To develop monoculture and mixed culture biofilms, A. niger, Nostoc sp., and S. maltophilia/B. subtilis were used to represent fungi, cyanobacteria, and bacteria, respectively. From each, one loopful of microbes was introduced to 250 mL CCM [13] broth to produce monocultures. Appropriate volumes of each monoculture was taken and inoculated to have two contrasting ratios [F:B:C 1:3:12 (α), and 12:4:1 (β)] in to 15 ml sterilized centrifuge tubes containing 10 ml of CCM medium to develop different biofilms; FBBs, FCBs, BCBs and FCBBs. In this manner, 14 different biofilms were developed; FBSα, FBBα, CBSα, CBBα, FCBSα, FCBBα, FBSβ, FBBβ, CBSβ, CBBβ, FCBSβ, FCBBβ, FCα and FCβ [C = Nostoc sp., F = A. niger, BS = gram (−) S. maltophilia, BB = gram (+) B. subtilis]. The cultures were incubated for seven days to form biofilms. Completely randomized design was employed with three replicates for each biofilm type.

The developed biofilms were observed under low and high power of the light microscope by staining with lacto-phenol cotton blue to characterize morphological differences among the different types of biofilms.

The extraction of EPS was performed by combining physical and chemical methods [14] . A NaCl solution was prepared by dissolving 5 g of NaCl in 100 mL of sterilized distilled water. Ten microliters of the solution was added to 15 ml centrifuge tubes that contained the developed biofilms. Then, they were subjected to ultra-sonication for 10 minutes, followed by centrifugation at 5000 rpm for 10 minutes. Finally, the supernatant in the centrifuge tube was taken for further analyses.

The data were analyzed using the statistical software Minitab version 17. FTIR absorbance data were analyzed using one-way analysis of variance (ANOVA), followed by Tukey’s HSD test. Probability ≤ 0.05 was used as the threshold for significance.

In the FBB, fungal filaments acted as the biotic surface for bacterial cells to colonize (Figure 1), as was also observed in previous studies [16] [17] . The attachment of bacterial cells to fungal filaments initiated within the first 48 hours of the biofilm formation and reached to its maximum attachment by 96 hours. The accumulation of EPS was clearly observed during the maturation of the FBB, and the highest quantity of EPS was observed on day 7 (Figure 1).

The same observations were made in CBBs. In the FCBs, fungal filaments were intertwined with cyanobacterial threads and they were surrounded by EPS. In the FCBBs, bacterial cells attached to fungal and cyanobacterial filaments surround by an extracellular matrix.

Production of mainly lipids, polysaccharides and proteins was observed in the biofilm-EPS (Figure 2). The highest amounts of lipids, proteins and polysaccharides were produced by the FCBSα biofilm, whereas the lowest amounts were produced by the CBBα, CBSβ and FCα, respectively (Figure 2).

In FCBBs, S. maltophilia performed better than B. subtilis in producing lipids, proteins and carbohydrates (Figure 2). In FBBs, B. subtilis did better than S. maltophilia in producing lipids (Figure 2), and S. maltophilia and B. subtilis performed similarly in producing proteins (Figure 2). Moreover, S. maltophilia produced higher amounts of carbohydrates than B. subtilis (Figure 2). In CBBs, S. maltophilia and B. subtilis performed similarly in producing lipids and proteins (Figure 2), but B. subtilis produced a higher amount of polysaccharides than S. maltophilia (Figure 2).

In FCBBs, the ratio α performed better than the ratio β in producing lipids, proteins and carbohydrates (Figure 2). In FCBs, the ratio β produced a higher amount of carbohydrates than the ratio of α (Figure 2). In FBBs and CBBs, both ratios performed similarly in producing lipids, proteins and carbohydrates (Figure 2).

Compound diversity of the fungal-bacterial biofilms (here the A. niger-S. maltophilia biofilm) was higher than that of the other biofilms (Figure 3). Possibly, it is the reason for the success of fungal-bacterial biofilms in industry [18] . Therefore, the A. niger-S. maltophilia biofilm was characterized further in order to identify the composition of its biofilm-EPS.

Production of mainly lipids, polysaccharides and proteins was observed in the biofilm-EPS (Figure 4). They are reported to play a crucial role in gene regulatory, protein and signaling networks which govern functioning and stability of biofilms [3] . Moreover, they also contribute to enhance functioning of ecosystems like soil and human body [7] .

In the FBB, number of high molecular weight metabolites (HMWMs) was significantly higher than that of low molecular weight metabolites (LMWMs) (P < 0.05, Figure 5). In natural systems, the HMWMs like multifunctional enzymes

work for the generation of diverse chemistry. The LMWMs participate in globally important processes which determine the physiological characteristics of Prokaryotes, Archaea and Eukaryotes, and also regulation of species-species relations [19] . Those processes influence biomineralization and even the ozone level in the stratosphere [20] .

In the A. niger-S. maltophilia biofilm, 214 biochemicals were detected from LC-MS, and only 5 of them were fully identified. In addition, 14 biochemicals were predicted with 90.3% - 98.9% probability. In all, only 9% of the biochemicals of the LC-MS spectrum was characterized.

Most of the biochemicals identified belong to compounds related to medicines, and the rest to agrochemicals, bioremediating agents and industrially important chemicals which are used individually in our society (Table 1 and Table 2). However, they collectively carry out many physicochemical and biological functions via nodes of the complex interactive biochemical networks in the natural environment. For example, plant growth promoting rhizobacteria (PGPR)

exert their action in the rhizosphere by producing a wide array of extracellular molecules for communication and defense, which play a crucial role in biochemical networks for beneficial plant-microbe interactions [42] . A similar functioning has been observed in metabolic networks of human gut microbiome [43] .

Microbial society of a complex biofilm is comparable to human society. The biofilm microbes produce the biochemicals (goods) they require for their functioning and health, which lead to sustenance. The biochemicals used by the biofilms are as same as those which are utilized by the human society. The striking difference is that humans use them individually with some adverse side effects and relatively low efficacy whereas the biofilm microbes utilize them simultaneously to improve the network interactions, which leads to better functioning in the environment, as has already been exemplified in agriculture [6] . This striking difference in utilization of the biochemicals and their enhanced effect when they act simultaneously needs further investigations. Moreover, some behavioral patterns and intelligence of microbes are analogous to those of humans [44] [45] . These similar features clearly show the historical evolution of life starting from microbes on the earth.

At present, the ecologically important biochemicals are depleted in the nature due to degradation caused by anthropogenic activities, and it leads to deteriorate ecosystem health [46] . When biochemicals are added to any degraded ecosystem,

its biodiversity will increase with simultaneous improvement of ecosystem functioning that leads to reinstate ecosystem health. The biofilm-EPS is a good candidate in supplying the biochemicals to any degraded ecosystem for its recovery, as depicted in the cartoon in Figure 6. This concept is valid for managed as well as natural ecosystems, including even human body ecosystem [7] [46] .