequencing, *Micromonospora* sp. was shown to be the "true" producer of keyicin in the co-culture. Keyicin (**30**) inhibited the growth of *Mycobacterium* sp. and *Rhodococcus* sp., as well as *B. subtilis* and methicillinsensitive *Staphylococcus aureus* (MSSA) at minimum inhibitory concentrations of 8 and 2 μg/mL, respectively. In contrast to many other anthracyclines, 20 *E. coli*-based chemical genomics studies revealed that keyicin's mechanism of action did not induce DNA damage.

In this section, it is shown that there are various types of co-culture between bacteria, and much remains to be discovered. Co-culture between bacteria has afforded interesting molecules and has made it possible to induce the production of new metabolites while increasing the production of known compounds or further enhancing their bioactivity, whether in small- to medium-sized liquid fermentation or in micro-fermentation scales. Co-culturing *Micromonospora* sp. and *Rhodococcus* sp. exclusively induced the production of the target metabolite keyicin (**30**), which was not afforded by the monocultures, although the biosynthetic gene cluster was found in *Micromonospora* sp.

#### *2.3. Co-Cultures between Two Fungal Strains*

In this last section, 14 pertinent articles were selected to describe the co-cultivation of two fungal species. Isolation of fungal samples from the marine environment has received increasing attention as a new source of interesting metabolites. As mentioned in the introduction, it is more difficult to differentiate a marine from a terrestrial fungus when compared to a bacterium. Unlike bacteria, marine fungi do not necessarily need sea salts in their culture medium to grow. Therefore, both obligate marine and marine-derived fungi will be included in this section.

Most of the literature has described fungal co-cultures between the genus *Penicillium* and/or *Aspergillus* and/or with other fungal genera. Bao et al. (2017) of the Qi group in Guangzhou, China, co-cultivated two marine gorgonian-associated fungi, *Aspergillus sclerotiorum* SCSGAF 0053 and *Penicillium citrinum* SCSGAF 0052, both isolated from *Muricella flexusa* collected from the South China Sea [37]. The development of a red pigment was only observed in the co-culture, which did not occur in either of the monocultures. Six new compounds (**31**–**36**) shown in Figure 7 were elucidated, including two furanone derivatives, sclerotiorumins A (**31**) and B (**32**); a novel oxadiazin derivative, sclerotiorumin C (**33**); a pyrrole derivative, 1-(4-benzyl-1*H*-pyrrol-3-yl)ethenone (**34**); and two complexes of neoaspergillic acid with aluminium (**35**) and iron (**36**) (Figure 7). Sclerotiorumin C (**33**) was the first naturally occurring 1,2,4-oxadiazin-6-one described. All compounds were tested for different activities—aluminiumneohydroxyaspergillin (**35**) showed significant selective cytotoxicity against human histiocytic lymphoma U937 cell line (IC50 = 4.2 μM) and strong toxicity towards brine shrimp (LC50 = 6.1 μM), and interestingly was able to increase the growth and biofilm formation of *S. aureus*.

Kossuga et al. (2013) of the Berlinck group in São Paulo co-cultivated a total of 50 fungal strains and 5 bacterial strains in 250 growth experiments. Analysis of the extracts indicated changes in the chemical profile of eight co-cultures when compared to their corresponding monocultures. However, most of the dereplicated metabolites are already known compounds. The co-cultures produced a minority of the secondary metabolites that contained novel carbon skeletons. *Penicillium* sp. Ma(M3)V co-cultured with *Trichoderma* sp. Gc(M2)1 produced a complex extract that yielded two new polyketides (Figure 8), (*Z*)-2-ethylhex-2-enedioic acid (**37**) and (*E*)-4-oxo-2-propylideneoct-7-enoic acid (**38**) [38]. Polyketides **37** and **38** are examples of unprecedented carbon skeletons.

**Figure 7.** Chemical structures of sclerotiorumins A-B (**31** and **32**), sclerotiorumin C (**33**), 1-(4-benzyl-1*H*-pyrrol-3-yl)ethanone (**34**), aluminiumneohydroxyaspergillin (**35**), and ferrineohydroxyaspergillin (**36**).

**Figure 8.** Chemical structures of (*Z*)-2-ethylhex-2-enedioic acid (**37**) and (*E*)-4-oxo-2-propylideneoct-7-enoic acid (**38**).

The group of Zhuravleva and Afiyatullov (2016) in Russia isolated known diorcinol congeners B to E (**39** to **42**) along with a new derivative, diorcinol J (**43**) (Figure 9), by the mixed fermentation of *Aspergillus sulphureus* KMM 4640 and *Isaria felina* KMM 4639 obtained from a muddy sand and a marine sediment, respectively [39]. Diorcinol J (**43**) was the only congener that showed cytotoxicity against Ehrlich carcinoma cells, by the membranolytic mechanism. In addition, more recently in 2018, the group of Zhuravleva and Afiyatullov isolated five new prenylated indole alkaloids (Figure 10) from the same co-culture. This included 17-hydroxynotoamide D (**44**), 17-*O*-ethylnotoamide M (**45**), 10-*O*acetylsclerotiamide (**46**), 10-*O*-ethylsclerotiamide (**47**), and 10-*O*-ethylnotoamide R (**48**) [40]. However, 10-*O*-ethylnotoamide R (**48**) was not detected in the original extract, and thus the authors postulated that **48** might not be a natural product, but an artefact obtained during the isolation process. Zhuravleva and Afiyatullov also investigated the effect of 17-hydroxynotoamide D (**44**), 17-*O*-ethylnotoamide M (**45**), and 10-*O*-ethylnotoamide R (**48**) on the viability of human non-malignant and prostate cancer cells, as well as on the formation of colonies of human prostate cancer cells 22Rv1. 17-*O*-Ethylnotoamide M (**45**) exerted a significant effect on reducing 22Rv1 colony formation at concentrations of 10 μM by 25%. 22Rv1 cells are known to be resistant to hormone therapy, as well as to the new second-generation drugs. Therefore, drugs that are active in these cell lines may be of potential interest for further investigation in the therapy of drug-resistant human prostate cancer. Although these new molecules were produced only in the co-culture, it is worth mentioning that brevianamide F (**49**) (Figure 10), a common precursor of the afforded alkaloids, had earlier been isolated from *A. sulphureus*.

**Figure 9.** Chemical structures of diorcinols congeners B to E (**39** to **42**) and diorcinol J (**43**).

**Figure 10.** Chemical structures of 17-hydroxynotoamide D (**44**), 17-*O*-ethylnotoamide M (**45**), 10-*O*acetylsclerotiamide (**46**), 10-*O*-ethylsclerotiamide (**47**), 10-*O*-ethylnotoamide R (**48**), and brevianamide F (**49**).

Ebada et al. (2014) of the Roth group in BioMar, Düsseldorf, co-cultured two marine algal-derived fungal strains of *Aspergillus* BM-05 and BM-05ML that yielded the cyclo-tripeptide psychrophilin E (**50**), along with five known compounds (protuboxepin A (**51**); oxepinamide E (**52**); and three mycotoxins, namely, sterigmatocystin (**53**), 5-methoxysterigmatocystin (**54**), and aversin (**55**)), as shown in Figure 11 [41]. Dalsgaard et al. (2004) of the Christophersen group in Copenhagen first reported the cyclotripeptide analogues, psychrophilins A to D (**56** to **59**), which were earlier isolated from a marinederived psychrotolerant fungal species of the genus *Penicillium* [42,43]. In contrast to previously described congeners, in psychrophilin E (**50**), the α-amino group in the tryptophan residue was acetylated into an *N*-acetyl moiety, which was oxidised in psychrophilins

A to D into a nitro group. In addition, the existence of other psychrophilins in the extract could not be detected, implying that this mixed fermentation may have played a role in modifying the biosynthetic pathway by an acetylation rather than an oxidation step. All the isolated compounds from the co-culture of *Aspergillus* BM-05 and BM-05ML were assessed for their in vitro anti-proliferative activities. Psychrophilin E (**50**), sterigmatocystin (**53**), and 5-methoxysterigmatocystin (**54**) exhibited selective anti-proliferative activities towards the HCT116 (colon cancer) cell line with IC50 values of 28.5, 10.3, and 4.4 μM, respectively, which were more active than cisplatin as a positive control (IC50 = 33.4 μM).

**Figure 11.** Chemical structures of psychrophilin E (**50**), protuboxepin A (**51**), oxepinamide E (**52**) sterigmatocystin (**53**), 5-methoxysterigmatocystin (**54**) aversin (**55**), and psychrophilin A to D (**56** to **59**).

The group of Zhu from Foshan University, China (2011), isolated aspergicin (**60**) with two know compounds (Figure 12), neoaspergillic acid (**61**) and ergosterol (**62**), from a coculture of two mangrove *Aspergillus* epiphytes [44]. Both aspergicin (**60**) and neoaspergillic acid (**61**) showed significant inhibitory effect on three Gram-positive bacteria, *Staphylococcus aureus*, *Staphylococcus epidermidis*, and *Bacillus subtilis*, and three Gram-negative bacteria, *Bacillus dysenteriae*, *Bacillus proteus*, and *E. coli*, but with lower MICs for neoaspergillic acid (**61**).

**Figure 12.** Chemical structures of aspergicin (**60**), neoaspergillic acid (**61**), and ergosterol (**62**).

During the last decade, several research teams in Guangzhou, China, led by She, Lin, (2010, 2011, and 2013), and Li (2014, 2015, and 2017), have been working on secondary metabolites produced by co-cultures of *Phomopsis* sp. K38 and *Alternaria* sp. E33, and were able to isolate many new compounds. All publications from the Guangzhou teams, except for Ding et al. [45], followed the same protocol. The Guangzhou research groups collected two mangrove-derived fungi, *Phomopsis* sp. K38 and *Alternaria* sp. E33, from the South China Sea coastline. The co-culture extract displayed higher cytotoxicity against Hep-2 and HepG2 cells than that of the K38 or E33 monocultures. Amongst the first molecules isolated was a new diimide derivative (Figure 13), (−)-byssochlamic acid bisdiimide (**63**) [46]. Unfortunately, the isolated purified bisdiimide exhibited weak cytotoxicity against Hep-2 and HepG2 cells, with IC50 values of only 45 and 51 μg/mL, respectively. The same fungal co-culture also yielded a new xanthone derivative (Figure 13), 8-hydroxy-3-methyl-9-oxo-9*H*-xanthene-1-carboxylic acid methyl ether (**64**) [47]. The new xanthone derivative (**64**) inhibited the growth of five tested microorganisms, *Gloeasporium musae*, *Blumeria graminearum*, *Fusarium oxysporum*, *Peronophthora cichoralearum*, and *Colletotrichum glocosporioides*. The co-culture also afforded a new polysubstituted benzaldehyde, ethyl 5-ethoxy-2-formyl-3-hydroxy-4-methylbenzoate (**65**) [48]. Wang et al. (2013) bioassayed the new polysubstituted benzaldehyde compound (**65**) against four plant pathogenic fungi, namely, *Fusarium graminearum*, *Gloeosporium musae*, *Rhizoctonia solani* Kuhn, and *Phytophthora sojae* Kaufmann and Gerdemann. Antifungal activities were observed at inhibitory zone diameters of 12.06, 11.57, 10.21, and 8.50 mm, respectively.

Later studies of the same co-culture strains E33 and K38 afforded two new cyclopeptides [49]. Huang et al. (2014) evaluated the in vitro antifungal activity of two cyclic tetrapeptides, cyclo (D-Pro-L-Tyr-L-Pro-L-Tyr) (**66**) and cyclo (Gly-L-Phe-L-Pro-L-Tyr) (**67**), against *C. albicans*, *G. graminis*, *R. cerealis*, *H. sativum*, and *F. graminearum*. Both peptides **66** and **67** showed moderate-to-high antifungal activities as compared with the positive control (ketoconazole), but cyclo (Gly-L-Phe-L-Pro-L-Tyr) (**67**) was more active than cyclo (D-Pro-L-Tyr-L-Pro-L-Tyr) (**66**). Li et al. (2014) found another cyclic tetrapeptide, cyclo-(L-leucyl-trans-4-hydroxy-L-prolyl-D-leucyl-trans-4-hydroxy-L-proline) (**68**), from the same fungal co-culture [50] and tested the compound against the same crop-threatening fungi except for *C. albicans*. Compound **68** showed moderate to high antifungal activities and afforded quite a comparable bioactivity to the positive control, triadimefon, against *H. sativum*. All isolated cyclopeptides **66** to **68** are shown in Figure 13.

From the same co-culture that produced the cyclic tetrapeptides, Wang et al. (2015) isolated a new 7-(γ,γ-dimethylallyloxy)-6-hydroxy-4-methylcoumarin (**69**) (Figure 13) [51], and this was also tested against the four plant pathogenic fungi. However, unlike the peptides, the compound showed no in vitro activity against these plant pathogens over a 6.00 mm inhibition zone at 250 μM.

More recently, Ding et al. (2017) elucidated a new nonadride derivative, (−)-byssochlamic acid imide (**70**) (Figure 13), from the same mangrove fungal co-cultures [45]. Chen et al. (2004) earlier reported the antifungal, phytotoxic, enzyme inhibitory, and cytotoxic activities of nonadride analogues [52]. The new nonadride derivative from the co-culture showed moderate inhibitory activity toward *Fusarium graminearum* and *F. oxysporum* with MIC values of 50 and 60 μg/mL, respectively, as compared with the positive control carbendazim (MIC 6.25 μg/mL). By adding salt to the media, changing the incubation period and

temperature, Ding et al. were able to generate a new analogue that is unrelated to cyclic tetrapeptide derivatives earlier targeted by their predecessors.

**Figure 13.** Chemical structures of (−)-byssochlamic acid bisdiimide (**63**), 8-hydroxy-3-methyl-9-oxo-9*H*-xanthene-1-carboxylic acid methyl ether (**64**), ethyl 5-ethoxy-2-formyl-3-hydroxy-4-methylbenzoate (**65**), cyclo (D-Pro-L-Tyr-L-Pro-L-Tyr) (**66**), cyclo (Gly-L-Phe-L-Pro-L-Tyr)s (**67**), cyclo-(L-leucyl-trans-4-hydroxy-L-prolyl-D-leucyl-trans-4-hydroxy-L-proline) (**68**), 7-(γ,γdimethylallyloxy)-6-hydroxy-4-methylcoumarin (**69**), and (−)-byssochlamic acid imide (**70**).

The last study presented in this section involved the co-culture of two fungal morphs of the marine algal-derived *Aspergillus alliaceus* [53]. The fungal co-culture consisted of identical strains but from different phases of development at their respective asexual and sclerotial morph stages. Both morphs produced distinct secondary metabolite patterns in monoculture, but their co-culture significantly changed the metabolic profile of the strain. Mandelare et al. (2018) of the Loesgen group in Oregon State isolated a new bianthrone dimer allianthrone A (**71**) from the co-cultures, while they observed elevated levels of nalgiolaxin (**72**) produced by the asexual morph. In parallel, Mandelare et al. found allianthrone A and its two diastereomers, allianthrones B (**73**) and C (**74**) (Figure 14), from the liquid fermentation culture at a ratio of 2:1:1. The two-week liquid cultures of the combined asexual and sclerotial morphs of *A. alliaceus* reliably produced the known compound nalgiolaxin at a higher yield. Allianthrone A showed weak cytotoxicity against the HCT-116 colon cancer and SK-Mel-5 melanoma cell lines. This study illustrated the first case of elicitation of new fungal chemistry by a co-culture approach of two different developmental stages of a homothallic *Aspergillus* species.

**Figure 14.** Chemical structures of allianthrone A (**71**), nalgiolaxin (**72**), allianthrone B (**73**), and C (**74**).

In recent publications, contrary to the previous section, it was observed that mixed fermentation between fungal species increased the discovery of new molecules, while the detection of enhanced biologically activity was less evident. Enhancing biomass yield in fungal cultures is in general easier to achieve in comparison to bacterial cultures due to their longer incubation period and life cycle. With higher biomass yield, the isolation of the metabolites is easier as well, and hence a higher rate of enhancing the production of targeted known bioactive compounds and at the same time a higher discovery rate of new compounds can be obtained but may not necessarily be active.

Nevertheless, as the utmost goal is to enhance the production yield, as well as to induce improved biological activity by stimulating the biosynthesis of new compounds, it can be discouraging that the generated new analogues may be rendered inactive. In most cases, the bioactivity seems to be at its optimum on one analogue, despite the occurrence of various new metabolites. A combination of metabolic engineering techniques needs to be considered, and this includes finding an efficient screening method to obtain the right elicitor to increase the probability of finding biologically active scaffolds. However, another limiting factor is the number of available assays in respective laboratories, and this could also be the main reason that despite the high number of new congeners being isolated, only one or two analogue(s) could be considered to exhibit potent bioactivity. The process of inducing the production of new bioactive compounds is dependent on conditions that a microorganism would need for a specific life mode to survive. There is yet much to be discovered about cryptic biosynthetic gene clusters and how they can be elicited for their expression. In this case, a full understanding of this microbial processing factory still needs to be explored.

#### **3. Different Techniques of Co-Culture Used**

In this last section, the techniques used by the different research teams presented in this literature review for various co-culture conditions are listed below in Table 1. The following are tabulated: the microorganisms used, their geographical origin, the culture medium(s) used, the conditions under which the cultures were made (static or under agitation, temperature, and incubation period), the experimental set-up, and the article from which the technique originated. The techniques are quite diverse, but generally the incubation time is longer for fungi than for bacteria and the temperature is often between 25 and 32 ◦C.

**Table 1.** Widely used co-culture techniques.

*Mar. Drugs* **2022**, *20*, 153

**Table 1.** *Cont.*

**Table 1.** *Cont.*

EY liquid medium: yeast 1.0 g, tryptone 5.0 g, FeCl3·6H2O 0.17 g, KH2PO4 0.12 g, sea salt 35 g, water 1 L. B liquid medium: soluble starch 20 g, KNO3 1 g, K2HPO4 0.5 g, MgSO4·7H2O 0.5 g, NaCl 0.5 g, FeSO4 0.01 g, water 1 L. Legend: LB medium = Luria–Bertani medium; YEME medium = yeast extract–malt extract medium; ISP2 medium = International *Streptomyces* Project-2 medium; ASW = artificial seawater medium.

Extremely low yielding target metabolites detected in axenic cultures may not be feasibly translated for isolation work. As illustrated by the literature reviewed in this paper [24–26,30], these metabolites could be detected but would not be quantifiable by mass spectrometry. These same metabolites would be below the limit of detection for a UV-HPLC-coupled system. In these cases, compound yields of these target metabolites in axenic culture were not measurable prior to coculturing. The authors of the reviewed papers demonstrated enhanced compound production by comparing extracted mass ion chromatograms of the target metabolites in axenic and co-culture conditions [24–26,30]. As demonstrated by references listed on Table 1, successful induction of compound production afforded a range of three- to fivefold increase in yield of the target metabolite. Except for the production of lobocompactol (**23**), Cho and Kim (2012) started with a quantifiable amount of 0.25 mg/L in axenic cultures, and they achieved a 10.4-fold increase by mixed fermentation [31]. In some publications, several techniques have been tested. The techniques that have generated the most conclusive results are shown in Table 1.

#### **4. Conclusions**

With new omics techniques and microbiological genetic data, it has been possible to identify a variety of previously unknown metabolic pathways. Microorganisms have a very high potential to produce metabolites with a high chemical diversity. As shown in this literature review, many of the microorganisms' biosynthetic genes remain silent or are not transcribed when they are grown under laboratory conditions. With co-culture or mixed fermentation for endogenous metabolite production, cryptic pathways can be stimulated through interaction and communication between microorganisms. Indeed, co-culture tries to mimic the environment where communications between microorganisms are abundant, dynamic, and complex.

From the various publications reviewed here, it was shown that co-culture can induce the production of new metabolites as well as increase the yields of respective target metabolites with pharmacological potential, and moreover can indirectly improve the biological activity of a crude extract. In parallel, mixed fermentation techniques may also suppress the production of metabolites that were found in axenic culture [34]. The diverse responses driven by the various co-culture methods could represent the potential counteracting response to the competition between the microorganisms. However, the afforded metabolites do not necessarily show activity against competing microorganisms, which indicates that the interactions could also be more complex than a simple question of competition.

The co-culture technique employed is essential, as it has been demonstrated that a single fermentation variable can have a very large impact on the production of specific metabolites [31]. When fungi and bacteria were co-cultured, it was essential to consider the incubation time needed for each to grow so that one does not take over the other. Physical contact may be necessary for the production of some metabolites, as for cytochalasin [24], but may not always be the case, as in the production of keyicin [36]. It is quite complicated to make any generalisations on the expected outcomes of certain co-culture techniques. However, techniques have been emerging, such as co-cultures in 96-well plates, which allowed for the testing of various combinations of media, buffers, and elicitors to be assessed. Innovative cultures also involved mixed fermentation of a single fungus from two different stages of development [53].

Furthermore, various other culture techniques could evolve to be associated with mixed fermentation methods, such as I-chip [54]. I-chip is a technique of in situ cultivation that utilises a device for the isolation of bacteria that are difficult to culture in the laboratory. This device contains hundreds of miniaturised diffusion chambers that are inoculated with a single bacterial cell. This device can then be placed in the natural environment of the organisms cultured in the diffusion chambers, be it in the sediment, the soil, or in the macro-organisms, as has recently been performed in a sponge [55]. I-chip allows microorganisms to grow in their natural environment, in addition to allocating microbial

interactions, and affords access to the discovery of new organisms supplementing novel molecules [54].

The use of microorganisms from different geographical origins would also broaden the possibility to increase diversity, for example in the mixed fermentation of macro-algae endophytes simply from various parts of the globe. Microorganisms from the marine environment are still considered to be underexploited, and many discoveries are yet to be made. This technique can be used routinely in laboratories associated with techniques such as one strain many compounds (OSMAC). Natural marine products and the technique of co-culture can allow the discovery of new molecules of pharmacological interest and address the problem regarding the expression of silent genes clusters.

**Author Contributions:** Conceptualisation, R.A.E.-E.; methodology, F.C.; formal analysis, F.C.; investigation, F.C.; resources, F.C.; data curation, F.C.; writing—original draft preparation, F.C.; writing—review and editing, R.A.E.-E. and N.T.-B.; visualisation, F.C. and R.A.E.-E.; supervision, R.A.E.-E. and N.T.-B.; project administration, N.T.-B.; funding acquisition, F.C and N.T.-B. All authors have read and agreed to the published version of the manuscript.

**Funding:** Student placement for F.C. was funded by the Mobilité Programme Erasmus+ of the European Union 2019-1-FR01-KA103-061405.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Acknowledgments:** F.C from the University of Perpignan via Domitia has carried out a mobility at the Strathclyde Institute of Pharmacy and Biomedical Sciences, Faculty of Science, University of Strathclyde, from 3 February to 23 June 2020 under the Erasmus programme—Student mobility for placements (SMP) to attain a Master 2 Certificate—Marine Science—Molécules Bioactives et Environnement Année universitaire 2019–2020.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


**Table 7.**

*Estimated VEC model 1.*

#### *Exchange Rate Volatility and Monetary Policy Shocks DOI: http://dx.doi.org/10.5772/intechopen.99606*

#### **Table 8.**

*Estimated VEC model 2.*

### *Exchange Rate Volatility and Monetary Policy Shocks DOI: http://dx.doi.org/10.5772/intechopen.99606*

*Notes: Standard errors are in ()and t-statistics are in [].*

#### **Table 9.**

*Estimated VEC model 2.*

exchange rate volatility in the short run. The results lead to the acceptance of null hypothesis that the treasury bills rate and the liquidity ratio have no significant nexus with the volatility in the exchange rate in the, but the impact of exchange rate on exchange rate volatility is significant in the short run, thus rejection of the null hypothesis concerning this nexus.

#### *4.1.4 Impulse response functions*

The results of the Impulse Response Functions (IRFs) of the first VEC model in graphical form are reported in the **Figure 1**.

The impulse response function of exchange rate volatility in the VEC model to a shock in itself shows that exchange rate volatility reacted positively to its innovations all through the five months of forecast. Similarly, it responded positively to innovations in monetary policy rate throughout the five months of forecast. However, exchange rate volatility responded negatively to a shock in money supply all through the five months of forecast.

**Figure 1.** *Impulse response functions. Source: Results extract from E-views 11.0*

**Figure 2.** *Forecast error decomposition functions. Source: Results extract from E-views 11.0.*

### *4.1.5 Forecast error variance decomposition functions*

The estimation of a VAR model firstly requires the explicit choice of lag length in the equations of the model. In this study, graphs of the forecast error variance decomposition functions are presented in **Figure 2**.

As shown in **Figure 2**, it can be observed from the variance decomposition of exchange rate volatility that own shocks contributed most [100%] to its variations in the first month of forecast but declined marginally to 91% in the fifth month of forecast. The proportions of variations in exchange rate volatility, due to shocks in monetary policy rate and money supply, are minimal throughout the five months of forecast. The two variables accounted for less than 6% in the entire periods of forecast. Hence, the variance decomposition of exchange rate volatility shows that own shocks predominantly determined variations in its variations throughout the months of forecast. Monetary policy rate and money supply accounted for less than an average of 6% variations in exchange rate volatility all through the five months of forecast. The proportions of variations in exchange rate volatility due to money supply shocks are marginally higher than those of monetary policy rate during the periods.

#### *4.1.6 Impulse response functions*

The results of the Impulse Response Functions (IRFs) of the second VEC model in graphical form are reported in the **Figure 3**.

The impulse response function of exchange rate volatility in the VEC model to a shock in itself shows that exchange rate volatility reacted positively to its innovations all through the five months of forecast. However, it responded negatively to innovations in treasury bill rate throughout the 5 months of forecast. With respect to a shock in liquidity ratio, exchange rate volatility responded positively all through the 5 months of forecast. Similarly, exchange rate volatility responded positively to shocks in exchange rate within the 5 months of forecast.


*Article* **Carbon Black/Polyvinylidene Fluoride Nanocomposite Membranes for Direct Solar Distillation**

**Marcello Pagliero 1,2,\*, Marina Alloisio 1, Camilla Costa 1,2, Raffaella Firpo 1,2, Ermias Ararsa Mideksa 1 and Antonio Comite 1,2**

	- 2 INSTM, Udr di Genova, Via Dodecaneso 31, 16146 Genova, Italy **\*** Correspondence<EMAIL_ADDRESS>
**Abstract:** Water reclamation is becoming a growing need, in particular in developing countries where harvesting the required energy can be a challenging problem. In this context, exploiting solar energy in a specifically tailored membrane distillation (MD) process can be a viable solution. Traditional MD guarantees a complete retention of non-volatile compounds and does not require high feed water temperatures. In this work, a suitable amount of carbon black (CB) was incorporated into the whole matrix of a polymeric porous membrane in order to absorb light and directly heat the feed. The mixed matrix membranes were prepared forming a uniform CB dispersion in the PVDF dope solution and then using a non-solvent induced phase separation process, which is a well-established technique for membrane manufacturing. CB addition was found to be beneficial on both the membrane structure, as it increased the pore size and porosity, and on the photothermal properties of the matrix. In fact, temperatures as high as 60 ◦C were reached on the irradiated membrane surface. These improvements led to satisfactory distillate flux (up to 2.3 L/m2h) during the direct solar membrane distillation tests performed with artificial light sources and make this membrane type a promising candidate for practical applications in the field of water purification.

**Keywords:** carbon black; photothermal; direct solar membrane distillation; PVDF; renewable energy

#### **1. Introduction**

Membrane distillation (MD) is a thermally driven separation process based on a hydrophobic porous membrane which contacts a hot concentrated solution and pure water. The temperature difference applied at each side of the membrane induces a vapor pressure difference across the membrane that acts as driving force for the process and establishes a water vapor flux from the hot to the cold section [1,2].

Membranes used for MD application must comply with important specific requirements. First, high porosity is mandatory to maximize the trans-membrane water flux since the mass transfer coefficient is proportional to the mean porosity of the membrane itself [3]. Another essential feature is good surface hydrophobicity. In fact, the pores need to remain dry during MD operation since their flooding can cause a decrease of the distillate flux, as well as a reduction of the separation ability of the membrane [4,5]. This property can be controlled either by decreasing the surface wettability or by developing membranes with small pores and a narrow pore size distribution. Smaller pores are indeed less prone to wetting phenomena [6].

MD membranes exist in two basic configurations: hollow fiber and flat sheet. They can be arranged in different modules [7,8] and are generally prepared using hydrophobic polymers, such as polypropylene [9,10], polyethylene [11], polytetrafluoroethylene (PTFE) [12,13] and polyvinylidene fluoride (PVDF) [14–17], or using modified ceramic materials [18–20]. Among all of the aforementioned materials, PVDF is particularly attractive

**Citation:** Pagliero, M.; Alloisio, M.; Costa, C.; Firpo, R.; Mideksa, E.A.; Comite, A. Carbon Black/ Polyvinylidene Fluoride Nanocomposite Membranes for Direct Solar Distillation. *Energies* **2022**, *15*, 740. https://doi.org/ 10.3390/en15030740

Academic Editor: Alessandra Criscuoli

Received: 17 December 2021 Accepted: 17 January 2022 Published: 20 January 2022

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

**Copyright:** © 2022 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/).

because it can be easily dissolved in most common organic solvents so membranes can be prepared via traditional processes, such as thermally induced phase separation (TIPS) and non-solvent induced phase separation (NIPS) [21].

Four main configurations of MD have been developed, namely direct contact MD (DCMD), sweeping gas MD (SGMD), air gap MD (AGMD) and vacuum MD (VMD). The main differences among these configurations reside in the distillate section of the equipment [22].

In this work, VMD configuration has been used to assess the performance of the prepared membranes. In this mode, a partial vacuum pressure is applied on the distillate side of the membrane to remove the water vapor molecules from the membrane module. Since the partial water vapor pressure is constantly maintained as low as possible, VMD guarantees the highest fluxes among all of the MD configurations and reduces the heat losses correlated to heat conduction through the membrane. However, in some conditions, the net pressure difference across the membrane can favor pore flooding, possibly causing a reduction of the process productivity and separation ability [2,23].

Nowadays, MD is a mature technology and it appears to be one of the best technologies for developing off-grid desalination/wastewater treatment plants due to its modular configuration, low power consumption and ability to harness low-grade energy resources [24].

An important problem affecting MD scale up is the heat loss associated to water evaporation in the feed section along the surface of the membrane module. The phase transition that takes place at the feed/pore interface reduces the temperature of water flowing along the module in the feed channel, gradually decreasing the effective driving force of the process [25]. This effect in practice limits the maximum length of the membrane module. An effective solution to contain the feed temperature reduction along the membrane module recently developed is the so-called membrane localized heating that can be obtained using several different techniques. Tan et al. [26], for example, harnessed the high thermal conductivity of nickel and its capacity to be inductively heated by placing a nickel foam in the feed channel of the membrane cell in place of a traditional plastic spacer. The advantage was the possibility of increasing the foam temperature by electromagnetic induction, and therefore heating the feed directly inside the membrane cell. Experimental tests proved that this arrangement was able to improve the energy efficiency and the productivity of the DCMD process. A similar solution was followed by Anvari et al. [27] who spray-coated a commercial PTFE membrane with a layer containing multiwall carbon nanotubes coated with iron oxide. This added layer had magnetic properties that allowed for providing inductive heating directly on the surface of the membrane. It was observed that increasing the nanoparticles loading and the feed residence time improved the distillate flux since heat transfer from the membrane to the feed solution was enhanced. Moreover, these membranes showed high efficiency for feed solutions containing highly saline solutions, proving that this process can be applied to traditional brine treatment.

A particular approach recently explored aims to achieve localized heating by harnessing solar energy. One of the main advantages of the direct solar membrane distillation (DSMD) layout is that the feed temperature is raised in the boundary layer in contact with the membrane surface. Therefore, temperature polarization effects are drastically reduced, and the effective driving force of the process is increased [28]. The DSMD process has been proposed by many researchers as an effective solution to developing larger MD plants [29–31]. The basic solar MD setup consists of a photovoltaic panel used to provide the electrical current required to run the feed water circulation system, and a solar collector that is used to heat the feed. However, this simple configuration is characterized by some flaws, namely the heat losses along the pipes connecting the feed tank to the membrane cell and the need for large solar collectors to reach an adequate feed temperature [32]. Said et al. [28] recently tested a DSMD small pilot plant, assessing its performance in real life operation conditions. The plant was completely powered by solar energy, with a photovoltaic panel providing the electricity for feed circulation and a 0.12 m2 photoactive membrane cell. During the testing time the plant mean distillate flux reached 0.55 L/m2h with a rejection factor of 99.8%.

The main research efforts on the development of photoactive membranes for DSMD have been focused at present on the surface modification of commercial hydrophobic membranes; many researchers have coated membrane surfaces with an additional layer containing different fillers, such as carbon nanotubes [33] or carbon black (CB) [28,34–36] and silica-gold nanoparticles [35]. In particular, Wu et al. [35] created a layer composed of polyvinyl alcohol containing carbon black or silica-gold nanospheres. CB-coated membranes showed an increase of 33% in distillate flux when irradiated with solar-like light, while membranes treated with silica-gold nanoparticles produced a more limited improvement of 17%.

In this work, a novel approach was adopted, i.e., hydrophobic photoactive membranes were autonomously prepared in our laboratory, including carbon black directly inside the starting bulk of the polymeric matrix rather than adding a supplementary layer on a preformed membrane. These original nanocomposite membranes were then produced easily with a common NIPS technique. The aim of this study was to explore the effect of the filler on the membrane photothermal properties, as well as on the membrane's internal structure. The influence of CB loading on the MD performance was investigated. To the best of our knowledge, intrinsically photoactive membranes of this particular kind, intended for direct solar membrane distillation, have not been prepared and studied before.

#### **2. Material and Methods**

#### *2.1. Dope Solution and Photoactive Membrane Preparation*

The membrane type studied in this work was prepared in our laboratory through a method described in a previous article [37]. CB (Vulcan XC72R, Cabot Corp—Boston, MA, USA, primary particle size: 30–60 nm [38]) was first dispersed in a green organic solvent (i.e., triethyl phosphate, TEP, Merck—Darmstadt, Germany). To this end, a precise amount of CB powder was placed inside a 50 mL bottle together with 15 g of TEP and was then sonicated with an ultrasonic bath for 30 min at 25 ◦C to improve the dispersion.

The membranes were prepared via the NIPS technique, by dissolving a commercial PVDF (Solef® 6010, Solvay Specialty Polymers—Bollate, Italy, Mw 300 kDa) dissolved in the CB/TEP dispersions to create a CB doped solution and provide the desired photothermal properties to the final membrane. A 300 μm thick film of dope solution was cast on a commercial PET non-woven support (Viledon® FO-2401, Freudenberg—Weinheim, Germany) and then immersed in a weak coagulation bath containing ethanol 96 *v*/*v*% (VWR International—Radnor, PA, USA). After 2 h, the membranes were removed from the nonsolvent, washed with deionized water to remove ethanol from the pores and finally dried overnight at room temperature. Table 1 summarizes the preparation conditions of all of the membranes assessed. In the sample name, the first part refers to the polymer concentration in the dope solution, while the second part refers to the CB concentration.

**Table 1.** Preparation conditions of the tested membranes.

1 with respect to PVDF mass; 2 with respect to the solution mass.

#### *2.2. Light Absorption Measurements*

Absolute hemispherical reflectance spectra of the membranes were acquired using an UV/VIS/NIR spectrophotometer (Lambda9, Perkin Elmer—Waltham, MA, USA) equipped with a 150 mm integrating sphere. The inspected spectral range was between 200 and 2700 nm. A TiO2 coated layer was used as a blank sample.

For each sample, four spectra were registered, rotating the specimen by 90◦ each time. The four spectra allowed for estimating the surface homogeneity of the sample and compensating for any possible influence related to defects in the sample and its surface roughness. The final results were reported as absorbance values, which were then calculated as the mean of the four measurements.

The photothermal properties of the dry membranes were assessed by measuring the surface temperature change over time after a light source was turned on with the setup schematized in Figure 1.

The samples (17.5 cm2 surface area) were put in a special container made of expanded PVC—used to avoid heat losses related to conduction through the membrane—and placed under the light source at a distance (D) of 9 cm. The surface temperature was determined using an infrared thermometer (RS-8662, RS PRO—Kuala Lumpur, Malaysia).

#### *2.3. Membrane Performance Evaluation*

The prepared membranes were tested using a DSMD setup expressly built in the laboratory for this application. VMD configuration was selected because of the high distillate flux that it guarantees in comparison to other MD modes. Figure 2 reports a scheme of this apparatus.

**Figure 2.** Sketch of the DSMD setup used to test the photothermal membranes.

The membrane cell was equipped with a transparent polymethylmethacrylate (PMMA) film that allowed the passage of simulated solar light. The feed channel was 3 mm thick and the peristaltic pump used for recirculating the liquid had a flow rate of 40 mL/min, therefore, the velocity inside the membrane cell was 53 cm/min and the residence time was almost 15 s. The concentrate was recirculated to the feed tank continuously. Two K type thermocouples, connected to a digital data logger (HD2128.1, Deltaohm—Selvazzano Dentro, Italy), were mounted at the feed inlet and at the concentrate outlet to measure the liquid temperature gradient along the membrane module.

The driving force was applied by connecting the support side of the cell (grey in Figure 2) to a vacuum pump that maintained the permeate side at 20 mbar (absolute pressure). Before the start of each test, a precise amount of deionized water (40 mL) was put in a closed tank equipped with a graduated pipette that was used to measure the distillate flux through the lowering of the liquid level after a set time interval (60 min).

The only heat source for these tests was a light source mounted 7 cm away from the membrane surface. Each membrane was tested using two different light sources: a traditional 75 W incandescent light bulb (OSRAM—Munich, Germany), and a 100 W solar spectrum LED light (Shenzhen Milyn Technology—Guangdong, China). A reference test was also performed in order to evaluate the membrane performance without any external light source. In this case, the membrane cell was covered with an aluminum foil during operation.

The irradiance of the two light sources at different distances ranging between 4 cm and approximately 40 cm was determined using a solar power meter (ISM 410, RS PRO—Kuala Lumpur, Malaysia). The measurements were performed by placing the lamp vertically above the sensor.

Table 2 summarizes the operating conditions used to test the DSMD performance of the prepared membranes.

#### **3. Results and Discussion**

*3.1. Light Source Characterization*

A major system characteristic to be investigated when using light sources to simulate the solar energy is the irradiated power per unit area, which is called irradiance. The irradiance then must be compared to the average yearly standard solar conditions at the sea level (about 1000 W/m2) [40]. Figure 4 reports the values collected at different distances for the two lamps employed in this work.

**Figure 4.** Irradiance of the two used lamps, LED and incandescent, W, through a PMMA window (**left**) and measuring setup scheme (**right**).

The traditional incandescent lamp (red) was able to provide higher power than the solar-like LED lamp (yellow). However, both light sources showed a hyperbolic-like decrease of the irradiance value as the distance between the source and the sensor was increased. This imposed a strict control on the distance between the lamp and the membrane surface in the successive VMD tests.

#### *3.2. Light Absorption of Membranes*

Common cells for traditional MD processes are generally made of non-transparent materials, such as stainless steel or plastic, owing to their ability to withstand the feed temperature and salinity. However, such materials are not transparent to UV and visible radiation. Since membranes for DSMD application must be able to absorb light and to transform the energy captured in the available heat, their surface should be allowed to be irradiated with light. Among all of the transparent material, PMMA was selected for this purpose during the VMD cell assembling due to its low absorbance in a broad wavelength interval, as reported in Figure 5.

**Figure 5.** Light absorbance of the VMD cell window made of PMMA (black) and ASTMG 173 AM1.5 solar emission spectrum (blue).

Spectral solar irradiance was shown and PMMA proved to be completely transparent in the majority of the wavelength interval of solar emission spectrum; it presented intense absorption bands only below 400 nm and over 1300 nm. In the interval 400–1300 nm, almost 90% of the whole solar irradiance is emitted. Therefore, PMMA was considered an ideal material to create the window required in this special VMD cell.

When the membrane surface is irradiated with light, the obvious key parameter for DSMD application is the membrane photoactivity that can be estimated by measuring the absorbance in the UV/visible wavelength interval. These measurements were performed using a Perkin Elmer Lambda9 UV/VIS/NIR spectrometer in the spectral range between 200 and 1400 nm (recall the comments on Figure 5). Figure 6 reports the registered spectra for the four membranes prepared at increasing CB loading as well as for the CB as such.

**Figure 6.** UV/VIS/NIR absorption spectra of the four membranes and of carbon black.

CB (red dotted line in Figure 6) showed a broad absorption band in the whole wavelength interval investigated with a wide maximum in the visible light range.

On the other hand, the pure PVDF membrane (blue line in Figure 6) was characterized by a far less intense absorption in the same wavelength interval. Most of the solar radiation evidently was reflected by the white surface of the membrane.

The introduction of CB inside the dope solution greatly increased the light absorption of the samples in the UV/VIS/NIR spectral region of interest. In particular, the absorbance showed an impressive enhancement, moving from 145\_0 sample to 145\_2 and then 145\_5, i.e., with an increase in CB concentration.

Figure 6 illustrates that the unloaded PVDF membrane had a very low ability to capture solar energy with a slight increase at higher wavelengths. The 145\_2 sample partly retains the effect of the PVDF matrix, showing an almost linear growth of the absorbance, but, in contrast, it reaches noticeable values at higher wavelengths. The samples 145\_5 and 145\_75 exhibited a sharp increment of absorbance at lower wavelengths and their spectra appeared quite similar to that of the CB as such. Moreover, there does not seem to be a substantial difference between the behaviors of the two membranes.

These features were reflected in the photothermal activity shown by the various samples. Figure 7 reports the surface temperature change over time when the membrane surface was exposed to the two different lamps (A: LED lamp, B: incandescent lamp).

**Figure 7.** Membrane surface temperature change using (**A**) LED lamp and (**B**) incandescent lamp.

The two lamps were characterized by different emission spectra (Figure 3), as well as a different irradiance (Figure 4). These tests were performed by placing the light source 9 cm above the membrane, meaning that the LED irradiance was around 90 W/m2, while the incandescent lamp provided about 435 W/m2 to the membrane surface. This difference was confirmed by the surface temperature measurements. Using the LED light (Figure 7A), the CB loaded membrane's temperature rose almost instantly from about 20 ◦C to 30 ◦C and continued to increase for several minutes reaching a stable value at around 35 ◦C. The incandescent lamp, thanks to its higher irradiance at an equal distance, showed a similar trend but higher temperatures were developed on the membrane surface that reached for all the membranes values of about 60 ◦C. In both cases A and B, adding CB to the dope solution generated a great difference between unloaded and loaded membrane and an almost 40% temperature increase was obtained for all of the samples. However, the CB concentration had only a marginal effect on the surface temperature of the membrane since, when irradiated under the same conditions, all of the CB-loaded membranes performed similarly. Table 3 reports some literature data of the maximum surface temperature reached by photoactive membranes irradiated with solar-like light sources.

**Table 3.** Literature data of surface temperature of photoactive membranes.

While it is almost impossible to find results obtained under identical conditions, the data reported in Table 3 highlight the excellent performance of the membranes developed in this work. In fact, despite being tested with a less powerful light source, the samples prepared directly including CB inside the PVDF matrix were able to reach temperatures higher than—or at least similar to—the ones obtained with coated membranes.

#### *3.3. Distillation Performance*

The membrane distillation performance was evaluated using the setup shown in Figure 2. The feed temperatures were registered over a one-hour test for each light source and the registered differences during this time span, ΔTm, are reported in Figure 8, and are expressed as follows:

$$
\Delta \mathbf{T\_m} = \frac{(\mathbf{T\_{out\_{60}}} - \mathbf{T\_{out\_0}}) + (\mathbf{T\_{in\_{60}}} - \mathbf{T\_{in\_0}})}{2} \tag{1}
$$

where the subscripts in60 and out60 indicate the liquid temperature at the cell inlet and outlet after 60 min of testing, respectively, while the in0 and out0 subscripts represent the same values recorded at the start of the tests.

**Figure 8.** Mean feed temperature difference between MD test end and start with no light (NL, black), LED lamp (yellow) and incandescent light bulb (W, red).

For all of the samples, the test performed without light (black in Figure 8) showed a decrease of the feed temperature over time. In the absence of any external energy supply the latent heat absorbed by water evaporation at the membrane surface caused an appreciable feed cooling.

The ΔTm values obtained when the membranes were irradiated with a light source exhibited a completely different trend. The LED lamp (yellow in Figure 8) was able to counteract the heat loss related to water evaporation but, since its irradiance was quite weak (see Figure 4), the feed temperature remained almost constant during the test. On the other hand, the incandescent lamp (red in Figure 8) provided more energy to the system and the feed temperature was increased during the time span of DSMD operation for all of the tested samples. These findings are in agreement with the data reported in Figure 7A,B.

The beneficial effect of the light irradiation obviously affected the distillate fluxes as expected. Figure 9 summarizes the results concerning the MD tests for all of the samples and all of the lighting options.

**Figure 9.** (**A**) mean distillate flux and (**B**) flux enhancement registered during the MD tests with no light (black), LED lamp (yellow) and incandescent light bulb (red).

The distillate fluxes (Figure 9A) obtained during the tests without light sources confirmed the results obtained during a previous work carried out in our laboratory [37]. That study also revealed that the addition of CB to the dope solution enhances both the pore size and porosity of the membrane and therefore can induce an increase in the membrane permeability. It was found that introducing a proper amount of CB led to an increment of both pore size—which passed from about 100 (sample 145\_0) to about 850 nm (sample 145\_75)—and overall porosity, which rose from 76% to 87%. Both of these parameters strongly affect the membrane mass transfer resistance; it is well known that larger pore size and porosity values provide higher transmembrane vapor fluxes during MD operation. Moreover, in the same study, it was demonstrated that higher CB loadings led to an enhancement of the matrix hydrophobicity, preventing negative phenomena, such as pore flooding. For all of these reasons, even without light irradiation the distillate flux of the 145\_75 sample was somewhat higher compared to the one registered for the pure PVDF membrane (145\_0).

In this work, using only light as the energy supply for feed heating was proved to be effective for all of the samples prepared. In fact, under irradiation, the distillate flux was always enhanced. This effect became more intense as the CB loading in the dope solution was raised from 2 wt% to 7.5 wt%. The flux enhancement (FE%) reported in Figure 9B was calculated using the following equation:

$$\text{FE}\% = \frac{\left(\text{J}\_{\text{Light}} - \text{J}\_{\text{NL}}\right)}{\text{J}\_{\text{NL}}} \cdot 100 \tag{2}$$

where JLight is the distillate flux obtained irradiating the membrane with a light source while JNL is the distillate flux without irradiation.

Increasing the CB concentration improved the light absorbance of the membranes (Figure 6) and boosted the feed temperature rise on the membrane surface. As a consequence, the vapor pressure at the pore entrance got higher and enhanced the driving force of the process. The distillate flux increment became more evident as the CB loading grew. It can be seen from Figure 9B that, for the 145\_75 sample, a distillate flux enhancement of up

to 60% and 70% was reached when the LED or incandescent lamps were used, respectively. Table 4 reports a VMD performance comparison between the membranes prepared in this work and a sample tested by Ma et al. [42] who coated a PTFE membrane with carbon nanotubes.

The two membranes were tested under similar conditions in terms of vacuum pressure, feed temperature and light irradiance and showed comparable distillate fluxes. These results confirmed the potential of mixed matrix membranes for DSMD application. The advantages of preparing a single layer membrane are, primarily, the easier procedure required and the more robust final structure. On the contrary, when a commercial membrane is coated with photothermal conversion materials, a poor adhesion between the support material and the surface skin can induce the delamination phenomena that can undermine the integrity and the performance of the membrane.

#### **4. Conclusions**

A simple and innovative preparation method to produce photoactive membranes was proposed, which involves incorporating the light absorbing filler directly inside the membrane matrix. The preparation procedure was based on a traditional NIPS technique and the filler was first dispersed in the polymeric dope solution. In line with the goals of an environmentally sustainable and safe membrane preparation process, a green solvent, triethyl phosphate, was used. The main novelty in this work was incorporating the selected filler, carbon black, inside the entire polymeric matrix. This particular formulation allowed to produce favorable outcomes on the internal membrane structure. Nearly all of the strategies to achieve surface heating MD are currently based on membrane surface modification by various techniques. However, based on our findings, inclusion of carbon black nanoparticles inside the whole membrane structure seems to have multiple beneficial effects on the membrane performance. In fact, the filler acts not only on the photothermal properties enhancing the light absorbance, but also on the membrane morphology increasing porosity and pores size. These latter characteristics, which are very important to reduce the membrane mass transfer resistance, were improved with the rising CB content.

In terms of photothermal properties, membranes prepared with a sufficient CB loading were able to reach satisfactory surface temperatures when exposed to light sources.

On the whole, as the carbon black loading was raised, the effectiveness during direct solar membrane distillation operation was improved and the membranes prepared with the highest CB concentration showed the best flux enhancement factor.

**Author Contributions:** Conceptualization, A.C. and M.P.; methodology, M.P.; investigation, M.A., R.F., E.A.M. and M.P.; writing—original draft preparation, M.P. and C.C.; writing—review and editing, A.C. and C.C.; visualization, M.P.; supervision, A.C. and C.C. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**