Chemosphere xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

Chemosphere
journal homepage: www.elsevier.com/locate/chemosphere

Review

Bioaugmentation and its application in wastewater treatment: A review

M. Herrero a,b, D.C. Stuckey a,⇑
a
Department of Chemical Engineering, Imperial College, London SW7 2AZ, UK
b
Department of Chemical Engineering and Environmental Technology, University of Oviedo, Spain

h i g h l i g h t s

 Community ecology and dynamics in bioreactors in WWTPs are complex processes.

 Progress in ecology, molecular biology, immobilization, bioreactor design are reviewed.
 Emerging research facilitates novel applications in bioaugmented bioreactors.
 Bioaugmentation should ultimately be assessed by data from field implementation.

a r t i c l e i n f o a b s t r a c t

Article history: Bioaugmentation (the process of adding selected strains/mixed cultures to wastewater reactors to
Received 31 January 2014 improve the catabolism of specific compounds, e.g. refractory organics, or overall COD) is a promising
Received in revised form 6 October 2014 technique to solve practical problems in wastewater treatment plants, and enhance removal efficiency.
Accepted 10 October 2014
The potential of this option can now be enhanced in order to take advantage of important advances in
Available online xxxx
the fields of microbial ecology, molecular biology, immobilization techniques and advanced bioreactor
design. Reports on bioaugmentation in WWT show the difficulties in evaluating the potential parameters
Keywords:
involved, leading frequently to inconclusive outcomes. Many studies have been carried out on the basis of
Bioaugmentation
Biological wastewater treatment
trial-and-error approaches, and it has been reported that reactors bioaugmented with pure cultures often
Microbial community dynamics fail to perform as well as the pure cultures under laboratory conditions. As an interesting technical
Advanced bioreactors challenge, the feasibility of bioaugmentation should ultimately be assessed by data from field implemen-
Immobilization tation, and this review highlights several promising areas to explore in the future.
Ó 2014 Elsevier Ltd. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
2. Community ecology and assembly . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
3. The aim of bioaugmentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
4. Selection criteria. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
5. Bioaugmentation failures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
6. Success of bioaugmentation strategies: key factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
6.1. Ecological basis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
6.2. Monitoring techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
6.3. Success in overcoming operational problems and plant management . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
6.4. Different successful selection criteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
6.5. Repeated or continuous application versus single inoculation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
6.6. Success of immobilization techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
6.7. Advantages of gene transfer methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
6.8. Success by using membrane bioreactors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
6.9. Success at full scale implementation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
7. Prospects. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

⇑ Corresponding author. Tel.: +44 (0)207 594 5591; fax: +44 (0)207 594 5629.
E-mail address: d.stuckey@imperial.ac.uk (D.C. Stuckey).

http://dx.doi.org/10.1016/j.chemosphere.2014.10.033
0045-6535/Ó 2014 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Herrero, M., Stuckey, D.C. Bioaugmentation and its application in wastewater treatment: A review. Chemosphere (2014),
http://dx.doi.org/10.1016/j.chemosphere.2014.10.033
2 M. Herrero, D.C. Stuckey / Chemosphere xxx (2014) xxx–xxx

8. Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

1. Introduction knowledge base has changed rapidly. Metagenomics (or

Ecogenomics – the application of genomics to ecological and envi-
Advanced bioreactor design and operation in wastewater treat- ronmental sciences – (for a review see Maphosa et al., 2010), other
ment plants (WWTPs) is essential in order to develop proper envi- molecular methods, microscopy and flow cytometry- based
ronmental conditions so that the most desirable microorganisms methods are providing an enormous source of information for
can be selected for and maintained under adequate physiological monitoring, detection, quantification and characterisation of
conditions. This extends to the contact with the stream to be trea- microorganisms. This opens up the possibility of exploring uncul-
ted, controlling mass transfer and reactor performance to achieve turable microorganisms, and exploiting biodiversity as a means
the treatment goal at full scale. Until recently, biological reactors to increasing biodegradation, even through genetic engineering
in wastewater treatment have been considered as ‘‘black boxes’’, (though the impact of genetically modified microorganisms on
their functionality depending only on empirical knowledge, and the environment is still unknown). The question is to determine
hence they are difficult to predict and control. Even now their per- not only the systematic and taxonomical structure of the microbial
formance can only be evaluated by applying material balances. consortia taking part in environmental bioprocesses (community
Now advanced bioreactor design can take advantage of compart- structure), but also to learn how these biological systems respond
mentalisation and the use of membranes to confine the key micro- to changes in the influent (understanding the biochemistry and
organisms in the process to a bioreactor (Barber and Stuckey, 1999; toxicity mechanisms involved), how the microbial population
Vyrides et al., 2010). However, it is also known that effective oper- dynamics evolve (community ecology and assembly), and in which
ation of biological treatment systems relies on highly active micro- way these changes can be related to bioprocess efficiency (commu-
organisms carrying out the process, and therefore on the ratio of nity function).
resistant/sensitive microorganisms to the contaminant being trea-
ted (depending on both its chemical nature and concentration).
Microorganisms can degrade a wide variety of organic contami- 2. Community ecology and assembly
nants and can adapt to many different inhospitable environments.
However, there are a variety of pollutants, both man made It should be noted that community assembly in biological reac-
(xenobiotic) and natural, that are not easy to degrade biologically, tors is a very complex process since different microorganisms com-
even over long periods of time -these are referred to as ‘‘refractory prising a multitude of cells work together to treat the effluent,
or recalcitrant’’. interacting by means of different types of cell signalling such as
In principle, poor bioreactor performance may be due to the quorum sensing and horizontal gene transfer (Verstraete, 2007).
lack of a sufficient number of a specific microorganism harbouring At the moment a complex community cannot be engineered
a key metabolic route to transform the target contaminant into less rationally (Curtis et al., 2003), since is still poorly understood
harmful end products. However, despite functional redundancy (Sloan et al., 2006). Theoretical ecologists such as MacArthur and
(i.e. different strains that can carry out the same or similar func- Wilson (1967) and May (1974) assumed microbial communities
tions), and metabolic versatility being common in environmental to be dynamic systems which were nonlinear, and predicted com-
bioprocesses, it is more likely that the specific target contaminant munity stability or instability. Lawrence and McCarty (1970)
(or mixture of compounds) can only be degraded by a very specific developed equations based on biochemistry and microbial kinetics
mixture of microorganisms (a consortium) harbouring the key that enabled key process variables to be predicted in two different
metabolic pathways, and cooperating in a synergistic way. The biological systems, aerobic and anaerobic. It has been postulated
importance of microbial consortia is such that, without their com- that dynamic population behaviour is probably innate to microbial
bination, many biodegradation processes could not be explained systems. Curtis et al. (2003) also suggest that it is possible ‘‘steady-
thermodynamically on the basis of free energy in a single chemical state’’ conditions never really exist in bioprocesses which have
reaction, explaining ultimately the shift in fermentation patterns been engineered, even in processes with constant inputs or no
(Melandri, 1997; Jorgensen and Gallardo, 1999). apparent variations in growth conditions. Immigration and chance
Despite non-specific bioaugmentation being used for years in are important in shaping the behaviour seen in prokaryotic com-
agriculture and wastewater treatment to reinforce the process, it munities (Sloan et al., 2006). Interestingly, it has been reported
is still considered a procedure with unpredictable outcomes. Even that biological communities in similar econiches may have con-
now bioaugmentation is considered less predictable and controlla- trasting dynamic behaviour depending on the specific chemical,
ble than other removal techniques which result in the direct physical, or biological conditions which the culture operates under
destruction of contaminants (Boon et al., 2000). The approach of (Ayala-del-Río et al., 2004).
bioaugmentation focuses on taking advantage of microbial consor- In order to advance functional organisation, the usefulness of
tia designed for the specific physico-chemical properties of the bio- Pareto–Lorenz diagrams have been applied to measure microbial
process, (Van der Gast et al., 2004) since this approach was shown diversity graphically to represent the structure of a bacterial com-
to be more efficient than using undefined inocula. Potentially munity. The Pareto 80/20 principle applied in macroeconomics has
higher efficiencies could occur in systems such as membrane bio- been established where 20% of the participants normally acquire
reactors which stop bacteria being ‘‘washed out’’, in contrast to 80% of all the energy (labour) flux (Dejonghe et al., 2001). By using
the natural environment where the environmental conditions can Pareto–Lorenz distribution patterns, Mertens et al. (2005) showed
be manipulated to enhance survival and prolong the activity of that only a small group of ammonia oxidising bacteria (AOB)
the exogenous population (El Fantroussi and Agathos, 2005). played a dominant role in nitrification, while the less dominant
Bioaugmentation of wastewater treatment has not been species remaining were postulated to embody a reserve of AOB
reviewed specifically for a number of years, while the current which could grow up to replace the species which dominated

Please cite this article in press as: Herrero, M., Stuckey, D.C. Bioaugmentation and its application in wastewater treatment: A review. Chemosphere (2014),
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 M. Herrero, D.C. Stuckey / Chemosphere xxx (2014) xxx–xxx 3

(Wittebolle et al., 2008). Wittebolle et al. (2005) also concluded allow the bioaugmented microorganism to shift their metabolism
that certain species which were not numerically very abundant to using other organic substrates rather than the target pollutant
seemed very active according to their RNA signal, so an rRNA eval- under real operating conditions. Thus, specific consortia rather
uation is useful when studying the performance and activity of a than a single strain may be more useful in ensuring successful bio-
biological WWT. augmentation, even in the presence of a single contaminant.
Frequently the presence of compounds with different levels of
toxicity complicates the biotreatment of chemically mixed waste-
3. The aim of bioaugmentation waters. Under these conditions bioaugmented bacteria may lose
their degradation ability by being inhibited by the more inhibitory
In order to establish bioreactors which can effectively treat pollutants prior to metabolizing the more easily degradable pollu-
chemically mixed wastes it is essential that they harbour and sta- tants. A ‘‘selective’’ bioaugmentation strategy can help to overcome
bly maintain key microbial consortia with sufficient activity to this problem in mixed wastewaters by separating (by adsorption)
degrade the contaminant compounds present (Van der Gast compounds showing different levels of toxicity. Following this line,
et al., 2004). Communities were identified that had the ability to a strategy combining selective adsorption and bioaugmentation
colonise such harsh niches with desired catabolic traits, and these has been reported for treating a mixed wastewater of nitrobenzene
provided an opportunity to develop specialised inocula that could and q-nitrophenol, passing through an adsorption column (Hu
be exploited by bioaugmentation (Van der Gast et al., 2002). et al., 2008). Without nitrobenzene inhibition, q-nitrophenol could
Bioaugmentation is using added microorganisms to ‘‘reinforce’’ be easily degraded through bioaugmentation. Nitrobenzene was
biological waste treatment populations so that they can effectively adsorbed by the resin, and then it could be desorbed and degraded;
reduce the contaminant load by transforming it into less dangerous desorption partly recovering the binding capacity of the resin. It
compounds. However, the introduced strain or strains may fail to was observed that below a minimum value, the nitrobenzene
grow or to be active in the bioreactor due to predation or compe- concentration was too low to support the growth of nitroben-
tition with the indigenous microbiota, presence of bacteriophages, zene-degrading bacteria.
or to a lack of acclimation to the environmental conditions. In
these cases the introduced microorganisms will not colonise or
show any population dominance in such a dynamic context which 4. Selection criteria
is subjected to changing conditions such as starvation periods, pH
or load variations and low temperatures. Microbial degradation of Different bioaugmentation options have been proven useful,
contaminants can also be enhanced by biostimulation, i.e. adding ranging from the use of commercial products, culture collections,
nutrients or electron acceptors to activate the indigenous microbi- and indigenous or exogenous strain/s or tailor-made consortia. A
ota. Obviously, a profound knowledge of the biosystem is required review of wastewater treatment bioaugmentation (Stephenson
to support decision making at any time in this process. and Stephenson, 1992) examined independent investigations (as
The most common options for performing bioaugmentation (El opposed to manufacturers reports) on the use of commercial
Fantroussi and Agathos, 2005) are; the addition of a pre-adapted products (developed as mixed populations including autotrophs,
pure bacterial strain, addition of a pre-adapted consortia, the heterotrophs, facultative anaerobes and aerobes, intended to serve
introduction of genetically engineered bacteria, or the addition of a wide range of purposes, marketed as ready-to-use frozen, freeze-
biodegradation-relevant genes that are packaged into a vector in dried or liquid preparations) at laboratory and full scale, along with
order to be transferred by conjugation into microorganisms a discussion of possible reasons for bioaugmentation failures.
already present in the biosystem. In this latter case, the technique Among them, the lack of acclimation (i.e., physiological adaptation
has the advantage of not depending on the survival or growth of mechanisms that allow microorganisms to survive and remain
the donor strain/s. However, some of these options have only been active under harsh environmental conditions) was highlighted.
performed at the laboratory scale (El Fantroussi and Agathos, The authors remarked that in order to properly evaluate the
2005). amount and type of bioaugmentation needed to enhance plant per-
When reporting on how effective bioaugmentation is, a clear formance, process conditions, such as flow and treatment technol-
definition of the treatment goal must be laid down in order to iden- ogies, and the type of microbial toxicity should be taken into
tify whether it aims to reduce a particular contaminant, or remove account. The main strategy used was based on isolation of natural
the total overall organic load (total organic carbon, TOC). A partic- environmental samples, selection of the most efficient strains after
ular contaminant may only amount to a small fraction of the total screening for their catabolic traits, further selection after the use of
TOC load, however, it may be the major cause of toxicity for the mutagenic agents, and finally fermentation tests for single strains
microbial community in the biosystem unless it can be reduced following traditional selective enrichment procedures
to the required effluent standard. Effective bioaugmentation strat- (Stephenson and Stephenson, 1992).
egies should achieve a quick diminution in toxicity to the microbial Several papers have postulated a large number of potential can-
community present, which in turn may help to select ‘‘co-opera- didates for bioaugmentation, and these can be found in a variety of
tors’’ for treating complicated wastewater by synergism (Wang sources such as Arctic or Antarctic environments, laboratory-scale
et al., 2009) in comparison with non bioaugmented reactors. reactors fed with samples taken from full scale plants, from aerobic
Nevertheless, this effect is not always guaranteed since it has been or anaerobic wastewater systems, culture collections, etc. (Singer
reported that effective bioaugmentation strategies can succeed in et al., 2005; Wang et al., 2009; Schauer-Gimenez et al., 2010).
removing a particular contaminant, for which the specific microor- Nevertheless, only limited understanding can be obtained on their
ganisms were selected for on the basis of their biodegradation biodegradation mechanisms in bioreactors (Wang et al., 2009),
capacity, but fail in enhancing the degradation of total organic car- their competitiveness, or their influence on indigenous communi-
bon (TOC) (Yu and Mohn, 2002). These authors explained that ties (Yu and Mohn, 2002), which are considerably more compli-
some of the characterised degraders are apparently nutritional cated processes than can be mimicked in pure culture assays.
specialists who can only use a small number of organics, lacking Furthermore, acclimation in scaling up is enormously important
the ability to degrade the majority of the organic compounds in for achieving effective bioaugmentation, as happens with the use
the wastewater; obviously, this situation does not fulfil all the of starter cultures in the food industry (in dairy or alcoholic
treatment goals. However, wider nutritional capacities would beverage fermentations at industrial scale). The complexity of

Please cite this article in press as: Herrero, M., Stuckey, D.C. Bioaugmentation and its application in wastewater treatment: A review. Chemosphere (2014),
http://dx.doi.org/10.1016/j.chemosphere.2014.10.033
4 M. Herrero, D.C. Stuckey / Chemosphere xxx (2014) xxx–xxx

engineered wastewater biotreatment (in terms of fluctuation in the appropriate catabolic genes to degrade the target compound
volume or composition) even requires specific efforts at this key (Thompson et al., 2005). Hence the efficacy of published bioaug-
stage. In a similar way, the interest in ‘‘priming’’ has been mentation results may frequently be inconclusive.
highlighted (Singer et al., 2005). Priming has been described as Members of the same genera are not all equally fit for certain
‘‘predisposing an isolate or population of microorganisms to future tasks, and hence some could be competitive under a broader range
conditions in which they are designed to perform a function’’ of conditions, while others may only be suited to very specialised
(Singer et al., 2005). Thus acclimation of the microbial consortia conditions (Thompson et al., 2005). Differentiation at species level,
should be required in the flow chart of the process to predispose even among those showing the same catabolic traits, has been
them better towards the physico-chemical conditions for which reported as the cause of failure, along with differences at strain
they were selected and designed for achieving efficient level related to persistence in the system after inoculation, obser-
performance. vations which could lead to more efficient bioaugmentation
As reported by Yu and Mohn (2002), microorganisms used in designs. Wenderoth et al. (2003) carried out bioaugmentation with
bioaugmentation should meet at bare minimum three criteria: different Pseudomonas spp., and with different strains of the same
firstly, to be catabolically able to degrade the contaminant, even species, all of which were capable of chloro- and dichloro-benzene
in the presence of other potentially inhibitory pollutants; secondly, degradation. With some strains, bioaugmentation resulted in little
they must persist and be competitive after their introduction into improvement, but interestingly, differentiation at strain level in
the biosystem; and thirdly, they should be compatible with the relation to persistence in the biosystem was observed. Both strains
indigenous communities present. Therefore, candidates should be Pseudomonas putida GJ31 and P. putida F1DCC could grow even
carefully selected, since only a few organisms are suited to bioaug- when the natural microbial community was present, stimulating
mentation, and these are not necessarily from the community the degradation of chlorobenzene. However, it was noted that
needing bioaugmentation (Yu and Mohn, 2002). An additional the P. putida GJ31 population decreased rapidly when the xenobi-
important point is that for obvious reasons, candidates should otics were depleted, while P. putida F1ACC survived even when the
not be closely related to human pathogens (same genus and spe- chlorobenzene had been degraded.
cies, as exemplified by some Pseudomonas aeruginosa strains) when Frequently, problems were encountered with: growth-limiting
used in field implementation (Singer et al., 2005). conditions due to low substrate concentration; the presence of
Strain selection for bioaugmentation is clearly a key issue. inhibitory substances in the stream to be treated, and even
Singer et al. (2005) proposed that substantial progress could be released by other microorganisms showing antagonistic effects
made through exploiting current knowledge, recent techniques in (such as antibiotics or bacteriocins); the presence of bacterio-
molecular biology, and worldwide access to culture collections. phages; poor biofilm forming ability (Fu et al., 2009), or as a result
At the same time, increasing information from published research of adverse operating conditions such as low temperatures
supports the most effective way of overcoming barriers in bioaug- (Stephenson and Stephenson, 1992). Suitable strains for bioaug-
mentation is to locate organisms from the same ecological niche as mentation could survive for a long time in a specific habitat, result-
the pollutant (El Fantroussi and Agathos, 2005). ing in slow but continuous degradation (Thompson et al., 2005), as
characterised by a low Vmax and Km. Apart from the traditionally
simplified dichotomy based on alive cells versus dead cells when
5. Bioaugmentation failures only culturability tests were applied, there is now evidence that
different microbial physiological states can be observed in environ-
Different workers have postulated a number of reasons for the mental bioprocesses (Díaz et al., 2010) such as metabolically active
failure of inocula when used for bioaugmentation (Stephenson cells, reproductive growing cells, damaged or permeabilised cells
and Stephenson, 1992; Vogel and Walter, 2001; El Fantroussi and (Nebe-von-Caron et al., 2000).
Agathos, 2005). The major claim is that the selected strains often Not only is selection a key factor in bioaugmentation, but also
fail to show under natural environmental conditions the abilities the way of introducing and maintaining the selected microorgan-
shown in the laboratory as pure cultures, even after showing resis- isms and/or their activities in the complex community. Obviously,
tance to starvation or lack of degradation repression in the pres- the biomass concentration introduced should be high enough to
ence of additional nutrients (Boon et al., 2000). Assays in test allow for the prevalence of the metabolically active cells bioaug-
tubes or in laboratory scale bioreactors using axenic cultures, even mented. The use of an enriching bioreactor for bioaugmentation
those strains with exceptional abilities, seem to be unable to mimic purposes was reported as an interesting strategy (Saravanane
the complexity innate to natural biosystems. The interaction of the et al., 2001). In this approach, the main reactor was bioaugmented
inoculated microorganisms with their new biological and non-bio- by the addition of acclimated cells periodically from a separate
logical environments in terms of activity, survival, and migration enrichment-reactor.
can be critical in the success of any bioaugmentation strategy (El As a result of a failed strategy when introducing a strain or con-
Fantroussi and Agathos, 2005). Bioaugmentation can change the sortia, it is possible to provoke undesirable effects or unbalanced
composition of the indigenous microbial community, for example ecosystems. With the intentional introduction of genetically engi-
by competition or inhibition (van Veen et al., 1997), and these neered bacteria, the greater risk is probably their escape from the
changes may be either positive or negative. It is frequently bioreactor to the environment, with unforeseeable results.
reported that, despite a significant increase in bioreactor perfor- Additionally, after inoculation with bioaugmented microorganisms
mance soon after bioaugmentation, very often the positive effects it is possible to unbalance the system. Bouchez et al. (2000)
observed are only maintained for a short time after inoculation reported on two nitrifying reactors operating under identical con-
(Boon et al., 2000; Patureau et al., 2001; Yu et al., 2010). ditions, except that aerobic denitrifying bacteria had been inocu-
Additionally, when different workers reported on bioaugmenta- lated twice into one of them. FISH showed that the bacteria
tion research, the difficulty in characterising, quantifying, and eval- which had been initially bioaugmented were rapidly consumed
uating all of the potential parameters involved could lead to by protozoa in the bioreactor. The second large inoculation in
general conclusions that may not be widely applicable one reactor resulted in the ecosystem becoming unbalanced lead-
(Stephenson and Stephenson, 1992). Some reported failures may ing to a rapid growth of protozoa and upsets in nitrification,
be due to choosing bioaugmentation in the first place, despite whereas in the non bioaugmented reactor both parameters were
the fact that the indigenous microbial community may contain stable. To solve this problem, two different inoculation strategies

Please cite this article in press as: Herrero, M., Stuckey, D.C. Bioaugmentation and its application in wastewater treatment: A review. Chemosphere (2014),
http://dx.doi.org/10.1016/j.chemosphere.2014.10.033
 M. Herrero, D.C. Stuckey / Chemosphere xxx (2014) xxx–xxx 5

were tested to improve the effectiveness of incorporating the bacte- characteristics of the bioprocess, a basic knowledge of the micro-
ria added to the indigenous culture. Firstly, chemicals which pro- bial ecology common to the target biosystem should be acquired
moted coagulation and flocculation were added to the reactor just in order to understand the reasons for poor bioreactor perfor-
after bioaugmentation. Secondly, which gave the best results, the mance. As reported by Dejonghe et al. (2001), it seems necessary
bioaugmented bacteria were encapsulated in alginate beads before to acquire this knowledge in order to understand the main meta-
being inoculated. Over time the beads eventually broke-up, bolic processes and, concomitantly, to find out whether a specific
although fragments of the alginate containing the bioaugmented species in a particular ecosystem belongs to the determinative
microorganisms were incorporated into the existing biological flocs, 20% (controlling 80% of the energy flux) or to the subsisting 80%
and colonisation of the alginate matrix by indigenous bacteria was of the community. These authors state that it is worthwhile explor-
also observed. Hence this demonstrated that immobilization in algi- ing methods of modifying the composition of the ruling fraction so
nate beads offered temporary protection against grazing, and even that other species (or other strains or catabolic genes) might par-
after breakage the fragments favoured the attachment of the bio- ticipate in the major conversion processes. With this approach,
augmented microcolonies to the existing flocs. the energy flux could be more evenly distributed over the commu-
It was also reported that after inoculation the bioaugmented nity, and even the suppressed bacteria could become active and
population decreased corresponding to dilution with the fresh fulfil an important role in the community (Dejonghe et al., 2001).
feed, eventually resulting in the population stabilising, but at a In this way it seems feasible to gain community flexibility in order
lower concentration (McClure et al., 1991). To overcome wash- to shift the electron and carbon flow through various alternative
out, bioaugmentation can potentially take advantage of compart- guilds which is a key factor in enhancing community function
mentalisation, or the use of membranes in advanced bioreactors, (Fernandez et al., 2000).
as a way of keeping the introduced microorganisms within the bio-
system. For example, in the anaerobic baffled reactor (ABR) the 6.2. Monitoring techniques
reactor is divided into eight compartments with little mixing
between the biomass in each compartment, promoting separation To support successful bioaugmentation strategies, along with
of the various trophic groups involved in the process while the gas enhancing biodegradation kinetics of the target pollutant, the use
phases remain separate (Barber and Stuckey, 1999). This design of molecular techniques is required (monitoring both the survival
allows for the migration of sensitive anaerobes, such as the meth- and/or activity of the added microorganisms). In the last few years
anogens, from the front of the reactor where unfavourable growth different techniques have been used such as PCR–DGGE (Guo et al.,
conditions may occur, to the more protected later compartments. 2010), PCR-temperature gradient gel electrophoresis (TGGE)
An aerated stage in the penultimate compartment may oxidise (Fouratt et al., 2003), T-RFLP (Tsutsui et al., 2010), ribosomal inter-
refractory COD and excess sulphide from the anaerobic stages, genic spacer analysis (RISA), (Qu et al., 2009), competitive PCR and
enabling nitrification to occur, especially if immobilized cells are Reverse transcription PCR (RT-PCR) (Muttray et al., 2001), quanti-
used. In the case of membrane-aerated biofilm reactors (MABRs) tative PCR and DGGE (Watanabe et al., 2002), quantitative PCR
(Ohandja and Stuckey, 2007), the membrane in the reactor sup- and RT-PCR (Morris et al., 2014), FISH (Patureau et al., 2001;
ports an active biofilm, maintaining a biofilm of active bacteria, Bartroli et al., 2011), and the use of marker genes such as the green
and the MABR can combine both anaerobic and aerobic layers in fluorescence protein (gpf-gene mark) (Boon et al., 2000; Yu et al.,
the biofilm. This enables processes to occur simultaneously in 2010). More detailed information on these techniques is given in
the same reactor when aerobic and anaerobic conditions are Table 1. Even the potential of applying microarrays to environmen-
needed, such as in the case of nitrification and denitrification pro- tal studies has been reported, highlighting the need to face several
cesses, or the degradation of chlorinated organics such as PCE challenges associated with specificity, sensitivity and quantifica-
(Ohandja and Stuckey, 2007). tion in these types of samples (Zhou and Thompson, 2002).
Failed bioaugmentation at full scale has been reported in coke However, it should be noted that high-throughput sequencing
WWT when using a consortium of a cyanide-degrading yeast (HTS) is causing a revolution recently by opening up new pathways
obtained from a culture collection (Cryptococcus humicolus) and in environmental microbiology (Logares et al., 2012). HTS will pro-
unidentified cyanide-degrading microorganisms (obtained from vide a new understanding of ecological processes and microbial
the activated sludge of a full-scale coke WWT facility) (Park community functioning. Applications of single-cell sequencing in
et al., 2008). After laboratory scale cultivation (up to 1.2 m3) combination with metagenomic analysis have been reviewed
enriched for two months with a huge supply of glucose, KCN and recently (Lasken, 2012). New powerful bioinformatic tools are
other nutrients, the consortium was inoculated into a fluidized- improving genome assembly and gene prediction in anonymous
bed type process (1280 m3). However, continuous operation of prokaryotic genomes, overcoming bottlenecks in the data analysis
the full-scale cyanide-degrading bioprocess showed poorer work.
removal efficiency than expected (slow biodegradation rate of fer-
ric cyanide) owing to the poor settling performance of microbial 6.3. Success in overcoming operational problems and plant
flocs, and lack of organic carbon sources within the wastewater. management
The concentration of activated sludge within the aeration tanks
decreased because of substantial wash out from the sludge settling Sometimes bioaugmentation is used in WWTPs to overcome a
tank. In addition, violent bubbling of air disturbed the formation of significant reduction in biotreatment efficiency due to seasonal
a biofilm on the carrier’s surface. The need for further studies was low temperatures, as typically occurs in nitrification during winter
highlighted in terms of how to solve operating problems in both months. In such cases, the reactor needs a greater biomass concen-
pilot and full scale bioaugmentation approaches. tration in the winter period than in the summer period. It has been
reported that bioaugmentation allowed for a reduction in the nec-
6. Success of bioaugmentation strategies: key factors essary volume needed for a stable process which could be main-
tained under conditions that would normally have prevented
6.1. Ecological basis nitrification, and moreover, its effects could be predicted by the
use of a simple model (Plaza et al., 2001).
Before making any decision regarding implementing Bioaugmentation was also useful in decreasing the recovery
bioaugmentation, along with the particular physico-chemical time of anaerobic digesters transiently overloaded (Tale et al.,

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6 M. Herrero, D.C. Stuckey / Chemosphere xxx (2014) xxx–xxx

Table 1
Examples of different monitoring techniques used in reported bioaugmentation procedures.

Treatment Process Strain/consortium/plasmid/genes Monitoring Contaminant(s) removed/activity detected Reference

techniques
Full scale municipal WWT, (SBR, Specialised consortium comprising PCR–DGGE Organics. Effluents quality: COD Guo et al.,
oxidation ditch, anoxic–oxic Proterobacteria, Bacterioides, Nitrospirales and BIOLOG (6660 mg L 1), NH4+–N (68 mg L 1
) 2010
activated sludge-A/O) at low Cyanobacteria, and the flocculent bacteria Bacillus
temperature sp. F2 and Bacillus sp. F6
Laboratory scale SBRs Commercial product, NBP PCR–TGGE Organics (synthetic wastewater), ammonia Fouratt
removal (until detection limit) et al., 2003
Laboratory scale activated sludge Plasmid pJP4 from Escherichia coli and T-RFLP Organics, (COD removal 84–86%) Tsutsui
reactors Pseudomonas putida (as donors) to indigenous et al., 2010
transconjugants
Laboratory scale anaerobic mcrA Gene and transcripts, coding the a subunit Quantitative PCR Methanogens dynamics, linking mcrA gene Morris
bioreactors of methyl coenzyme M reductase and RT-PCR copy number to methane flux et al., 2014
Laboratory scale membrane Sphingomonas xenophaga QYY RISA Bromoamine acid (dye) in synthetic Qu et al.,
bioreactor (MBR) wastewater, COD removal 30–50%, 2009
decolorization ratio (supernatant and
membrane effluent) 80–90%
Laboratory-scale batch/ Pseudomonas abietaniphila BKME-9 Competitive PCR Dihydroabietic acid (DhA), degradation rate Muttray
chemostat and RT-PCR two times faster than control et al., 2001
Activated sludge, municipal phc genes coding for multicomponent phenol Quantitative Phenol, average value phenol-oxygenating Watanabe
treatment plant hydroxylase mPH, PhcKLMNP and its competitive PCR activity 15.3 ± 0.8 U g 1 et al., 2002
transcripcional regulators, PhcR, and PhcT
(phcTRKLMNP)
SBR anaerobic–aerobic Microvirgula aerodenitrificans (free cells, different FISH N removal (33–43%); or N-oxides (10.2– Patureau
inoculation levels/continuous supply/ 13.8 mg L 1), a concomitant phosphate et al., 2001
immobilised in alginate beads) concentration (0–2 mg L 1 ; no ammonia
Pilot scale, continuous airlift Nitrifying activated sludge (from a pilot plant) FISH TAN oxidation near 100%, max nitrifying Bartroli
reactors, activated carbon for capacity at steady-state conditions et al., 2011
biofilm formation 1.3 gN L 1 d 1 (synthetic wastewater)
Laboratory scale, SCAS reactors Comamonas testosterone I2gfp chromosomally Autofluorescence, 3-Chloroaniline (3-CA) (synthetic Boon et al.,
gfp-marked) PCR–DGGE wastewater) initially complete removal, 2000
50% in further operation
Laboratory scale, SBR Pseudomonas putida ONBA-17gfp Autofluorescence, o-Nitrobenzaldehyde (ONBA) complete Yu et al.,
PCR–DGGE degradation, and COD (96.28%) (synthetic 2010
wastewater)

SBR: sequencing batch reactor; SCAS: semicontinuous activated sludge; PAO: phosphorus accumulating organisms; TAN: total ammonia nitrogen.

2011), or exposed to a transient toxic event (Schauer-Gimenez results may be due to differences in the original microbial commu-
et al., 2010). In the latter case, results varied depending on the nities in the two sets of digesters. Therefore, it could be observed
source of the inoculum to start-up each set of digesters. These that the microbial community structure within an existing biolog-
authors claimed that production and distribution of individual bio- ical system is just as important as the community structure of any
augmentation cultures, enriched to degrade a specific substrate, culture added when bioaugmentation is implemented.
would be time consuming. Using a novel approach, it was proposed Successful bioaugmentation strategies can also be essential to
that it could be more practical to target a key, ubiquitous interme- achieve better WWTP management when the existing facilities
diate that accumulates during toxic events. To this aim, hydrogen become insufficient to treat increasing wastewater volumes. As
(H2) was chosen since its degradation is often a rate-limiting step reported by Ma et al. (2009), as both the amount and type of
in methane production from many complex substrates, so it is pos- petrochemical products increase, the existing anoxic–oxic (A/O)
sible that the more rapid H2 utilisation, the more complete the con- activated sludge process in the WWTP could not meet the demands
version of propionate and other substrates to methane. Two sets of of the increasingly complicated petrochemical wastewater, so it
laboratory-scale digesters were transiently exposed to the model was urgent to develop innovative technologies for proper treat-
toxicant (oxygen): the sets differed only in the source of the ment. The bioaugmentation option chosen to upgrade the existing
original inoculum employed to start operation. In one set, original facilities was proven to be efficient.
biomass was taken from a mesophilic anaerobic digester fed syn-
thetic municipal sludge. The other set was originally started with
biomass from a mesophilic anaerobic digester fed synthetic indus- 6.4. Different successful selection criteria
trial wastewater. The methanogenic culture used for bioaugmenta-
tion was developed using biomass from a mesophilic municipal As mentioned above, the strategy of using tailor-made consortia
anaerobic digester after enrichment. The archaeal community in seems to be a promising approach. Van der Gast et al. (2004) inves-
the bioaugmented culture was analysed, showing sequences simi- tigated the effectiveness of a bacterial consortium composed of
lar to those of H2-utilising methanogens. However, one set of four species on the basis of their ubiquity in waste metal-working
digesters produced lingering high propionate concentrations, and fluids (MWFs), their degradation ability, and their tolerance to the
bioaugmentation resulted in significantly shorter recovery periods, fluctuating conditions. The inoculum was found to represent a sig-
while the second set of bioaugmented digesters did not display nificant component of the community in bioreactors both with and
lingering propionate and recovered in the same time as the non- without the presence of indigenous MWF populations. The reduc-
bioaugmented controls. As stated, the contradiction between these tion in the COD by the consortium was approximately 85% of the

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 M. Herrero, D.C. Stuckey / Chemosphere xxx (2014) xxx–xxx 7

total pollution load, and was 30–40% more effective than any other indicating that, even with a strain originating from the ecosystem
treatment. and able to grow effectively on a selective substrate, bioaugmenta-
However, tailoring inocula for every case and application would tion was not permanent and would probably require regular
just not be practicable (Thompson et al., 2005). At least some of the resupplementation.
broad commercial bioaugmentation consortia have been proven to As mentioned earlier, an enriching bioreactor in the flow chart
be efficient. For example, a generic commercial consortium was of the process may be useful to guarantee enough acclimated cells
successfully added by Duran et al. (2006) to enhance the conver- to be continuously or intermittently introduced into the main reac-
sion of biosolids to methane and remove odorous compounds, con- tor. The enricher-reactor can operate not only separately, but also
taining selected strains of bacteria from the genera Bacillus, under different conditions from the main reactor. The biomass pro-
Pseudomonas, and Actinomycetes, along with ancillary organic com- duced within this enricher-reactor where optimum growth condi-
pounds containing various micronutrients. It is known that certain tions were maintained and growth-supporting substrates were
commercially available products containing enzymes such as added, turned out to be viable in the main reactor for several gen-
lipases, proteinases, cellulases, and hydrolases enhance enzymatic erations, increasing the biomass concentration. Results demon-
break down of macromolecules. Despite the non-specific nature of strated that this continuous or intermittently transferred
this consortium, its addition enhanced methane production (29%), inoculant overcame the difficulty of growing adequate biomass
and reduced propionate levels (50%) compared to the control. A in the main reactor on inhibitory or toxic substances (Saravanane
commercial product suited for nitrification (nitrifying bioaugmen- et al., 2001). Interestingly, with this strategy it is possible to sepa-
tation product, NBP, presented as a mixed consortium) was useful rate the propagation and acclimation stages from the biodegrada-
in enhancing the activity and process efficiency in nitrification. tion stage, ensuring high cell densities for inoculation.
Nevertheless, after efficient bioaugmentation with NBP at 1%, it Another interesting strategy is using the slow release of immo-
was not possible to correlate the observed increase in activity with bilized bioaugmented cells (growing in moulded agar) into the
a detectable shift in the population by means of TGGE (with higher activated sludge by means of encapsulation in 4 mm diameter
detection level, requiring at least 5%), as reported by Fouratt et al. open-ended silicone tubes (3 cm long) (Boon et al., 2002). The
(2003). tubes containing the immobilized bacteria represented about 1%
In any case, a crucial issue for any commercial bioaugmentation of the volume of the mixed liquor. The bioaugmentation activity
consortium is the preservation and storage of cultures under the of a reactor containing the immobilized cells was compared to a
best conditions to achieve higher survival/activity at inoculation reactor inoculated with suspended cells, revealing that from
time. To this aim it has been reported that there is considerable approximately 30 days after inoculation the reactor with sus-
interest in adding cryoprotectants to freeze-drying in air methano- pended cells failed to completely degrade the contaminant because
genic cultures to achieve higher activities (Bhattad et al., 2010). of a decrease in metabolic activity. However, slow release of the
Bioaugmentation with exogenous genes (phc, which code for growing embedded cells from the agar into the activated sludge
phenol hydroxylase and its transcriptional regulators) transferred medium resulted in a higher number of active degrading cells,
to the indigenous species dominating the biosystem turned out and was responsible for nearly 90% degradation.
to be a successful strategy (Watanabe et al., 2002). With this
approach it seems possible to enhance the activity of the already 6.6. Success of immobilization techniques
established population, showing good abilities to tolerate the pre-
vailing conditions in the target niche. The introduction of the target Immobilization techniques of exogenously added bacterial cells
catabolic genes into indigenous microorganisms that are already might be a solution to generate protective barriers around micro-
adapted to survive and proliferate in the environment better guar- organisms, and also to increase metabolic activity. Entrapment
antees persistence versus the technologically challenging survival can be an efficient way to protect microorganisms from grazing
of the exogenous strains. Genes from Comamonas testosteroni R5, by protozoa, as well as reducing biomass loss caused by washout.
were introduced into an indigenous strain, Comamonas sp. rN7, Although attachment by adsorption has been the preferred method
which constituted the dominant catabolic population of the acti- of immobilization for wastewater treatment, much higher cell
vated sludge community. The high phenol-oxygenating activity concentrations can be obtained by encapsulation, despite the
showed by the introduced transformant, rN7(R503) could be estab- attachment methods requiring no chemical addition. It has also
lished within the sludge, improving resistance to phenol-shock been reported that nitrification rates were generally higher for
loading compared to sludge inoculated with no cells, or to the host polyvinyl alcohol (PVA) encapsulation than for attachment systems
rN7 or to the foreign phenol-degrading strain R5. Nevertheless, it (Rostron et al., 2001). Laboratory-scale continuously stirred tank
was observed that the expression of Phc mPH in rN7(R503) cells reactors containing freely suspended and immobilized biomass
was less efficient in the sludge than in pure culture, highlighting were operated with a high-strength synthetic ammonia wastewa-
the importance of studying factors that govern gene expression ter. Nitrifiers are slow growing and have a low yield, and hence
in natural ecosystems. So it is feasible that dominant populations without long retention times they will be washed out of a contin-
in wastewater reactors can be good candidates as hosts for desired uous reactor unless they are kept in by immobilization. A high cell
catabolic activities that are to be exogenously introduced to accel- concentration is possible with immobilization, so the volumetric
erate the natural gene exchange, and recombination events seeking efficiency is greatly increased. This can lead to relatively small
to spread degradation genes. reactors, and may afford protection from toxic shocks and adverse
temperatures. The freely suspended nitrifiers were washed out of
6.5. Repeated or continuous application versus single inoculation the reactors at a 1 d hydraulic retention time (HRT), whereas the
reactors containing adsorption particles and PVA-encapsulated nit-
Interestingly, a key factor ensuring a successful strategy is that rifiers continued partially nitrifying down to 12 h. At that time all
bioaugmentation must be performed on a regular basis due pri- reactors suffered a loss of nitrification, with the PVA reactor main-
marily to the temporary stability of the newly introduced strains taining the highest nitrification rate and 30% full nitrification to
(Boon et al., 2000). In Boon’s work it was considered that the nitrate.
natural level of the indigenous strain was too low to degrade the Other matrices have also been used successfully. Whole-cell
target contaminant. After the strain was introduced, community immobilization of selenate-respiring Sulfurospirillum barnesii in
structure was analysed using DGGE of the 16S rRNA genes polyacrylamide gels was used to treat selenate contaminated

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8 M. Herrero, D.C. Stuckey / Chemosphere xxx (2014) xxx–xxx

synthetic wastewater with a high molar excess of nitrate (1500 metal contaminated sites (for a review see Top et al., 2002;
times) and sulphate (200 times). To validate the bioaugmentation Tsutsui et al., 2010). The authors concluded that successful cases
success under microbial competition, gel cubes with immobilized suggest that the strategy could indeed work under specific condi-
S. barnesii cells were added to an upflow anaerobic sludge bed tions, such as when the in situ degradation potential is absent,
(UASB) reactor, resulting in earlier selenate and sulphate removal and the pollutant degrading transconjugants can grow and become
and higher nitrate/nitrite removal efficiencies compared to a non- numerically dominant populations in the bacterial community.
bioaugmented control reactor. The selenate reducing activity was Obviously, further studies in this area are needed to improve
maintained during long-term operation, and molecular analysis current knowledge on the efficiency of gene dissemination as an
showed that S. barnesii was present in both the sludge bed and effective tool under real operating conditions.
effluent. It could be demonstrated that gel immobilization of spec-
ialised bacterial strains can survive wash-out and out-compete 6.8. Success by using membrane bioreactors
newly introduced strains in continuous bioaugmented systems
(Lenz et al., 2009). Membrane bioreactors (MB) have been used successfully for
An anaerobic sequencing batch biofilm reactor (AnSBBR) was bioaugmentation purposes (Qu et al., 2009; see Table 1). The intro-
inoculated with enriched sulphate reducing bacteria (SRB) in an duction of Sphingomonas xenophaga QYY (a specialised degrader of
alginate-immobilized matrix for the enhanced treatment of sul- the target compound) into the MBR system significantly increased
phate bearing chemical wastewater. Following a sound approach, contaminant removal in comparison to the non-inoculated MBR.
firstly, the technological and ecological base of the failure was Also, bioaugmentation could accelerate start-up of the MBR and
acknowledged. Poor bioreactor performance could be assessed by enable it to run well despite facing sudden toxic pollutant shock
the accumulation of volatile fatty acids, and subsequently this loads. These authors highlighted the fact that the introduced spec-
effect was attributed to process inhibition due to the presence of ialised strain was compatible with the indigenous populations.
sulphate, and the non-existence of sulphate reducing bacteria Also, the long-term performance of a bioaugmented MBR for treat-
(SRB) and methanogenic bacteria. Consequently, the reactor was ment of textile wastewater containing different azo dyes has been
augmented with an enriched SRB consortia entrapped in an algi- reported (Hai et al., 2011). Stable decolourization along with signif-
nate matrix; after augmentation the reactor showed significant icant TOC removal over 7 months under extremely high dye load-
enhancement in overall performance (COD removal efficiency ings demonstrated the feasibility of the process. Thus, the use of an
and sulphate reduction). Microbial diversity in a non-augmented MBR in combination with bioaugmentation techniques can help to
reactor showed the dominance of acetogenic bacteria over the overcome the frequent claim that bioaugmentation is effective but
methanogenic and SRB. After augmentation, the microbial distri- ephemeral in bioreactors.
bution varied significantly: the introduction of an enriched SRB
consortia resulted in competition between anaerobic bacteria in 6.9. Success at full scale implementation
the system being altering leading to an improvement in process
performance. The entrapping matrix protected the consortia from Application of bioaugmentation to existing industrial WWT
possible predation from the new environment prior to adaptation, facilities has rarely been reported. Bioaugmentation with mixed
giving sufficient time for the inoculated consortia to acclimatise to cultures of specialised bacteria targeting various refractory organ-
the new environment. Interestingly, it was concluded that biofilm ics was successfully applied to upgrade a full-scale activated
configured systems were generally more favourable for augmenta- sludge system into a contact oxidation system (Ma et al., 2009).
tion due to an increased cell density, the high potential for cell to In this case, application of bioaugmentation was combined with
cell contact, reduced mass transfer limitations, and good retention immobilization through the contact oxidation process taking
of the added consortia (Mohan et al., 2005). advantage of compartmentalisation. The petrochemical WWTP
influent was a mixed waste stream from an oil refinery and various
6.7. Advantages of gene transfer methods petrochemical industries, and the wastewater contained numerous
highly toxic and refractory organics (such as petroleum hydrocar-
Since introduced microorganisms often do not survive follow- bons, benzene hydrocarbons, aniline, nitrobenzene, phenols as well
ing bioaugmentation, scientists have investigated the use of as their derivatives). The consortium (mainly consisting of
naturally occurring horizontal gene transfer processes for the Pseudomonas, Bacillus, Acinetobacter, Flavobacterium and
introduction of catabolic genes to treat wastes. It is known that Micrococcus) was enriched from the activated sludge of various
genes that encode for the degradation of both naturally occurring petrochemical WWTPs and then acclimated. After bioaugmenta-
and xenobiotic organic compounds are often located on plasmids, tion, the start-up time was shorter than in the non-bioaugmented
transposons or other mobile and/or integrative elements (Top system; besides the rapid upgrade period, the bioaugmented
et al., 2002). Recent advances in genome sequencing are revealing system also improved the degradation efficiency of recalcitrant
the substantial role that horizontal gene transfer has played in compounds and the resistance to shock loadings. Rapid removal
microbial development and adaptation in the environment was facilitated by temporal and spatial multiple stages accom-
(Ochman et al., 2000). Horizontal gene transfer may occur via plished by the collaborative functions of the bacterial communities
uptake of naked DNA by competent cells, which has reached this formed in each compartment. In conclusion, the authors high-
particular physiological stage in their life cycle (by transforma- lighted that success relied on the survival of the specialised consor-
tion), mediation by bacteriophage (general or specialised transduc- tia as the most significant factor, along with adjustment of DO
tion, in the latter case when transferred DNA is adjacent to the concentration in the biological tank, which were considered to
phage attachment site), or physical contact and exchange of create the optimum operational conditions for the growth and
genetic material such as plasmids or conjugative transposons reproduction of the inoculated bacteria.
between microorganisms (conjugation). The potential advantage
of using gene bioaugmentation over traditional cell bioaugmenta- 7. Prospects
tion approaches relies on no requirement for long-term survival
of the introduced host strain. The transfer of plasmids, via conjuga- With the recent advent of membrane reactors in WWT, the
tion, is the technology most studied with respect to bioaugmenta- ability to keep microbial consortia in bioreactors has been
tion, and gene bioaugmentation may also have applications for enhanced substantially, which should lead to better performance

Please cite this article in press as: Herrero, M., Stuckey, D.C. Bioaugmentation and its application in wastewater treatment: A review. Chemosphere (2014),
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 M. Herrero, D.C. Stuckey / Chemosphere xxx (2014) xxx–xxx 9

and better quality effluents. However, increasing interest in bio- 8. Conclusions

augmentation ought to be assessed by full scale data which to date
is still very scarce. Community assembly, ecology, and microbial dynamics in bio-
Emerging areas of research should help in developing new reactors treating wastewater are complex processes. Basic knowl-
approaches for novel applications in bioaugmented bioreactors. edge of the biosystem ecology is required to establish a clear
Protein engineering has a wide potential for technological appli- definition of the treatment goal to achieve. Not only should strain
cations. It is known that immobilization techniques can give rise (or tailor-made consortium) selection, but also the way of intro-
to higher enzymatic rates by achieving higher concentrations and ducing and maintaining the selected microorganisms and/or their
even promoting conformational changes. It should also be consid- activities in the community, and acclimation in scaling up and bio-
ered that many of the enzymes and substrates do not function in reactor design must be considered. Emerging areas of research
solution. Instead, cells use a form of ‘solid-state’ biochemistry, should help in developing new bioaugmented bioreactors: protein
and it has been pointed out that chemical reactions are often engineering; in vitro compartmentalisation; nanomaterials for
‘channelled’ through these structures so that substrates are not enhanced reactivity, surface area, and/or sequestration characteris-
freely diffusible and pass from one modifying enzyme or enzyme tics; and, single cell analysis and cell–cell interactions under real
complex to neighbouring functional assemblies, exhibiting much operating conditions.
higher efficiencies than is possible to mimic in solution in a test
tube (Ingber, 2010). This idea could be further investigated to Acknowledgements
enhance bioaugmentation strategies by cell-to-cell contact (flocs,
biofilms). An in vitro compartmentalisation (IVC) technique has M.H. acknowledges the support of the Research Vice
been developed by creating cell-like structures such as water- Chancellor’s Office, University of Oviedo, for her stay in the
in-oil emulsions (droplets) where single genes are encapsulated Department of Chemical Engineering, Imperial College, London.
in an artificial membrane along with the components required
to transcribe and translate them, adding other molecules if References
required (for a review see Taly et al., 2007). An equivalent proce-
dure is to compartmentalise bacteria into droplets, allowing each Ayala-del-Río, H.L., Callister, S.J., Criddle, C.S., Tiedje, J.M., 2004. Correspondence
between community structure and function during succession in phenol- and
droplet to work as an individual microbioreactor, not limited by phenol-plus-trichloroethene-fed sequencing batch reactors. Appl. Environ.
the fitness of the organism expressing the enzyme of interest, Microbiol. 70, 4950–4960.
thus permitting conditions that would be detrimental to most liv- Barber, W.P., Stuckey, D.C., 1999. The use of the anaerobic baffled reactor (ABR) for
wastewater treatment: a review. Water Res. 33, 1559–1578.
ing organisms.
Bartroli, A., Carrera, J., Pérez, J., 2011. Bioaugmentation as a tool for improving the
Nowadays, nanotechnology is an emerging area of research start-up and stability of a pilot-scale partial nitrification biofilm airlift reactor.
with considerable potential. Nanomaterials have been extensively Bioresour. Technol. 102, 4370–4375.
used for rapid or cost effective clean-up of wastes (Shan et al., Bhattad, U.H., Maki, J.S., Struble, C.A., Schauer-Gimenez, A.E., Zitomer, D.H., 2010.
Culture conditions and cryoprotectant addition influences methanogenic
2009). Their benefits of application in wastewater treatment are activity after freeze-drying in air. In: Proceedings of International Water
derived from the nanoparticle characteristics: enhanced reactivity, Association (IWA) 12th World Congress on Anaerobic Digestion, Guadalajara,
surface area, sub-surface transport, and/or sequestration charac- Mexico, October 31st–November 4th, 5p.
Boon, N., Goris, J., De Vos, P., Verstraete, W., Top, E.M., 2000. Bioaugmentation of
teristics (Brar et al., 2010). As a practical application, reverse micel- activated sludge by an indigenous 3-chloroaniline-degrading Comamonas
lar extraction (RME) has the advantages of ease of scaling-up and testosteroni strain, I2gfp. Appl. Environ. Microbiol. 66, 2906–2913.
continuous operation. In this case, biomolecules can be recovered Boon, N., De Gelder, L., Lievens, H., Siciliano, S.D., Top, E.M., Verstraete, W., 2002.
Bioaugmenting bioreactors for the continuous removal of 3-chloroaniline by a
from the reverse micellar phase by exploiting the disassembling slow release approach. Environ. Sci. Technol. 36, 4698–4704.
nature of RMEs in aqueous media (George and Stuckey, 2010). A Bouchez, T., Patureau, D., Dabert, P., Juretschko, S., Doré, J., Delgenès, P., Moletta, R.,
priori, new approaches supported by the current knowledge could Wagner, M., 2000. Ecological study of a bioaugmentation failure. Environ.
Microbiol. 2, 179–190.
be useful not only for engineered nanomaterials detoxification of a Brar, S.K., Verma, M., Tyagi, R.D., Surampalli, R.Y., 2010. Engineered nanoparticles in
wide variety of contaminants in waters by adsorptive removal wastewater and wastewater sludge – evidence and impacts. Waste Manage. 30,
(pesticides, residual antibiotics, pharmaceutical compounds and 504–520.
Curtis, T.P., Head, I.M., Graham, D.W., 2003. Theoretical ecology for engineering
endocrine disruptors), but maybe potentially as delivery systems
biology. Environ. Sci. Technol. 37, 64A–70A.
to facilitate the introduction of gene products in bioreactors treat- Dejonghe, W., Boon, N., Seghers, D., Top, E.M., Verstraete, W., 2001.
ing highly toxic, refractory or recalcitrant compounds in WWT. Bioaugmentation of soils by increasing microbial richness: missing links.
However, engineered nanomaterials could also become water con- Environ. Microbiol. 3, 649–657.
Díaz, M., Herrero, M., García, L.A., Quirós, C., 2010. Application of flow cytometry to
taminants so concerns regarding the use of persistent nanoparti- industrial microbial bioprocesses. Biochem. Eng. J. 48, 385–407.
cles that, on ingestion, will accumulate within the body will Duran, M., Tepe, N., Yurtsever, D., Punzi, V.L., Bruno, C., Mehta, R.J., 2006.
require new methodologies for their evaluation and for establish- Bioaugmenting anaerobic digestion of biosolids with selected strains of
Bacillus, Pseudomonas, and Actinomycetes species for increased
ing adequate criteria for risk assessment (Brar et al., 2010). methanogenesis and odor control. Appl. Microbiol. Biotechnol. 73, 960–966.
Single cell analysis (SCA) will be crucial for elucidating cellular El Fantroussi, S., Agathos, S.N., 2005. Is bioaugmentation a feasible strategy for
diversity and heterogeneity (Wang and Bodovitz, 2010). A deeper pollutant removal and site remediation? Curr. Opin. Microbiol. 8, 268–275.
Fernandez, A., Hashsham, S., Dollhopf, S.L., Raskin, L., Glagoleva, O., Dazzo, F.B.,
knowledge at the single cell level (considering the biochemistry Hickey, R.F., Dazzo, F., Criddle, C., Tiedje, J., 2000. Flexible community structure
and toxicity mechanisms of contaminants) also complemented at correlates with stable community function in methanogenic bioreactor
the community level is still needed to understand the success communities perturbed by glucose. Appl. Environ. Microbiol. 66, 4058–4067.
Fouratt, M.A., Rhodes, J.S., Smithers, C.M., Love, N.G., Stevens, A.M., 2003.
and key factors controlling bioaugmentation under real operation Application of temperature gradient gel electrophoresis to the
conditions. It seems evident that a close interaction between engi- characterization of a nitrifying bioaugmentation product. FEMS Microbiol.
neering and biotechnology approaches is essential to promote Ecol. 43, 277–286.
Fu, S., Fan, H., Liu, S., Liu, Y., Liu, Z., 2009. A bioaugmentation failure caused by phage
higher removal efficiencies. It is also necessary that bioreactor
infection and weak biofilm formation ability. J. Environ. Sci. 21, 1153–1161.
design facilitates the transition from laboratory to full scale opera- George, A.D., Stuckey, D.C., 2010. Extraction of monoclonal antibodies (IgG1) using
tion. Finally, the last step is to develop proper models, usable at all anionic and anionic/nonionic reverse micelles. Biotechnol. Prog. 26, 1352–1360.
scales, which are capable of predicting the function of microbial Guo, J., Wang, J., Cui, D., Wang, L., Ma, F., Chang, C.-C., Yang, J., 2010. Application of
bioaugmentation in the rapid start-up and stable operation of biological
populations in biological treatment under different process processes for municipal wastewater treatment at low temperatures. Bioresour.
conditions. Technol. 101, 6622–6629.

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Hai, F.I., Yamamoto, K., Nakajima, F., Fukushi, K., 2011. Bioaugmented membrane Schauer-Gimenez, A.E., Zitomer, D.H., Maki, J.S., Struble, C.A., 2010.
bioreactor (MBR) with a GAC-packed zone for high rate textile wastewater Bioaugmentation for improved recovery of anaerobic digesters after toxicant
treatment. Water Res. 45, 2199–2206. exposure. Water Res. 44, 3555–3564.
Hu, X., Li, A., Fan, J., Deng, C., Zhang, Q., 2008. Biotreatment of q-nitrophenol and Shan, G., Yan, S., Tyagi, R.D., Surampalli, R.Y., Zhang, T., 2009. Application of
nitrobenzene in mixed wastewater through selective bioaugmentation. nanomaterials in environmental science and engineering: review. Pract. Period
Bioresour. Technol. 99, 4529–4533. Hazard. Toxic Radioact. Waste Manage. 13, 110–119.
Ingber, D.E., 2010. From cellular mechanotransduction to biologically inspired Singer, A.C., Van der Gast, C.J., Thompson, I.P., 2005. Perspectives and vision for
engineering. Ann. Biomed. Eng. 38, 1148–1161. strain selection in bioaugmentation. Trends Biotechnol. 23, 74–77.
Jorgensen, B.B., Gallardo, V.A., 1999. Thioploca spp.: filamentous sulfur bacteria with Sloan, W.T., Lunn, M., Woodcock, S., Head, I.M., Nee, S., Curtis, T.P., 2006.
nitrate vacuoles. FEMS Microbiol. Ecol. 28, 301–304. Quantifying the roles of immigration and chance in shaping prokaryote
Lasken, R.S., 2012. Genomic sequencing of uncultured microorganisms from single community structure. Environ. Microbiol. 8, 732–740.
cells. Nat. Rev. Microbiol. 10, 631–640. Stephenson, D., Stephenson, T., 1992. Bioaugmentation for enhancing biological
Lawrence, A.W., McCarty, P.L., 1970. Unified basis for biological treatment design wastewater treatment. Biotechnol. Adv. 10, 549–559.
and operation. J. Sanit. Eng. Div. Am. Soc. Civ. Eng. 96, 757–775. Tale, V.P., Maki, J.S., Struble, C.A., Zitomer, D.H., 2011. Methanogen community
Lenz, M., Enright, A.M., O’Flaherty, V., Van Aelst, A.C., Lens, P.N.L., 2009. structure–activity relationship and bioaugmentation of overloaded anaerobic
Bioaugmentation of UASB reactors with immobilized Sulfurospirillum barnesii digesters. Water Res. 45, 5249–5256.
for simultaneous selenate and nitrate removal. Appl. Microbiol. Biotechnol. 83, Taly, V., Kelly, B.T., Griffiths, A.D., 2007. Droplets as microreactors for high-
377–388. throughput biology. ChemBioChem 8, 263–272.
Logares, R., Haverkamp, T.H., Kumar, S., Lanzén, A., Nederbragt, A.J., Quince, C., Thompson, I.P., Van der Gast, C.J., Ciric, L., Singer, A.C., 2005. Bioaugmentation for
Kauserud, H., 2012. Environmental microbiology through the lens of high- bioremediation: the challenge of strain selection. Environ. Microbiol. 7, 909–
throughput DNA sequencing: synopsis of current platforms and bioinformatics 915.
approaches. J. Microbiol. Methods 91, 106–113. Top, E.M., Springael, D., Boon, N., 2002. Catabolic mobile genetic elements and their
Ma, F., Guo, J.B., Zhao, L.J., Chang, C.C., Cu, D., 2009. Application of bioaugmentation potential use in bioaugmentation of polluted soils and waters. FEMS Microbiol.
to improve the activated sludge system into the contact oxidation system Ecol. 42, 199–208.
treating petrochemical wastewater. Bioresour. Technol. 100, 597–602. Tsutsui, H., Anami, Y., Matsuda, M., Inoue, D., Sei, K., Soda, S., Ike, M., 2010. Transfer
MacArthur, R., Wilson, E., 1967. The Theory of Island. Biogeography. Princeton of plasmid pJP4 from Escherichia coli and Pseudomonas putida to bacteria in
University Press, Princeton, NJ. activated sludge developed under different sludge retention times. J. Biosci.
Maphosa, F., Vos, W.M., Smidt, H., 2010. Exploiting the ecogenomics toolbox for Bioeng. 110, 684–689.
environmental diagnostics of organohalide-respiring bacteria. Trends Van der Gast, C.J., Knowles, C.J., Starkey, M., Thompson, I.P., 2002. Selection of
Biotechnol. 28, 308–316. microbial consortia for treating metal working fluids. J. Ind. Microbiol.
May, R.M., 1974. Stability and Complexity in Model Ecosystems. Princeton Biotechnol. 29, 20–27.
University Press, Princeton, NJ. Van der Gast, C.J., Whiteley, A.S., Thompson, I.P., 2004. Temporal dynamics and
McClure, N.C., Fry, J.C., Weightman, A.J., 1991. Genetic engineering for wastewater degradation activity of a bacterial inoculum for treating waste metal-working
treatment. J. Inst. Water Environ. Manage. 5, 608–616. fluid. Environ. Microbiol. 6, 254–263.
Melandri, B.A., 1997. Vectorial bioenergetics. In: Graber, P., Milazzo, G. (Eds.), Van Veen, J.A., Van Overbeek, L.S., Van Elsas, J.D., 1997. Fate and activity of
Bioenergetics. Birkhauser Verlag, Basel, Switzerland, pp. 95–138. microorganisms introduced into soil. Microbiol. Mol. Biol. Rev. 61, 121–135.
Mertens, B., Boon, N., Verstraete, W., 2005. Stereospecific effect of Verstraete, W., 2007. Microbial ecology and environmental biotechnology. ISME J. 1,
hexachlorocyclohexane on activity and structure of soil methanotrophic 4–8.
communities. Environ. Microbiol. 7, 660–669. Vogel, T.M., Walter, M.V., 2001. Bioaugmentation. In: Hurst, C.J., Crawford, R.L.,
Mohan, S.V., Rao, N.C., Prasad, K.K., Sarma, P.N., 2005. Bioaugmentation of an Knudsen, G.R., McInerney, M.J., Stetzenbach, L.D. (Eds.), Manual of
anaerobic sequencing batch biofilm reactor (AnSBBR) with immobilized Environmental Microbiology. American Society for Microbiology Press,
sulphate reducing bacteria (SRB) for the treatment of sulphate bearing Washington, DC, USA, pp. 952–959.
chemical wastewater. Process Biochem. 40, 2849–2857. Vyrides, I., Santos, H., Mingote, A., Ray, M.J., Stuckey, D.C., 2010. Are compatible
Morris, R.L., Schauer-Gimenez, A.E., Bhattad, U., Kearney, C., Struble, C., Zitomer, solutes compatible with biological treatment of saline wastewater? Batch and
D.H., Maki, J.S., Maki, J.S., 2014. Methyl coenzyme M reductase (mcrA) gene continuous studies using submerged anaerobic membrane bioreactors
abundance correlates with activity measurements of methanogenic H2/CO2 (SAMBRs). Environ. Sci. Technol. 44, 7437–7442.
enriched anaerobic biomass. Microb. Biotechnol. 7, 77–84. Wang, D., Bodovitz, S., 2010. Single cell analysis: the new frontier in ‘omics’. Trends
Muttray, A.F., Yu, Z., Mohn, W.W., 2001. Population dynamics and metabolic activity Biotechnol. 28, 281–290.
of Pseudomonas abietaniphila BKME-9 within pulp mill wastewater microbial Wang, M., Yang, G., Min, H., Lv, Z., Jia, X., 2009. Bioaugmentation with the nicotine-
communities assayed by competitive PCR and RT-PCR. FEMS Microbiol. Ecol. 38, degrading bacterium Pseudomonas sp. HF-1 in a sequencing batch reactor
21–31. treating tobacco wastewater: degradation study and analysis of its
Nebe-von Caron, G., Stephens, P.J., Hewitt, C.J., Powell, J.R., Badley, R.A., 2000. mechanisms. Water Res. 43, 4187–4196.
Analysis of bacterial function by multicolour fluorescence flow cytometry and Watanabe, K., Teramoto, M., Harayama, S., 2002. Stable bioaugmentation of
single cell sorting. J. Microbiol. Methods 42, 97–114. activated sludge with foreign catabolic genes harboured by an indigenous
Ochman, H., Lawrence, J.G., Groisman, E.A., 2000. Lateral gene transfer and the dominant bacterium. Environ. Microbiol. 4, 577–583.
nature of bacterial innovation. Nature 405, 299–304. Wenderoth, D.F., Rosenbrock, P., Abraham, W.-R., Pieper, D.H., Hofle, M.G., 2003.
Ohandja, D.-G., Stuckey, D.C., 2007. Biodegradation of PCE in a hybrid membrane Bacterial community dynamics during biostimulation and bioaugmentation
aerated biofilm reactor. J. Environ. Eng. 133, 20–27. experiments aiming at chlorobenzene degradation in groundwater. Microb.
Park, D., Lee, D.S., Kim, Y.M., Park, J.M., 2008. Bioaugmentation of cyanide-degrading Ecol. 46, 161–176.
microorganisms in a full-scale cokes wastewater treatment facility. Bioresour. Wittebolle, L., Boon, N., Vanparys, B., Heylen, K., De Vos, P., Verstraete, W., 2005.
Technol. 99, 2092–2096. Failure of the ammonia oxidation process in two pharmaceutical wastewater
Patureau, D., Helloin, E., Rustrian, E., Bouchez, T., Delgenes, J.P., Moletta, R., 2001. treatment plants is linked to shifts in the bacterial communities. J. Appl.
Combined phosphate and nitrogen removal in a sequencing batch reactor using Microbiol. 99, 997–1006.
the aerobic denitrifier, Microvirgula aerodenitrificans. Water Res. 35, 189–197. Wittebolle, L., Vervaeren, H., Verstraete, W., Boon, N., 2008. Quantifying community
Plaza, E., Trela, J., Hultman, B., 2001. Impact of seeding with nitrifying bacteria on dynamics of nitrifiers in functionally stable reactors. Appl. Environ. Microbiol.
nitrification process efficiency. Water Sci. Technol. 43, 155–163. 74, 286–293.
Qu, Y.-Y., Zhou, J.-T., Wang, J., Xing, L.-L., Jiang, N., Gou, M., Uddin, M.S., 2009. Yu, Z.T., Mohn, W.W., 2002. Bioaugmentation with the resin-acid degrading bacteria
Population dynamics in bioaugmented membrane bioreactor for treatment of Zoogloea resiniphila DhA-35 to counteract pH stress in an aerated lagoon
bromoamine acid wastewater. Bioresour. Technol. 100, 244–248. treating pulp and paper mill effluent. Water Res. 36, 2793–2801.
Rostron, W.M., Stuckey, D.C., Young, A.A., 2001. Nitrification of high strength Yu, F.-B., Ali, S.W., Guan, L.-B., Li, S.-P., Zhou, S., 2010. Bioaugmentation of a
ammonia wastewater: comparative study of immobilization media. Water Res. sequencing batch reactor with Pseudomonas putida ONBA-17, and its impact on
35, 1169–1178. reactor bacterial communities. J. Hazard. Mater. 176, 20–26.
Saravanane, R., Murthy, D.V.S., Krishnaiah, K., 2001. Bioaugmentation and treatment Zhou, J.Z., Thompson, D.K., 2002. Challenges in applying microarrays to
of cephalexin drug-based pharmaceutical effluent in an up-flow anaerobic environmental studies. Curr. Opin. Biotechnol. 13, 204–207.
fluidized bed system. Bioresour. Technol. 76, 279–281.

Please cite this article in press as: Herrero, M., Stuckey, D.C. Bioaugmentation and its application in wastewater treatment: A review. Chemosphere (2014),
http://dx.doi.org/10.1016/j.chemosphere.2014.10.033