Sea water desalination is an increasing practice all over the world. Urban populations that live close to coastal areas can acquire significant quantities of fresh water through desalination [1]. Reverse osmosis (RO) membranes perform an effective desalination method in terms of permeability, packing density, and fouling control [1] [2] [3]. Conversely, membrane fouling reduces membrane lifetime and increases cost-effective maintenance. In particular, organic fouling could result in water flux decline through RO membranes so complex structures are formed by dissolved organic matters in combination with other substances [4] [5] [6]. Unlike inorganic fouling caused by salt precipitation, organic fouling consists of marine organisms and their metabolic residues such as extracellular polysaccharides, proteins and lipids. Bacteria cells are enclosed in self-produced extracellular polymeric substances that adhere the bacteria to osmosis membranes [7] [8] [9].

Bacteria fouling is difficult to eradicate with pre-treatment methods, which has huge implications on operational costs. Several approaches based in dissecting fouled-RO membranes have given greater understanding of the specific origin and composition of biofilms.

Bacteria analyses of RO membranes have identified bacteria from phyla β-proteobacteria, γ-proteobacteria and α-proteobacteria, with representative genus of Sphingomonas, Pseudomonas and Acidovorax [10] [11]. This has allowed science to discover the types of bacteria that adhere, grow and participate in forming the biofilm on RO membranes. Biochemical analysis of fouled-RO membranes has also described conspicuous information on bacteria communities, although only between 0.01% and 3% of the population in natural environments has been described.

Notwithstanding the above, molecular identification based on 16s rRNA gene and the specific genes that encode both fouling and anti-fouling proteins can also reveal important information and allow for the development of biotechnological applications in the future. We hypothesised that appropriate identification of the dominant bacteria from biofouling, along with the study of gene behaviour has to improve RO-membranes designs in order to prevent biofouling. The objective of this work was to molecularly characterise bacteria associated with several kinds of RO membranes with culture-dependent and culture-independent techniques, based on region 16s rRNA.

The microbial community of biofouling in RO membranes is barely known since many papers are based on bacteria which are easily cultivable on a nutrient rich artificial medium. As a benchmark, this paper focuses on isolates using approaches based on culture-dependent and culture-independent techniques through the clone library. Differences between the four reverse osmosis membranes analysed were not observed.

Although bacteria were able to grow in all the culture media—namely R2A Agar, seawater supplemented with agar, tryptone yeast agar prepared with seawater, and marine agar-, R2A Agar media showed the highest recovery of bacteria colonies and was used to differentiate bacteria. Thus, six different colonies were characterised and selected in order to obtain pure culture (Table 3).

Biochemical tests performed with API 20 NE are shown in Table 4. Substrate assimilations were read after 24 and 48 h. The results were interpreted after 48 h using the identification software from the BioMérieux web site [19]. When required, API tests were carried out two or three times to corroborate ambiguous sugar results that were inconclusive at 42˚C. The results agreed with those previously reported in bacteria from sugar-starved habitats that were able to develop alternative metabolic pathways for sugars [20]. The API results also only showed gram-negative bacteria colonies (Table 4).

Although it was easy to grow the bacteria, the presence of gram-negative bacteria suggested that the natural bacteria communities were not fully represented. Hence, this work was also supported by culture-independent techniques. Khambhaty and Plumb [21] reported that a culture-based approach favoured the presence of bacteria such as proteobacteria over other bacteria groups. Moreover, over 99% of bacteria in seawater reverse osmosis membranes cannot be cultivated on nutrient rich artificial media [22].

Moreover, the API test allowed us to identify four out of six bacteria colonies at species level based on a >90% confidence level reported by the manufacturer (Table 5).

As Table 5 shows, the bacteria species identified were Vibrio metschnikovii, Aeromonas salmonicida ssp. salmonicida w., Brevundimonas vesicularis and Sphingomonas paucimobilis. These biofouling bacteria were reported on the four RO membranes analysed, indicating that they probably adapted to attaching on RO membrane surfaces as they grew on different types of membranes. Although all these species were recognised as marine species, Sphingomonas sp. also constituted the dominant bacteria colonies. Sphingomonas has been described as the dominant bacteria on RO membranes of desalination plants and in water purification processes, as related with the formation of biofilms by this bacterium [11]. Sphingomonas also facilitates the adherence of other bacteria and encourages the maturing of the biofilm [23]. Thus, these results open the door to investigating bacteria communities involved in biofouling and their role in triggering a biofilm network.

Several Sphingomonas colonies were molecularly identified using the 16s rRNA gene. Using maximum likelihood, the analysis recovered a higher resolution

and grouped 16s rRNA Sphingomonas with another 43 16s rRNA gene sequences annotated as belonging to this genus (Table 6).

There were a total of 1023 positions in the final dataset. The phylogenetic analysis positioned the Sphingomonas sequences, from colonies determined biochemically, within the Shingomonas sp. genus. This result (Figure 1) demonstrated that the Sphingomonas obtained in this study could be regarded as representing a species within the genus.

On the other hand, sequencing of 16s rRNA gene amplification fragments, assumed as operational taxonomic units (OTU) from independent cultures, revealed forty 16s rRNA gene fragments which were identified. Thirty percent of the total number of amplification fragments (total number of OTU, 40) was related to Pseudomonas species and 3 OTUs to Sphingomonas. The dominance of Pseudomonas may be explained by the association of these organisms with submerged surfaces. The remaining sequences were unrelated to other known bacteria and these were identified as non-representative. A total of 25 OTUs were detected as possible chimeras and were excluded from analysis.

Evolutionary analysis using the Maximum Likelihood method involved 24 nucleotide sequences (Table 7) with a total of 687 positions in the final dataset.

The phylogenetic analysis positioned the Pseudomonas sequences in a clade within Pseudomonas marincola (Figure 2). Pseudomonas marincola has been recently annotated as a marine species [22].

All in all, assuming the criterion for differentiating bacteria with a 16s rRNA gene sequence similarity value of over 95% [24], the phylogenetic trees of Sphingomonas sp. and Pseudomonas marincola were consistent with the molecular characterisation and their affiliations to the respective genus.

From an application point of view, it is worth mentioning that Sphingomonas and Pseudomonas have been described as containing an open reading frame coding for enzymes that are necessary for the initial stages of biofilm development [13]. Therefore, these molecular identifications open the door to studying the gene expression levels that encode the attachment protein and the biofouling potential of these bacteria. Gene expression levels of fouling and antifouling proteins under different experimental conditions (i.e. high-pressure water, permeate flow) can help to: 1) indicate a functional inclination to form biofilm on a reverse osmosis membrane, 2) discover how the membrane surface is colonised, and 3) determine how extracellular polysaccharides can help to initiate biofilm formation.

In conclusion, Sphingomonas sp. and Pseudomonas sp. assigned as P. marincola, have been identified on fouled marine reverse osmosis membranes. Culture-dependent and culture-independent approaches (clone library) showed that although the bacteria communities were not all identical, two dominant bacteria were commonly observed on the four RO membranes analysed.

P.G.J. conceived, designed and wrote the manuscript. M.C.A. conducted the phylogeny analysis. C.E.P and I.L.A. carried out microbiological assays. J.A.H.M. and J.R.B.R. analysed microbiological data. All the authors read and approved the manuscript.

This research was supported by the collaboration of Ministerio de Ciencia, Innovación y Universidades and from the Universidad de Las Palmas de Gran Canaria. (Grant CGL2016-78442-C2-2-R, GOBESP2017-04 ULPGC) to PGJ, and co-funded by ERDF funds, INTERREG MAC 2014-2020 programme, within the DESAL+ project (MAC/1.1a/094)”. M.C.A was supported by a predoctoral fellowship granted by the University of Las Palmas de Gran Canaria (ULPGC2016).

The authors declare no conflict of interest.

Garcia-Jimenez, P., Carrasco-Acosta, M., Payá, C.E., López, I.A., Rodríguez, J.R.B. and Melián, J.A.H. (2019) Composition and Molecular Identification of Bacterial Community in Seawater Desalination Plants. Advances in Microbiology, 9, 863-876. https://doi.org/10.4236/aim.2019.910053