Journal of Membrane Science 545 (2018) 240–249

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Journal of Membrane Science

journal homepage: www.elsevier.com/locate/memsci

Microbial diversity in biofilms from reverse osmosis membranes: A short MARK

review
Olga Sánchez
Departament de Genètica i Microbiologia, Facultat de Biociències, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain

A R T I C L E I N F O A B S T R A C T

Keywords: Biofilm occurrence in reverse osmosis (RO) membranes constitutes a key problem in water plants, leading to a
Reverse osmosis membranes loss of performance and a decrease in treatment efficiency. Knowledge on the microbial community composition
-Omics and the links to operational conditions is therefore crucial to understand biofilm formation and develop proper
Wastewater microbiology biofouling management strategies. The advent of molecular methodologies in the last years has evidenced the
Biofouling
failure of culture-dependent techniques to assess the great level of diversity present in RO membranes. More
recently, the application of –omics has unveiled a new horizon to study microbial diversity and function in
wastewater treatment. This manuscript focuses on the latest discoveries in prokaryotic diversity in RO mem-
branes, highlighting the importance of operating conditions and cleaning strategies in community structure. The
current knowledge of biofilm dynamics in these systems is also reviewed. However, despite the enormous
possibilities that metagenomics and metatranscriptomics can offer, studies using these methodologies in RO
membranes are still scarce, and further efforts to uncover a core microbial composition and expression patterns
of relevant genes are still needed.

1. Introduction
Membrane technologies, and particularly reverse osmosis (RO),
constitute an interesting choice for water treatment and purification,
especially in those areas which suffer from water scarcity and opt to the
reuse of reclaimed water as an alternative to conventional freshwater
sources. Apart from drinking water supply and wastewater purification,
RO applications include deionized water production and desalination,
since this technology enables the efficient removal of a wide variety of
contaminants, such as organic compounds, total dissolved solids or
microorganisms from a variety of streams of different qualities (sea-
water, natural, or industrial), offering a high quality product water
[1,2]. Yet, fouling occurrence in RO membranes represents a major
obstacle for their performance and economic viability; it shortens
membrane life by increasing transmembrane pressure and decreasing
membrane flux as well as pollutants retention rate [3,4], ultimately
leading to a loss of a capacity of the water plant. Membrane fouling can
be caused by different factors, such as organic matter deposition, mi-
croorganisms, and inorganic components, and it is mainly affected by
aspects like feed water chemistry or membrane properties [4]. Among
these, biofouling has drawn researchers’ attention in the last decades
because it is the principal reason for the deterioration of membrane
operation, being microbial deposition and the extracellular polymeric
substances (EPS) excreted by bacteria the main elements of membrane
biofilms [4]. The EPS matrix offers important advantages for embedded
microorganisms [5], but decreases the membrane flux and rises the
fluid resistance near the membrane surface, while microbial deposition
causes an increase in osmotic pressure and a reduction in permeate flux
and salt rejection rate [4].
Microbial antifouling strategies should then be based on a correct
identification of the cause of the problem and on an effective mon-
itoring of biofilm development for further prevention. Therefore, a deep
comprehension of microbial populations in biofilms is essential for
proper biofouling management strategies. Biofilms are composed of
complex communities, and preliminary studies using culture-dependent
methods in RO membranes [3,6] showed to be insufficient to ascertain
the composition of microorganisms in these systems, since the fraction
of culturable bacteria is very low compared to total cell counts [7]. The
advent of modern molecular techniques, such as genetic clone libraries,
fluorescence in situ hybridization (FISH), and nowadays, the new
–omics, overturned this scenario and circumvented cultivation and its
underlying selectivity. Knowledge on these intricate microbial assem-
blages has therefore been improved, and a more accurate representa-
tion of microbial diversity is currently presented. In this manuscript, a
review of the latest discoveries concerning the diversity of prokaryotic
microorganisms in RO membranes is presented, with special emphasis
on the influence of operating conditions and cleaning measures on
microbial diversity and the role of certain bacteria in biofilm dynamics.

E-mail address: olga.sanchez@uab.es.

http://dx.doi.org/10.1016/j.memsci.2017.09.082
Received 15 December 2016; Received in revised form 19 July 2017; Accepted 26 September 2017
Available online 28 September 2017
0376-7388/ © 2017 Elsevier B.V. All rights reserved.
O. Sánchez
Table 1
Some studies showing the phylogenetic groups identified in biofilms from RO membranes using culture-dependent techniques, which in some cases were combined with molecular
methods. Abundance numbers make reference to viable counts, % of isolates, % of OTUs (clone libraries) or % of probe positive cells (FISH). The most abundant groups are highlighted in
grey for each study.
2. Microbial diversity in RO biofilms
2.1. Methods to study microbial diversity
The first approaches dealing with microbial diversity in RO mem-
branes used cultivation methods for quantifying and identifying viable
bacteria and fungi present in the autopsies of RO membranes (Table 1).
The study of Ridgway et al. (6) was also accompanied by microscopy
observations to examine biofilm structure, and revealed that biofilms
were composed of several layers of compacted bacterial cells, which
formed an extensive network of extracellular polymeric fibrils. Some
works have combined the use of culture-dependent techniques with
molecular methodologies (Table 1) such as genetic clone libraries of
16S rRNA gene or Fluorescence In Situ Hybridization (FISH), while
others have utilized exclusively culture-independent methods to assess
microbial diversity (Tables 2 and 3).
In general, the use in the recent years of tag sequencing techniques
based in the analysis of 16S rRNA gene has widened our knowledge of
microbial diversity in wastewater microbiology [9,10]. Compared to
fingerprinting techniques or genetic clone libraries, these methodolo-
gies can provide for the first time thousands of sequence reads, evi-
dencing that they can offer a better assessment of the bacterial
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Journal of Membrane Science 545 (2018) 240–249
community present in wastewater treatment systems. However, with
the exception of FISH, all these methods are not free of the bias asso-
ciated to PCR amplification and primer sets [9]. By contrast, FISH
overcomes this problem, but is limited by the available number of
probes. On the other hand, the current rise of –omics has allowed the
study of phylogenetic and potential functional diversity of microbial
communities, circumventing the use of PCR and culture-dependent
methods. Millions of sequence reads can be obtained at a reasonable
low cost, making possible the study of all the genes present in a mi-
crobial population (metagenomics), or the analysis of RNA transcripts
and therefore gene expression (metatranscriptomics). Nevertheless,
metagenomics requires substantial improvements in computational in-
frastructure and appropriate bioinformatic tools to handle and manage
the massive amounts of sequence data generated for making sense of
the information.
2.2. Microorganisms in RO biofilms
The common bacteria isolated and identified in the first studies of
RO membranes included Acinetobacter (Gammaproteobacteria),
Pseudomonas (Gammaproteobacteria), Alcaligenes (Betaproteobacteria),
Corynebacterium (Actinobacteria), Flavobacterium (Bacteroidetes), and
O. Sánchez Journal of Membrane Science 545 (2018) 240–249

Table 2
Phylogenetic groups identified in biofilms from RO membranes with molecular methods (DGGE, clone libraries and FISH) in studies reported from the last decade. Abundance numbers
make reference to % of OTUs (clone libraries) or % of probe positive cells (FISH). The most abundant groups are highlighted in grey for each study.

Bacillus (Firmicutes) among others [3,6]. However, the microbial di-
versity retrieved with culture-dependent methods do not necessarily
reflect the diversity of biofilms, since only a small fraction of bacteria
can be cultivated under standard laboratory conditions [7], a short-
coming that can be addressed using molecular-based techniques. In
general, the degree of concurrence between culturing and the different
molecular methods is low, since some microorganisms characterized
with molecular tools are not recovered using conventional isolation
methods. For example, this is the case for some representatives from
Betaproteobacteria, or a number of phylotypes related to yet uncultured
organisms from Alphaproteobacteria, Bacteroidetes, Planctomycetes
and candidate divisions TM6 and TM7 [11].
In virtually all the molecular studies referenced in Tables 1–3, in-
dependently on the source of influent waters [secondary effluents from
wastewater treatment plants (WWTPs) or effluents from industrial,
purification or desalination plants], there was a constant supremacy of
the phylum Proteobacteria, particularly members of the Alpha, Beta
and Gammaproteobacteria. Within the Alphaproteobacteria, the order

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Table 3
Phylogenetic groups identified in biofilms from RO membranes with tag sequencing methods in studies reported from the last five years. Abundance numbers make reference to % of
OTUs. The most abundant groups are highlighted in grey for each study.
Rhizobiales stand out particularly in several studies [10–17], suggesting
an important role of this group in biological fouling of membrane
processes. Genera such as Rhodopseudomonas, Afipia, Methylocystis,
Ochrobactrum, Bosea, Oligotropha, Methylocella, Hyphomicrobium, Nor-
della, Devosia, Mycoplana, Bradyrhizhobium, Ensifer, Rhizobium or Si-
norhizobium, among others, have been found in biofilms from RO
membranes (Table 4). In fact, Pang and Liu [11] demonstrated through
the analysis of carbon substrate utilization patterns that the Rhizobiales
strains were metabolically versatile under aerobic conditions, a phe-
notypic trait that may be critical because readily biodegradable organic
carbon sources can become limiting in the RO environment due to
upstream biological pretreatment. According to these authors, these
organisms could adapt to this environment by switching substrate types
to avoid direct competition with other biofilm populations. Further-
more, some Rhizobiales isolates have been reported to degrade che-
micals such as parathion, quaternary ammonium alcohols, substituted
thiophens, metolachtor, polyacrilamide and 4-chlorobenzoic acid [11].
In addition, several genera of Rhizobiales, including Rhizobium, Shi-
norhizobium or Bradyrhizobium, can potentially secrete EPS and con-
tribute to biofilm formation.
On the other hand, two other alphaproteobacterial families,
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Journal of Membrane Science 545 (2018) 240–249
Rhodobacteraceae and Sphingomonadaceae, frequently appeared in
different surveys where microbial diversity of RO membranes was
characterized (Table 4). Members of the genera Ruegeria, Roseobacter,
Rhodobacter, Donghicola, Antarctobacter, Citreicella, Pelagibaca or Sulfi-
tobacter, belonging to Rhodobacteraceae, seem to be associated to
mature biofilms in these systems, where nutrient competition is estab-
lished [18]. In contrast, Sphingomonadaceae, and particularly the
genus Sphingomonas, have often been reported to be involved in biofilm
formation as pioneers [13,19], probably due to their well-known ca-
pacity to colonize solid surfaces favored by their swarming and
twitching mobility and the excretion of abundant exopolymers [20];
additionally, they are metabolically well adapted to substrate limitation
in the initially clean RO system, and are also able to survive at high
nutrient concentrations, utilizing a broad range of organic compounds
and environmental contaminants [12].
Within the Betaproteobacteria, the order Burkholderiales is also
frequently retrieved in RO biofilms, with genera like Acidovorax,
Hydrogenophaga, Polaromonas, Xylophilus or Achromobacter among
others (Table 4). Concerning the Gammaproteobacteria, several studies
confirmed the presence of the genus Pseudomonas in RO membranes
[3,6,12–14,16,21–24]; in turn, Pseudomonas sp. has been used as a
 O. Sánchez

Table 4
Main families and genera belonging to the most abundant groups retrieved with molecular tools in biofilms from RO membranes (groups highlighted in grey in Tables 1–3).

Ref. Alphaproteobacteria Betaproteobacteria Gammaproteobacteria Bacteroidetes Firmicutes

[10] Rhodopseudomonas palustris, Afipia sp. Methylocystis

parvus, Magnetospirillum sp.
[11] Ochrobactrum sp. Bosea sp., Oligotropha sp., .
Rhodopseudomonas sp. Methylocella sp.,
[54] Ruegeria sp. Roseobacter sp., Rhodobacter sp., Donghicola
sp., Sphingomonadaceae
[12] Sphingomonas sp., Caulobacter gingengsoli,
Hyphomicrobium sp., Nordella sp.
[13] Sphingomonas sp., Sphingopyxis sp., Azospirillum sp., Candidatus "Nitrotoga antarctica", Nitrosomonas sp.,
Ancanthamoeba endosymbiont, Hyphomicrobium sp. Burkholderiales
[14] Acanthamoeba endosymbiont, Azospirillum sp., Acidovorax sp., Aquamonas fontana, Aquaspirillum
Hyphomicrobium sp., Devosia insulae, Mycoplana sp., autotrophicum, Hydrogenophaga sp., Polaromonas
Bradyrhizobium sp., Novosphingobium sp., Sphingopyxis rhizosphaerae, Xylophilus sp., Burkholderiales,
sp., Sphingomonas sp. Nitrosomonas sp., Candidatus "Nitrotoga antarctica"

244
[15] Hyphomicrobiaceae, Parvularcula sp., Rheinheimera, Ectothiorhospiraceae, SAR86 Brumimicrobium sp.,
Phyllobacteriaceae, Rhizobiales, Rhodobacteraceae, clade Cryomorphaceae,
SAR11 clade, Rhodospirillales, OCS11 clade, Micavibrio Flavobacteriaceae,
sp., Rickettsiales Haliscomenobacter sp.
[16] Afipia felis, Bradyrhizobium sp., Ensifer sp., Rhizobium etli,
Roseomonas sp., Sinorhizobium sp., Sphingomonadales
[18] Rhodobacteraceae
[17] Sphingomonadales, Rhizobiales Burkholderiales
[21] Cyclocasticus sp., Colwellia sp., Spongiibacter
sp., Marinobacter sp., Pseudomonas stutzeri,
Pseudidiomarina sp.
[22] Rhodobacteraceae
[23] Neisseriaceae, Hydrogenophaga sp., Acidovorax sp.,
Achromobacter sp.
[24] Rhodobacteraceae Acinetobacter sp., Pseudomonas sp.
[27] Streptococcus sp.,
Enterococcus sp.,
Lactobacillus sp.,
Lactococcus sp.
Journal of Membrane Science 545 (2018) 240–249
O. Sánchez
model microorganism to study biofilm formation in RO membranes
[25].
Interestingly, the phylum Bacteroidetes appeared to be abundant in
some biofilms of RO membranes feeded with seawater or secondary
effluent from WWTPs [15,17,23,24]. The members of this group are
supposed to play an important role in the degradation of polymeric
organic matter [26], and can be found associated to surfaces, probably
degrading biopolymers in membranes [15]. In contrast, Firmicutes was
the main group found in biofilms from milk processing membranes
[27].
Other prokaryotes, including Actinobacteria, Acidobacteria,
Planctomycetes, Verrucomicrobia, Nitrospirae, Deltaproteobacteria or
Archaea, as well as fungi, can also be found in RO biofilms. However, in
most cases their contribution is low [10,12–14,27] and probably they
are part of the rare biosphere, a term used to describe those species
represented by a low number of individuals [28,29]. Besides, there is
also a great proportion of sequences that remain as “uncultured”, a fact
that shows that further efforts should be directed to characterize those
microorganisms comprising microbial communities in RO membranes.
3. Influence of operating conditions on microbial diversity
Biofouling causes loss of performance in RO membranes, a problem
that depends upon biofilm composition, which is itself dependent upon
environmental conditions. There is a large set of operating factors that
may affect community structure in RO biofilms, such as the nature of
the influent, its temperature, the properties of the membrane surface,
the materials used in the pretreatment process, as well as the membrane
flux (Fig. 1). However, literature dealing with the interactions between
biofilm microbial diversity and environmental conditions is still scarce.
Instead, most of the studies have provided indirect evidences of the
effect of operating factors on microbial diversity by focusing on biofilm
development through the use of microscopy techniques or model mi-
croorganisms.
3.1. Influent content
Thus, concerning the effect of the influent content, Flemming and
Schaule [30] stated that the adhesion process carried out by the fast
adhering species Pseudomonas from tap water was greatly improved if
cells were not subject to nutrient limitation; when starvation occurred,
colonization of the surface membrane was not complete, a result sup-
ported by other works [31,32]. McEldowney and Fletcher [33] also
suggested that changes in nutrient concentrations may influence the
attachment of bacteria to solid surfaces differently, hence affecting the
population structure of developing biofilms. Besides, a correlation be-
tween organic matter and microbial membrane fouling was extensively
reviewed by Ke et al. [4]. More recently, Chamberland et al. [27], using
molecular tools to analyze microbial communities found on spiral-
wound membranes in the dairy industry, concluded that the type of
influent largely explained the variance observed between communities
in membranes, therefore suggesting that the nature of the fluid was an
important element to consider influencing microbial composition. Thus,
nutrient availability appears to be a driver of biofouling and as a
Influent
Temperature
content
Pretreatment
process Membrane
flux
Membrane
type RO membrane
Fig. 1. Main operating factors that influence microbial diversity in RO membranes.
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Journal of Membrane Science 545 (2018) 240–249
consequence, control of available organic carbon may be a good point
to consider for constraining biofilm formation.
3.2. Temperature and pH
On the other hand, Farhat et al. [34] evaluated the effect of different
water temperatures on biofilm development and structure in a mem-
brane fouling simulator flow cell, with the same type of membrane,
water type and nutrient concentration, and inferred that with in-
creasing feed temperature the biofilm activity (due to an increase in
nutrient diffusion) and drop pressure concomitantly raised, while bio-
film thickness decreased. This study is particularly relevant, since it
used feed water with a mixed bacterial population. Previous works
addressed to evaluate the effect of temperature on bacterial attachment
were performed with single strain bacteria or a mixture of two or three
bacterial species, with results that stated a better or worse adhesion
depending on the strain at high temperatures [35–38]. Overall, as
Farhad et al. concluded [34], these changes in temperature may point
out indirectly to alterations in community structure, related to varia-
tions in bacterial cell attachment and EPS production and viscosity.
High temperature leads to a reduction of the EPS matrix viscosity re-
sulting in higher detachment rates [39], and also to more spatially
heterogeneous biofilms with a less cohesive structure [40].
On the contrary, pH modifications seemed to have little effect on
adhesion of model bacteria [31,41], while the influence of ion con-
centration was not clear, with cases in which adhesion to membranes
increased when increasing electrolyte concentration [42] and others
with no significant correlation between adhesion and ionic strength
[31,41].
3.3. Pretreatment of feed water
An extensive pretreatment process, with activities as coagulation,
flocculation, sand and cartridge filtration and ultrafiltration is required
in RO systems in order to control membrane fouling, although micro-
organisms may survive and end up with colonization of RO membranes.
In this context, there are also evidences that changes in the biological
support media used in the pretreatment process result in differences in
biofilm community in RO membranes. Ferrera et al. [23], utilizing tag
sequencing tools (454 pyrosequencing), demonstrated that biofilms
from two water reclamation plants fed with secondary effluent from a
WWTP, which differed in the material used in the filtration process,
were phylogenetically different and consequently likely also function-
ally distinct.
3.4. Membrane type
Likewise, different studies have been reported concerning the effect
of membrane type on biofouling [30,31,41]. Since the first RO appli-
cations, several polymeric materials for RO membranes have been ap-
plied, such as polyetherurea, polysulfone, polyethersulfone, cellulose
acetate, cellulose diacetate, polyamide or polyvinyl among others
[43,44], whose choice depends on the process (e.g., type of influent,
operating conditions) and economic considerations. Ridgway et al. [41]
concluded that mycobacteria were found to adhere to a cellulose
acetate membranes much more effectively than Escherichia coli, and
that their adhesion abilities were correlated with relative surface hy-
drophobicities, an important observation which advances the role of
hydrophobic interactions between bacterial cell surface components
and membrane surfaces at the initial stages of bacterial adhesion. These
results were confirmed by Sadr Ghayeni et al. [31], who demonstrated
that membrane properties like hydrophobicity or roughness influenced
adhesion of model microorganisms. On the other side, Flemming and
Schaule [30] detected a certain affinity of different membrane materials
towards particular bacteria, having polyetherurea a significantly lower
biological affinity than other materials like polisulfone,
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polyethersulfone or polyamide, a result confirmed by the work from
Jenkins and Tanner [45], who proved that a composite membrane
formed by a polyamide-urea barrier was superior in RO operation
compared to a polyamide membrane due to its fouling-resistant prop-
erties.
3.5. Membrane flux
Membrane flux constitutes another important operating character-
istic that has a great impact on membrane fouling, and several models
have been developed for predicting the critical flux from which fouling
will establish [46]. However, literature dealing with the correlation
between this factor and microbial diversity in RO membranes is vir-
tually non-existent. Despite that, studies in membrane bioreactors op-
erated at different fluxes have showed that a high membrane flux
generates a faster biofilm development and leads to different pre-
dominant populations in the biofilms [20], which were analyzed with
genetic clone libraries; in contrast, highly similar biofilms were found
associated to membrane bioreactors with low flux operation.
In conclusion, membrane fouling is a complex mechanism where
different factors, such as the quality of influent water, the physico-
chemical properties of the membrane, and other operating conditions
all play a role. Ke et al. [4] inferred that effluent organic matter
membrane fouling and microbes were the main drivers causing bio-
fouling, and they were significantly correlated. In any case, biofouling
will always be a part of the membrane filtration process, and an ef-
fective biofouling mitigation strategy involves the use of an appropriate
process design and operation with an adequate feed pretreatment and
membrane cleaning.
4. Effect of cleaning on microbial diversity
Apart from feed pretreatment, chemical agents such as chlorine or
ozone, which are capable of inactivating microorganisms due to their
powerful oxidation effects, have also been used to restore membrane
fluxes. However, they only have a temporary effect, since their appli-
cation does not ensure that membrane biofouling will not occur, and in
several studies, it has been demonstrated that biofouling is severely
enhanced (see revision from Matin et al. [47]). Though, other cleaning
strategies have arised as possible antibiofouling alternatives, like
membrane surface modification [47–51], addition of reducing [50] or
oxidant [53] agents, as well as biological methods [54,55].
Nevertheless, the effect of cleaning on microbial diversity in RO
membranes has been hardly investigated. As in the case of operational
factors, the result of cleaning procedures has been reported only as a
change in pressure drop and membrane flux, and in several works ob-
servations on biofilm development were accompanied by microscopy
techniques [56] or by the use of simplified laboratory models which
employed one or a few bacterial strains [49,52,53]. These models, al-
though offering preliminary results, have several drawbacks, since the
single strains do not represent the complex microbial community pre-
sent in the real influent, and the chemical components used might be
remarkably different from those found in the actual feed, which is in
fact a mixture of different organic matters, ions, and solids.
4.1. Biological methods and microbial diversity
Actually, only a few studies have addressed the impact of cleaning
on microbial diversity. For instance, Belgini et al. [54] investigated in
some bacterial isolates the effect of phages on biofilm formation, and
highlighted their potential as a good alternative for biofilm prevention.
Xiong and Liu [57] expressed some concerns related to the specific
parasitic characteristics of phages, which could pose difficulties to
apply in large scale wastewater treatment. Nevertheless, beyond their
specific litic action, phages can produce enzymes able to degrade EPS
causing biofilm slough off [58] and in the work of Belgini et al. [54] the
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Journal of Membrane Science 545 (2018) 240–249
authors concluded that the reduction of biofilms of some isolates was
related to the EPS desestabilization by this kind of enzymes rather than
to cell lysis, so that there was a clear effect of this cleaning strategy on
microorganisms. Furthermore, as the pore size of RO membranes is
generally smaller than phages, some of them can left attached to the
membrane surface and can be available to continue infection of the
oncoming bacteria without interfering in the filtration process [59]. In
any case, studies reporting the use of phages for the control of complex
bacterial communities from RO systems are scarce and further research
is needed to successfully apply this strategy.
4.2. Chemical agents and microbial diversity
In contrast, other studies have utilized molecular methodologies in
order to assess microbial diversity to investigate the effect of different
cleaning treatments. Thus, Bereschenko et al. [14] explored the impact
of a conventional chemical treatment (sequential washing steps of RO
permeate, sodium bisulfite and a mixed acid detergent descaler) along
time using FISH, DGGE and clone libraries, as well as microscopy
techniques, concluding that chemical procedures facilitate establish-
ment and subsequent maturation of biofilm structures on membranes
and other surfaces of the system, and highlighting the fact that only
when the dead biomass is effectively removed in the cleaning process,
the control of biofilm formation can be achieved. This study confirmed
the failure in removing developed biofilms from RO membranes using
conventional chemical treatment. The majority of the bacterial popu-
lation disappeared due to the toxic effect of the chemicals, but the
network of biofilm-associated EPSs appeared to be remarkable stable,
so that there was a collapse of the three-dimensional biofilm structure
but not an effective complete biofilm removal. Only the upper layer was
affected, while the structural integrity of the base layer was hardly
changed. In this context, only Sphingomonas species, which typically
located at the biofilm base, survived the cleaning process.
More recently, a few works have addressed the influence of different
cleaning treatments on microbial diversity using high-throughput
techniques. Therefore, Barnes et al. [60] examined the impact of nitric
oxide on single and mixed model species biofilms, as well as on biofilms
of RO membranes, by using confocal microscopy and 454 pyr-
osequencing; they concluded that the addition of this compound re-
sulted in an induction of biofilm dispersal in almost all the bacteria
tested and the mixed species, in addition to a substantial reduction of
the rate of biofouling in RO membranes, together with a decline in
polysaccharides, proteins and microorganisms. This effect was also as-
sociated to a decrease in biofilm surface coverage and average thick-
ness, presenting strong evidence for the application of nitric oxide for
biofouling control. Interestingly, community sequence analysis re-
vealed that the cleaning treatment did not significantly modify the
microbial population composition of membrane biofilms, being the
families Chitinophagaceae (Bacteroidetes), Shingobacteriaceae (Bac-
teroidetes), Comamonadaceae (Betaproteobacteria), Oxalobacteraceae
(Betaproteobacteria), Enterobacteriaceae (Gammaproteobacteria) and
Rickettsiales (Alphaproteobacteria) those that dominated biofilms of
the treated and untreated membranes. On the contrary, Khan et al.
[24], by means also of high-throughput techniques to analyze the mi-
crobial community of fouled membranes, reported the incapability of
chlorination on preventing biofilm formation, due possibly to three
main factors: plugging of membrane fiber which led to inaccessibility of
chlorine, its consumption by organics accumulated on the feed region
fibers, or chlorine adaptation of certain bacterial populations.
In summary, depending on the cleaning protocol, the biofilm com-
munity assemblage seems to be affected in a different way. Hence,
chemical conventional strategies, although reducing the number of
microorganisms, do not seem to have a substantial effect in biofilm
removal, since some bacteria are able to survive and the EPS structure
remains unaltered. Other strategies such as the use of phages or nitric
oxide appear to be more promising alternatives for biofouling control of
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RO membranes, although further investigation in this sense is required.
5. Biofilm dynamics
5.1. Stages in biofilm formation
The formation of a biofilm goes through different stages, which
include (i) the adsorption of suspended particles and organic species
from the feed water on the RO membrane, (ii) the transport of the
microbial cells, mainly due to fluid dynamic forces, (iii) the attachment
of microorganisms to the membrane surface, affected by factors such as
membrane or feed water characteristics, (iv) the development of at-
tached microbes, which relies on microbe-dependent properties like
species composition, population density, growth phase, nutrient status,
hydrophobicity, charges, and physiological responses, and (v) the
achievement of a steady state fouling resistance and cell dispersion
[61]. The multiplication of microorganisms is produced at the expense
of the nutrients supplied by the feed water or those organics adsorbed
on the membrane surface. In this process, microorganisms excrete the
EPS, which favours the colonization of other organisms and is greatly
responsible for flux decline.
5.2. Cell properties and biofilm formation
Different studies carried out with model microorganisms have pro-
vided clues about the dynamics of biofilm formation on RO membranes,
as well as about the genetic traits of these model bacteria. When dif-
ferent strains were compared, biofilm development occurred to a dif-
ferent extent depending on cell properties such as hydrophobicity,
surface charge or motility [31,62]. Hydrophobic interactions seem to be
important in the initial stages of bacterial adhesion; an electrostatic
double layer repulsion occurs between a bacterium with a negative
charge and a hydrophobic membrane (RO membranes tend to have a
negative charge at pHs around 7), and those bacteria having more ne-
gative charges would experience greater repulsion [62]. In this sense,
Pang et al. [62] demonstrated that bacteria like Rhodopseudomonas sp.
or Dermacoccus sp. would adhere more easily due to their lower cell
surface charge. Once adhesion has initiated, those microorganisms
which exhibit twitching or swarming motility could colonize membrane
surfaces more rapidly, like Shingomonas sp.
In an interesting study, the model bacterial strain Pseudomonas
aeruginosa was used to investigate the biofouling mechanisms of RO
membranes which led to a decline in membrane performance, attrib-
uted to an increase in both the hydraulic resistance and the trans-
membrane osmotic pressure [63]; the authors reported that the increase
in hydraulic resistance was due to the EPS produced by bacterial cells,
while the rise in trans-membrane osmotic pressure was assigned to the
deposited bacterial cells, which improved the concentration polariza-
tion effects of salt close to the membrane surface. Furthermore, Herz-
berg and Elimelech [25] also compared biofilm formation in planktonic
cells and biofilms of P. aeruginosa by means of transcriptomic techni-
ques and concluded that attachment and motility related genes were
repressed in the membrane biofilm; conversely, motility and chemo-
taxis phenotypes were observed in suspended cells. The authors sug-
gested that the increase in nutrient concentration close to the mem-
brane could enhance biofilm formation by means of chemotaxis
response of the suspended cells, which could move towards the mem-
brane surface.
5.3. The role of Sphingomonas
On the other hand, different works attempted to investigate which
bacteria are responsible for biofouling initiation. In this sense, it has
been reported the presence of Sphingomonas species in many biofilms of
diverse RO water purification processes [12–14,16,17,64]; for instance,
Bereschenko et al. [12] reported that 27% of the total clones from a RO
247
Journal of Membrane Science 545 (2018) 240–249
biofilm was related to Sphingomonas spp. The genus Sphingomonas is
widely distributed in the environment due to its ability to metabolize a
great variety of organic compounds and to survive under oligotrophic
or starvation conditions [65,66]; besides, they seem also to persist at
high nutrition concentrations that occur near the membrane surface in
RO units as a consequence of the concentration polarization effect and
the deposit of nutrients in the biofilm matrix [12]. These character-
istics, added to the possession of twitching and swarming mobility,
point to Sphingomonas as a good candidate to facilitate colonization on
the surface of RO membranes.
Sphingomonas sp. has been isolated from RO membranes [62], and
has been used as well to study the contribution of its glycosphingolipids
(GSL) from the outer membrane and its EPS to the initial colonization
and development of biofilms [19]. In this last work, the authors showed
noticeable differences between the adhesion properties of S. wittichii
compared to E. coli and P. aeruginosa, with the former exhibiting 50%
higher adhesion to polyamide coated crystals and a greater flux decline,
likely due to a combination of GSL presence and EPS contribution. In
other works focusing on the study of the dynamics of the whole mi-
crobial community in biofilms instead of presenting results with model
microorganisms, the use of molecular tools has also evidenced the un-
ique role of Sphingomonas spp. in the initial development and sub-
sequent maturation of biofilms [13]; their unique capabilities for
spreading and producing a layer of EPS may outcompete other micro-
organisms like Pseudomonas, which can exist as free-floating aggregates
in the feed water [13].
5.4. Biofilm kinetics and microbial composition
In membrane fouling kinetic studies, it has been shown a pro-
gressive increase in the abundance of the foulant substances, although
their relative proportion changed over the age of the biofilm layer,
concomitantly with microbial composition [18]. Thus, initial cell ad-
hesion was accompanied with a preferential coating of humic like
material, followed by formation of microcolonies as a result of micro-
bial growth in an intermediate phase where polysaccharides were
abundant and Gammaproteobacteria (with the presence of Methylo-
phaga and Pseudidiomarina homiensis) were the predominant group. In
contrast, the biofilm advanced phase was characterized by the forma-
tion of macrocolonies, release of EPS and the presence of Alphapro-
teobacteria [18], being this stage dominated by Antarctobacter sp. The
increase in representativeness of Alphaproteobacteria along time might
be associated with the concomitant changes in the chemical composi-
tion of the biofilm.
6. Conclusions and future directions
6.1. Are there universal fouling organisms in RO membranes?
Although the phylum Proteobacteria is always recovered in di-
versity studies from RO membranes, variations in operating conditions
lead to differences in community structure at the level of genus or
species, a fact that is evident for the numerous studies presented in this
review, where different fouling complex patterns are described de-
pending on the composition of the feed stream, type of membrane or
environmental parameters. This suggests that up to now there are not
real universal fouling organisms. However, in some studies, such as the
one from Zhang et al. [64], examination of five seawater RO mem-
branes from desalination plants located in different parts of the world
showed that although biofilm bacterial communities were not identical
to each other, some dominant bacteria were observed. In this work, the
authors pointed to the filamentous bacterium Leucothrix mucor and
Ruegeria sp., both Gamma and Alphaproteobacteria respectively, as
dominant microorganisms in these membranes. Yet, other studies did
not retrieve these bacteria, and pointed out to Sphingomonas as initial
colonizer [12–14,16,17,64].
O. Sánchez
Differences in community structure has also been reported in
membrane bioreactors (MBRs) even when using an identical experi-
mental design [67]. Vanysacker et al. [68] identified a list of candidates
that attached to the membrane surfaces during the early stages of fil-
tration in MBRs, and concluded that the control of biofilm formation
could focus upon these pioneer bacterial assemblages rather than the
total microbial community. Nevertheless, this list provides a broad
range of phyla and species, and again the biofilm species composition
seems to be dependent on a great number of factors. Among the dif-
ferent phyla, the authors listed mainly Proteobacteria, Bacteroidetes
and Firmicutes members from a wide range of influent types.
On the other hand, the differences found could simply reflect the
distinct molecular approaches used to characterize microbial diversity
rather than being real modifications in microbial community compo-
sition. Thus, the 454 platform generally gives longer sequencing reads
with lower error rates compared to Illumina, and hence, the taxonomic
assignments may vary. Also, differences on the region of the 16S rRNA
gene amplified and sequenced may lead to differences in the identified
OTUs. Similarly, the use of different databases when analyzing results
can provide different taxonomic outputs. As a consequence, divergence
in community structure may not always be attributable to differences in
operating conditions. Besides, an additional point or consideration is
how well sampled and replicated these studies actually are, as it is not
so common to use experimental replicates in these systems. Thus, there
may be quite a lot of noise in the data that do not give an accurate
picture of the true community.
6.2. Novel findings to mitigate RO membrane biofouling
Biofouling is a universal phenomenon, and in comparison to other
environments, membrane systems provide a unique habitat for biofilm
development. However, major advances in antifouling strategies have
been made in recent years to mitigate its occurrence. Among these,
membrane surface modification appears to be a very promising field
compared to other methods such as feed pretreatment or biocide ap-
plication, due to the fact that the former is based on bacterial adhesion
prevention or microbial inactivation while the latter are aimed at re-
moving microorganisms. Particularly, nanotechnology has broadened
the options for the development of antifouling RO membranes, and
diverse constituents like metals, metal oxides, zeolite, boehmite or
carbon-based materials have been incorporated into membranes or on
membrane surfaces, becoming an encouraging option for next-genera-
tion antifouling membranes due to their antimicrobial properties [50].
A great variety of this kind of membranes have been successfully pro-
duced in laboratories, although only a few of them have been applied at
a large scale. Some drawbacks need first to be overcome, for example
the poor-term retention of nanoparticles which leads to a loss of anti-
bacterial activity, the possible degradation of the membrane layer, or
the need for a continuous UV light source in the case of photocatalysts
[47]. Therefore, there is still a long way to go for developing an easy
and more controllable method to supply RO membranes with stable
antifouling characteristics, and this type of research, apart from the
need of cooperative contributions from engineering and materials sci-
ence, goes through the study of microbial diversity and functionality to
understand the mechanisms that drive biofilm formation.
A totally different strategy consists on the application of quorum
quenching bacteria, which produce enzymes that can degrade or dis-
rupt N-acyl homoserine lactones (AHLs), small diffusible signal mole-
cules synthesized by several species of Gram-negative bacteria that
allow them to coordinate gene expression and regulate different phe-
notypes such as biofilm formation, secretion of EPS and virulence. A
variety of quorum quenching mechanisms have been used over the past
few years as potent tools for inhibition of membrane biofouling which,
combined with advances in membrane fabrication, may allow the im-
provement of antibiofouling efficacies [69,70]. The AHLs-mediated
quorum quenching can be found in several Proteobacteria and should
248
Journal of Membrane Science 545 (2018) 240–249
be evaluated as an alterative strategy for combating biofilms. In this
sense, the use of metagenomics and metatranscriptomics could be ex-
tremely useful for the identification of those genes and pathways that
participate and increase their activity in biofilm formation.
6.3. The –omics techniques
Metagenomics can provide evidence about the phylogenetic di-
versity of populations, but also about the genomic potential of biofilms
without bias; nevertheless, there is a lack of information in this sense in
RO membranes, a fact that contrasts with other fields in wastewater
microbiology such as the characterization of activated sludge in was-
tewater treatment plants or the study of populations dwelling in con-
structed wetlands [8,9]. On the other hand, although microbial com-
position is usually linked with function, they are not necessarily related,
since some functions are restricted to certain groups but others are
spread in different phylogenetic lineages. As a consequence, meta-
transcriptomics appears to be a crucial tool to understand the function
and behaviour of bacteria present in biofilms, for instance providing
knowledge about the effect on different pathways and genes of certain
factors, such as nutrient availability, quorum sensing, biofilm forma-
tion, or EPS production among others, as well as offering some clues
about the integration of cleaning strategies in RO membranes. Thus, the
future challenge lies behind the combination of metagenomics and
metatranscriptomics to deepen into the analysis of microbial commu-
nities and their functioning.
Another question that still remains to be elucidated focus in the
study of the microbiome of RO biofilms, that is, the set of micro-
organisms present within similar environments. Knowledge of the key
microbial components in different stages of biofilm formation can offer
information about how the process functions across complex microbial
assemblages. The different –omics can provide insights into this ques-
tion when combined with mathematical models to predict population
composition and population functioning under different operational
conditions. For now, however, they only provide a more refined view of
microbial diversity, which is of course of ecological interest, but in a
future, they will help to inform on strategies to prevent or remove
biofilms from membrane surfaces.
Acknowledgements
The author thanks her institution and lab colleagues for support.
This work was supported by the spanish grant REMEI (CTM2015-
70340-R) from the Ministerio de Economía y Competitividad.
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