microorganisms

Review
Current Trends in Bioaugmentation Tools for Bioremediation: A
Critical Review of Advances and Knowledge Gaps
Olga Muter

Faculty of Biology, University of Latvia, LV-1004 Riga, Latvia; olga.mutere@lu.lv

Abstract: Bioaugmentation is widely used in soil bioremediation, wastewater treatment, and air
biofiltration. The addition of microbial biomass to contaminated areas can considerably improve
their biodegradation performance. Nevertheless, analyses of large data sets on the topic available
in literature do not provide a comprehensive view of the mechanisms responsible for inoculum-
assisted stimulation. On the one hand, there is no universal mechanism of bioaugmentation for
a broad spectrum of environmental conditions, contaminants, and technology operation concepts.
On the other hand, further analyses of bioaugmentation outcomes under laboratory conditions and
in the field will strengthen the theoretical basis for a better prediction of bioremediation processes
under certain conditions. This review focuses on the following aspects: (i) choosing the source of
microorganisms and the isolation procedure; (ii) preparation of the inoculum, e.g., cultivation of
single strains or consortia, adaptation; (iii) application of immobilised cells; (iv) application schemes
for soil, water bodies, bioreactors, and hydroponics; and (v) microbial succession and biodiversity.
Reviews of recent scientific papers dating mostly from 2022–2023, as well as our own long-term
studies, are provided here.

Keywords: bioaugmentation; contamination; microbial succession

1. Introduction
Citation: Muter, O. Current Trends in
Bioaugmentation Tools for
Bioremediation encompasses a broad range of environmental biotechnologies, which
Bioremediation: A Critical Review of
require multidisciplinary approaches through implementation of innovative tools to the
Advances and Knowledge Gaps. natural biological processes occurring in soil, water, and air. The addition of microbial
Microorganisms 2023, 11, 710. biomass (bacteria, fungi, and their secreted enzymes) to contaminated areas, i.e., the process
https://doi.org/10.3390/ of bioaugmentation, can be adapted to the green environment and can notably improve an
microorganisms11030710 area’s pollutant removal efficiency (RE), as well as reduce their removal time and costs [1].
However, bioaugmentation under controlled conditions in the field remains challenging, due
Academic Editors: Guangli Cao and
to the biodiversity of a whole system, competition between microbial agents and indigenous
Yong Sun
microorganisms, substrate competition, climatic conditions, remediation cycles, and other
Received: 15 February 2023 factors. To select an optimal bioaugmentation strategy, further studies on the interactions
Revised: 6 March 2023 of different functional bacteria related to their resistance to multiple stress factors, enzyme
Accepted: 8 March 2023 activity and system robustness are needed [2]. Rigorous research and critical analyses of
Published: 9 March 2023 available databases, as well as the incorporation of genetic engineering, nanotechnology, and
systems biology can bring bioremediation to a more advanced level [3].
Bioaugmentation is a site-specific approach. Thus, recent research publications and
reviews on bioaugmentation have focused on the following aspects: thermophilic reduc-
Copyright: © 2023 by the author.
Licensee MDPI, Basel, Switzerland.
tive dechlorination [4,5]; the psychrophilic treatment of groundwaters [6]; microbially
This article is an open access article
induced calcium carbonate precipitation techniques to mitigate the wind-induced erosion
distributed under the terms and of calcareous desert sand [7]; hydrocarbon biodegradation in freshwater sediments from
conditions of the Creative Commons historically contaminated lakes [8]; the bacterial remediation of pesticide-polluted soils [3],
Attribution (CC BY) license (https:// emerging trends in the remediation of organic contaminated soils as a whole [9]; mecha-
creativecommons.org/licenses/by/ nisms of microbial activity in heavy metal removal [10]; comparisons of autochthonous
4.0/). and allochthonous bioaugmentation [11]; the stimulation of plant growth in bioaugmented

Microorganisms 2023, 11, 710. https://doi.org/10.3390/microorganisms11030710 https://www.mdpi.com/journal/microorganisms

Microorganisms 2023, 11, 710 2 of 13

hydroponic systems [12]; and the bioaugmentation of wastewaters (WWs) with yeast in the
presence of antimicrobials [13], among others.
The aim of this review was to gather different aspects related to bioaugmentation ap-
proaches. Bioaugmentation has received increasing interest from the scientific community
over the last five years. Indeed, a total of 160 articles and 12 reviews on this topic were
published in 2017, while in 2022 these numbers had increased to 1447 and 148, respectively
(database SCOPUS, keyword “bioaugmentation”). In this respect, to avoid possible repeti-
tions, this review focused mostly on the scientific articles and review papers published in
2022–2023, as well as the authors’ own research results.

2. Sources of Microorganisms for Bioaugmentation

There are abundant data available on the variations and successions of microbial
communities upon bioaugmentation-assisted remediation where the introduced micro-
bial “agent” disappears during the remediation process. However, positive effects on
biodegradation dynamics have been detected.
Our recent studies have demonstrated a dominant abundance of the Proteobacteria
and Firmicutes phyla in various biodegradation processes assisted by autochthonous and
allochthonous bioaugmentation.
In a study of the bioremediation of hydrocarbon-contaminated lake sediments, a
32-day batch incubation in (i) biostimulated (N), (ii) biostimulated and bioaugmented
(NB), and (iii) unamended (K) sets resulted in an increasing abundance of Proteobacteria
from 48.8% in the raw sediments to 59.3%, 58.0%, and 61.7%, respectively, mainly due
to an increase in Betaproteobacteria. The relative abundance of the Pseudomonas genus
(Gammaproteobacteria) was the highest in the (N) set at 35.8%, compared to 17.5% and
21.0% for the (K) and (NB) sets, respectively. The genera that dominated in the inoculum
(allochthonous) were identified as Citrobacter (29.5%), Klebsiella (24.5%), Pseudomonas
(15.5%), and Aeromonas (11.3%). The concentration of hydrocarbons in the lake sediments
during the 32-day incubation decreased on average from 465 mg/kg to 165 mg/kg and
117.5 mg/kg in the (N) and (NB) sets, respectively [8].
Another study focused on the effect of two allochthonous bacterial consortia on the
growth of Mentha aquatica in a hydroponic system. After the 47-day hydroponic greenhouse
experiment, the structure of the bacterial communities attached to expanded clay pellets
was represented mostly by Proteobacteria at the phylum level (80–90%). Bioaugmentation
provided significant (p < 0.05) stimulation for the growth of M. aquatica, although the
metagenome analysis of the rhizosphere did not reveal any abundance of bacterial strains,
which were introduced into the hydroponic media at the beginning of the experiment [12].
The bioaugmentation of municipal sewage sludge/straw substrate with an autochthonous
bacterial consortium resulted in a marked shift in the microbial community structure. Particu-
larly, the raw sewage sludge contained Firmicutes/Proteobacteria/Actinobacteria at levels of
4.4%/60.2%/26.5%, respectively, while the inoculum for bioaugmentation, which was pre-
pared using a selective broth, contained these phyla in proportions of 92.13%/1.7%/5.1%, re-
spectively. After 16 days of batch incubation, the proportions varied as follows: (32.7–53.8%)/
(30.6–54.3%)/(5.3–13.7%). All treated samples were characterised by an increased abundance
of Firmicutes. Yet, an increased abundance of ungrouped reads of Pseudomonas putida was
detected in all bioaugmented sets; however, it was not detected in the inoculum. Compared
with non-bioaugmented sets, the combination of a wheat straw amendment to a sewage
sludge with bioaugmentation showed the highest and most stable microbial respiration in-
tensity, the lowest ammonia emissions, and the highest stimulation effect on cress seedling
growth [14]. More recent studies have also shown a positive effect of bioaugmentation on
manure composting, which influenced the bacterial response, matter transformation, and
metal immobilisation [15].
The effect of the bioaugmentation of activated sludge with viable brewing spent yeast
biomass on microbial community structure was studied in the presence of benzalkonium
chloride. The added yeast biomass remained viable during 10-day treatment and reduced
Microorganisms 2023, 11, 710 3 of 13

an inhibitory effect of BAC on Bacilli in activated sludge. Yet, the bioaugmentation stim-
ulated bacterial growth and microbial respiration. At the phylum level, two dominant
taxa, i.e., Firmicutes and Proteobacteria, were found in the activated sludge, with their
abundance in the control (non-incubated) and all incubated samples ranging between
27–35% and 22–36%, respectively [13].
Recently, Ref. [16] reported that Proteobacteria, Firmicutes, Bacteroidetes, Actinobacte-
ria, and Acidobacteria were the dominant phyla during the biodegradation of crude oil.
Some key enzymes related to the biodegradation of petroleum products have been de-
tected in Bacillus megaterium (alkane hydroxylase, catechol 1,2-dioxygenase, protocatechol
3,4-dioxygenase), Bacillus pumilus (esterases and lipase), Pseudomonas aeruginosa (catechol
1,2-dioxygenase, protocatechol 3,4-dioxygenase), and Stenotrophomonas maltophilia (catechol
2,3-dioxygenase) [17]. The use of manganese-oxidising Pseudomonas sp. QJX-1 with humic
acids as the sole carbon source has been proposed for the removal of pharmaceuticals
(caffeine) from drinking water via sand filtration [18]. Furthermore, Rhodococcus spp. are
known to play an important role in the biodegradation of organic contaminants, as well
as in the recovery of the nitrification performance in the presence of antibacterial agents
in activated sludge and other processes [19–21]. The catabolic activity of rhodococci in-
volves catabolizing short- and long-chain alkanes, as well as aromatic (halogenated and
nitro-substituted), heterocyclic, and polycyclic aromatic compounds. The high adaptability
of rhodococci with respect to substrates has previously been reviewed by [22], with an
emphasis on hyperrecombination evolutionary strategies. Linear plasmids in the large
Rhodococcus genomes store multiple copies of many biodegradative genes [22]. Bacteria
of the genera Pseudomonas, Bacillus, and Rhodococcus can be found in a broad range of
ecosystems exhibiting extraordinary activities in the breakdown of natural pollutants and
xenobiotics and taking part in microbial consortia and/or endophytic cooperation.
Although the overall microbial community structure in organics-polluted sites commonly
depends on the geographic location [23], some bacterial genera are often predominant.
On the one hand, Bacillus spp., Pseudomonas spp., and Rhodococcus spp. appear to
be dominant because of microbial succession upon biodegradation. On the other hand,
researchers frequently use these bacteria as an inoculum for bioaugmentation [1,24].
Searching for “Pseudomonas bioaugmentation”, “Bacillus bioaugmentation”, and “Rhodococ-
cus bioaugmentation” in the SCOPUS database for studies published in 2022 revealed 52, 37,
and 23 sources, respectively. Some recent studies are summarised in Table 1.

Table 1. Studies on biodegradation with the application of Bacillus spp., Pseudomonas spp., and
Rhodococcus spp. as bioaugmentation agents.

Culture for Bioaugmentation

Type of Removal Efficiency (RE), Environment, Co-Cultures
Species/ Reference
Genus Contaminant
Strain
B. megaterium and RE in solution: 99% of CLP in a solution with an initial concentration of 10 mg/L after 60
Chlorpyrifos (CLP) [25]
B. safensis days. RE in soil: 61–65% after 100 days.
RE: 1.73 mg/kg soil day for 56 days.
B. firmus Phenanthrene [26]
Soil, anaerobic nitrate-reducing environment.
B. subtilis,
B. licheniformis, RE: 92% of nitrate at the laboratory and 62% outdoors. A plug flow system; Arthrobacter
Nitrogen in WW [27]
B. megaterium, sp., Acinetobacter parafneus, Corynebacterium sp., and Streptomyces globisporus
B. cereus
Bacillus

RE: 62.76% after 29 d. Secondary salinized soil, pot experiment. Improved NO3 -
B. megaterium Nitrogen in WW [28]
removal rate.
RE: up to 10.51% after 21 days. Initial conc. 200 mg/kg soil. Staphylococcus sp. and
Bacillus sp. Decachlorobiphenyl [29]
Acinetobacter sp. Consortia better than individual strains.
Bacillus spp. and B. Algal–bacterial bioflocs and microbe–rice bran complexes, Scenedesmus dimorphus and
Aquaculture WW [30]
aryabhattai Chlorella sp. 1:1
Sulfamethoxazole
B. paramycoides Biochar-immobilized. After five rounds of reuse, RE: 43.24% for SMX and 50.34% for Zn2+ [31]
(SMX) and Zn2+
Indoleacetic acid (IAA)-production, assimilation of soluble salt, condensation and
Secondary
B. safensis aromatization of humus, accumulation of dissolved organic nitrogen and carbon. [32]
composting
Corynebacterium stationis subsp. safensis.
Microorganisms 2023, 11, 710 4 of 13

Table 1. Cont.

Culture for Bioaugmentation

Type of Removal Efficiency (RE), Environment, Co-Cultures
Species/ Reference
Genus Contaminant
Strain
RE: two-fold increase of elemental sulfur generation. Establishment of a stronger biofilm
Pseudomonas sp. Sulphide [33]
structure. Granular sludge bed reactor. Arcobacter, Azoarcus
P. mendocina, Nitrogen removal in RE: 92.4% of NH4 +-N; 79.8% of total nitrogen. Heterotrophic nitrification, aerobic
[34]
P. putida petroleum WW denitrification. Brucella sp., Paracoccus sp.
P. plecoglossicida Crude oil 76.7% ability to degrade crude oil in a liquid broth. 14 days. [11]
RE: 53% and 79% for synthetic and real WW with 5 mg/L glyphosate. Continuous
P. stutzeri Glyphosate photobioreactors. Acclimation to glyphosate from 5 to 50 mg L-1. Comamonas [35]
Pseudomonas

odontotermitis; Sinomonas atrocyanea, Chlorella protothecoides.

Benzene, toluene,
RE: 20 mg/L in a mineral salt medium for 6 h.
Pseudomonas sp. ethylbenzene, and [36]
Variovorax paradoxus
p-xylene (BTEX)
RE: 73.6%, 69.3%, 50% and 50% in soils polluted with 1%, 10%, 20% and 30% oil,
Oil-contaminated
P. songnenensis respectively, after 6 months. [37]
desert soil
Actinotalea ferrariae, Arthrobacter ginsengisoli, Dietzia cinnamea, Dietzia papillomatosis
RE: 77.54%, 99.39%, and 67.25% for F- , NO3 - , and Ca2+ , respectively, for 8 h.
Pseudomonas sp. Fluoride Microbially induced calcium precipitation (MICP). Self-assembled fungus-flexible fibre [38]
composite microspheres. Phoma sp.
RE: 97% during 22–30 cycles. 25 mg/L PCMX within 180 min in activated sludge system.
Chloroxylenol
R. ruber Aerobic 100 mL batch, 4% inoculum (v/v). R. ruber became dominant within 30 cycles. [19]
(PCMX)
Detoxify nitrification system.
Quinoline and its
RE: for quinoline, 100% for 1.5 h. For 2-HQ, max. appearance of 0.025 mM within 0.5 h,
toxic intermediate
R. ruber 100% degradation after 1 h. Aerobic 100 mL batch, 15% inoculum (v/v), initial [20]
2-hydroxyl
concentration of quinoline of 0.25 mM. Accelerated nitrification and enriched Nitrospira.
quinoline (2-HQ)
R. erythropolis
Rhodococcus

RE: total organic carbon 63–89%; NH4–N 72–82%; total nitrogen 63–87%; chemical oxygen
(Genetically Nanofiltration
demand 81–95%, dependent on treatment mode. Integrated system of advanced oxidation
engineered concentrate of [21]
processes cooperated with rRho-NM. Bioaugmentation to the aerobic fluidized reactor (2
expressing Nirs and landfill leachate
L), inoculum 106 /mL.
AMO (rRho-NM).)
Di-(2-
RE: 89.94% of DEHP within 84 h. Initial DEHP conc. 5 mg/L DEHP in 10 mL municipal
R. pyridinivorans ethylhexyl)phthalate [39]
WW (batch). Aerobic denitrifying phosphate-accumulating bacterial strain RL-GZ01.
(DEHP)
Biphenyl and
RE: biphenyl 96% within 5 days, PCB31 92% for 3 days. Initial conc. biphenyl 500 mg/L,
R. biphenylivorans polychlorinated [40]
aerobic 5 mL batch cultivation, pH 7.0.
biphenyl (PCB) 31
3-Methylindole RE: >99% for 24 h. Initial skatole conc. 60 mg/L. Degradation performance in consortium.
Rhodococcus sp. [41]
(skatole) Inoculum 2%.

3. Obtaining Microorganism Degraders: Sources and Methods

Functional consortia of microorganisms with a high degradation activity can be iso-
lated from contaminated sites, agricultural waste, activated sludge, and other sources
which are characterised by a relatively high microbial biodiversity. Specific conditions
chosen for the isolation and further cultivation of these microorganisms include a selective
pressure, which allows the formation of a distinct subpopulation of bacteria. Alternatively,
intact nutrient- and microorganism-rich substrates such as compost and activated sludge
can be applied to bioaugmentation (without isolation procedures). Furthermore, genetically
modified organisms can also be used. Some of following bioaugmentation sources were
reported in 2022:
-Vermicompost allowed the enrichment of resilient and degrading microorganisms
which could be extracted through an aerated aqueous extraction process. The authors
suggested a simple and fast isolation of microbial consortia by aerated aqueous extrac-
tion [42]. The application of vermicompost on the moderately weathered soil polluted with
heavy linear alkanes resulted in five-fold and two-fold increases in the amounts of available
phosphorus (P) and exchangeable potassium (K), respectively. Hydrocarbon degradation
was increased by up to 34.4% relative to the control [43].
-Activated sludge. The effect of the introduction of exogenous activated sludge on
the activity and composition of the microbial consortium carrying out the nitritation–
anammox process in a sequencing batch bioreactor was investigated by [44]. The addition
Microorganisms 2023, 11, 710 5 of 13

of exogenous activated sludge after the stable nitrogen removal mode was reached (day 53)
increased the efficiency of nitrogen removal by 21–35%, and this difference was maintained
until the end of the experiment (90 days) [44].
-Plant growth-promoting rhizobacteria (PGPR) as a bioaugmentation tool for treating
soils contaminated by hydrocarbons [45], polyesters [46], and heavy metals [47]. PGPR pro-
duce multiple types of biosurfactants and diverse oxygenases in variable bacterial species,
e.g., Pseudomonas, Acinetobacter, Mycobacterium, Haemophilus, Rhodococcus, Paenibacillus, and
Ralstonia [45,48]. A recent study of the effect of the rhizobacteria Bacillus subtilis on cad-
mium bioavailability and distribution in soil planted with ryegrass (Lolium multiflorum L.)
demonstrated a reduction in Cd bioavailability by 39.1%, followed by alterations in the
microbial community structure, e.g., enrichment of Proteobacteria [47]. The effect of rice
assisted with a PGPR consortium (three isolates of Bacillus sp., Agrobacterium sp.) on the re-
mediation of a multi-compound (i.e., di (2-ethylhexyl) phthalate, Cd, and Zn)-contaminated
site was recently studied by [46]. The treatment resulted in the removal of di (2-ethylhexyl)
phthalate, Cd, and Zn by 86,1%, 76.0%, and 92.2%, respectively, within 30 days [46].
-Genetic bioaugmentation. Genetically modified organisms have also been shown to
improve stability and resistance to environmental stressors, and their prolonged viability
results in greater effectiveness [49–51]. Bioaugmentation with Pseudomonas putida KT2440,
harbouring the transferrable triclocarban-catabolic plasmid pDCA-1-gfp-tccA2, rapidly
converted 50 µM triclocarban in WW into 3,4-dichloroaniline and 4-chloroaniline, which
were further mineralised more easily [52]. In genetic bioaugmentation for pollutant removal,
a donor bacterium harbouring a catabolic plasmid will transfer the plasmid to a recipient
cell (transconjugant) and both the donor and transconjugant can express degradation
genes for the removal of the contaminant. Varner et al. [53] explored the effect of the
ecological growth strategies of plasmid donors and recipients on the conjugation and
naphthalene degradation of two PAH-degrading plasmids, pNL1 and NAH7 [53]. Bokade
et al. [3] reviewed the suggested mobile genetic elements mediating the horizontal transfer
of pesticide degradation genes, i.e., plasmids, transposons, genomic islands, transcription
sequences, and integron gene cassettes.
Contaminated sites can act as a repository of highly adapted diverse populations
which must be harnessed to score different degraders. Bokade et al. [3] reviewed different
enrichment approaches employed in the isolation of microorganisms from diverse environ-
ments, i.e., magnetic separation, differential centrifugation, micromanipulation, dilution
to extinction, concentration to extinction, toxicity to extinction, heat pretreatment, and
dilution-to-stimulation/extinction. Enrichment techniques offer an effective strategy for
selectively isolating the microorganisms of interest. In this technique, specific environ-
mental conditions are simulated to increase the abundance of organisms to a detectable
level. Usually, this method involves the use of specific growth media and conditions that
favour the growth of a specific microorganism over others. Several techniques including
micromanipulation, magnetic separation, differential centrifugation, dilution-to-extinction,
concentration-to-extinction, and toxicity-to-extinction have been applied for the enrichment
and isolation of specific degrader microorganisms, depending on the intended purpose [3].
Functional consortia can be developed by collecting different isolates with certain
target properties. More specifically, [54] developed the following microbial consortium
for composting: protein-degrading bacteria (Brevibacillus brevis), starch-degrading bac-
teria (Acinetobacter johnonii), ammonia-oxidising bacteria (Ureibacillus terrenus), oil de-
graders (Aneurinibacillus thermoaerophilus, Bacillus hisahii, Candida tropicalis), and mixed
lignocellulose-degrading microorganisms [54]. In a study using an aquaculture WW treat-
ment, an inoculum consisting of algae and bacteria was tested. This consortium provided
the most compact biofloc structure (0.59 g/L), high settleability (71.91%), and a large
particle diameter (4.25 mm) [30]. The mixed salt-tolerant bacteria system composed of
ammonia-, nitrite-, and nitrate–nitrogen-utilising bacteria was artificially constructed for
the salt-tolerant aerobic granular sludge. The flocculent consortium was aggregated by
Aspergillus tubingensis mycelium pellet regions [2].
Microorganisms 2023, 11, 710 6 of 13

The effects of bioaugmentation by single cultures and consortia on the biodegradation

of highly chlorinated compounds, i.e., decachlorobiphenyl (PCB-209), were compared
by [29]. The application of a consortium resulted in the highest PCB-209 removal poten-
tial [29]. Another study showed that consortia and individual strains had similar crude
oil-degrading capacities [11]. Moreover, [37] reported that the oil biodegradation effi-
ciency of single strains and consortia (Actinotalea ferrariae, Arthrobacter ginsengisoli, Dietzia
cinnamea, Dietzia papillomatosis, and Pseudomonas songnenensis) was similar, and that the
REs of bioaugmented and non-bioaugmented sets in an oil-saturated desert soil after a
6-month experiment were also similar, exhibiting similar predominant bacterial species,
e.g., Ar. ginsengisoli [37].
Regarding synergistic, syntrophic, antagonistic, neutral, or other types of microbial
interrelations, Foster and Bell suggested that adaptation to other microbial species typically
results in competitive rather than cooperative phenotypes. The positive effects in one
direction, where one species gains at the expense of another, i.e., through predator–prey-
like interactions, were not excluded [55].

4. Preparation of the Inoculum

The adaptation mechanisms of microbial consortia require a better understanding to
optimise bioremediation conditions and predict/control the outcome of bioaugmentation.
Studies of complex adaptive systems have previously revealed characteristic behaviours
such as resilience and regime shifts. Hosoda et al. [56] proposed a population–reaction
model based on a combination of microbial experimental ecosystems and a hierarchical
dynamic model.
In our earlier study, it was hypothesised that eight bacterial cultures belonging to
the Pseudomonas spp. and Stenotrophomonas maltophilia groups, originating from distinct
soil samples of the same oil-contaminated site and representing a close phylogenetic
relationship, could considerably increase their activity by serial batch cultivations (seven
days each) with a stepwise increase (1%, 3%, 5% w/w) in diesel oil concentration [57]. The
inoculum was prepared by mixing aliquots of the eight individual strains, which were
pre-grown in a Bushnell-Haas (BH) broth supplemented with 0.5% molasses for 24 h and
justified by optical density, thus achieving an equal initial cell concentration of each isolate
at the beginning of the cultivation. The overall count of colony-forming units (CFUs) and
enzyme activity in cultures was higher in the presence of 0.5% molasses as compared to
that with 0.05% molasses, irrespective of the diesel oil concentration and the step of the
serial batches. In the sets with 0.05% molasses, the highest cell biomass was detected after
step 2, i.e., in the presence of 3% diesel oil. Visual changes in culture turbidity and colour
indicated the formation of biosurfactants after just 48 h of incubation. The negative effect
of the serial batches with increasing diesel oil concentrations from 1% to 5% was detected
for fluorescein diacetate hydrolysis activity at 0.05% molasses, and the same was detected
for dehydrogenase activity at both molasses concentrations tested. Conversely, urease
activity was shown to increase gradually with increasing diesel oil concentrations in the
broth amended with 0.5% molasses in three serial batches [57].
The adaptation of inoculum has recently been described by [58] for nitrifiers at en-
hanced concentrations of ammonium (1.6–150 mgN/L/d) and NaCl (2–30 g/L). Ammonium-
oxidising bacteria prepared with 15 and 30 g/L salinity demonstrated a higher resistance
to salinity changes than ammonium-oxidising archaea and comammox. The authors rec-
ommend separately preparing cultures for freshwater (low salinity) and brackish marine
uses [58].
In another study, the application of aqueous aerated extracts from a biomixture ac-
climated with ibuprofen, diclofenac and triclosan was examined. The dissipation of 90%
of the diclofenac and triclosan required 60 and 108 days less, respectively, than did the
controls [42].
The amount of inoculum is another important factor which can influence biodegrada-
tion performance. According to the data reviewed by [59], the inocula concentrations can
Microorganisms 2023, 11, 710 7 of 13

range from 107 –109 CFU/mL for the enhanced bioremediation of diesel–biodiesel-polluted
soils. Other studies have reported that an increased microbial activity did not always result
in effective degradation [59].

5. Application of Immobilised Cells

Planktonic bacteria are sensitive to environmental variations, thereby limiting their
application. Bioaugmentation in both drinking water treatment reservoirs and biological
filters can be challenged by the washout/loss of bioaugmented bacteria. Numerous new
immobilization methods have been developed to solve problems associated with the ap-
plication of enzymes, such as directional immobilisation, co-immobilisation of multiple
enzymes, and new immobilisation carriers [9]. Among promising carriers for biofilm devel-
opment, biochar is an excellent carrier with potential for decontamination and bacterial
adsorption via rich functional groups, a large specific surface area, high porosity, and
excellent biocompatibility. Microbes can be immobilised on biochar via different fixation
methods such as adsorption, entrapment, cross-linking, and covalent bonding or a combi-
nation of two methods [31,60]. Saeed et al. [45] recently reviewed advances in biochar and
plant growth-promoting rhizobacteria engineering systems for hydrocarbon degradation
and showed that the synergistic effect of biochar and PGPR depends on the concentration
of biochar and PGPR used [45]. Recent studies which have applied immobilised cells for
biodegradation processes are summarised in Table 2.
The use of quorum sensing (QS) in a biofilm reactor has recently gained significant
research interest due to its vital role in biofilm formation, especially acyl-homoserine
lactone (AHL) [61]. However, it is not economical or practical to regulate QS in a biofilm
reactor through dosing pure AHL. Dosing AHL-producing bacteria might instead be more
reasonable [61]. Therefore, in the context of QS, bioaugmentation approaches are also
very important.

Table 2. Engineered materials for improvement in biofilm performance and in the shelf-life of
desiccated microbial strains.

Carrier, Technique Microorganism Effect Reference

Protector: albumin-trehalose. Stabilized desiccated
Shell encapsulation Bradyrhizobium cells for 4 months at high relative humidity, [62]
increased the glass transition temperature
Vertical flow mesocosm-constructed wetland.
Bark biochar Activated sludge [63]
Removal (>40%) of irbesartan and carbamazepine
10% adapted thermophilic
Anaerobic digestion. Methane yield increased up
Biochar Methanosarcina thermophila on [64]
to 35%
2 g/L biochar
Perlite 1010 CFU/g Anaerobic digestion. Biodegradation of olive cake [65]
1010 cells/g of biocomposite
Soil microcosm. 80% herbicide MCPA
Fe-modified zeolite grains (selected microbial [66]
(2-methyl-4-chlorophenoxyacetic acid)
consortium)
Moving bed biofilm reactor (MBBR) at 5 ◦ C.
Higher chemical oxygen demand and NH4 +-N
removal rate (93% and 75%). Co-culture with
High-density polyethylene Sphingomonas rubra BH3T as a Nitrospira. The increased biofilm thickness
[61]
(HDPE) carrier quorum-sensing bacteria (60.23%) during the whole operating time,
accompanied by more potent adhesion force
(61.59%), was related to increased polysaccharides
and proteins in the biofilm
Gel-immobilization enhanced Ammonia-tolerant Continuous biogas reactor. Long-term
[67]
with biochar methanogens ammonia resistance
Microorganisms 2023, 11, 710 8 of 13

Table 2. Cont.

Carrier, Technique Microorganism Effect Reference

Aerobic granules with Aerobic granular sludge from Continuous flow granular reactors. Removed up
[68].
microalgae aquaculture WW to 77% and 80% of ammonium and nitrite
Decrease in removal of cyanotoxins like
Injection of
Algal organic matter (AOM) microcystin-LR (MC-LR). Decreases in MC-LR
contaminant-degrading [69]
and humic substances (HS) biodegradation rate of 1.6- and 3.4-fold in the
microorganisms
presence of AOM and HS, respectively

6. Bioaugmentation Strategy
It is important to note that bioaugmentation schemes, which are described in different
studies, can be reproducible but are sometimes not comparable because of differences in
experimental setup, feedstock/soil/water composition, measurement units, the physiologi-
cal state of the inoculum, etc. Nevertheless, rigorous analyses of various bioaugmentation
experiments should be useful for choosing the most suitable treatment scheme.
The dosing ratio and dosing time play a vital role in the coordination and adaptive
potential of the functional flora in the bioaugmentation system. The dosing strategies for
the bioaugmentation of seafood processing WW in a sequencing batch bioreactor using
artificially constructed mixed bacteria systems have been described by [2]. The reactor was
operated with an 8 h cycle including 5 min of settling, 5 min of decanting (a volumetric
exchange ratio of 50%), 5 min of filling, 105 min of anaerobic reaction, and 360 min of aerobic
reaction. The use of a bacterial agent as a dosing compound in the batches (supplementing
2.5% on day 1 and day 10, respectively) dramatically increased the removal of NH4+-N
and total nitrogen of seafood processing WW in winter from 66 89% and 52.77% to 79 0.02%
and 69.97%, respectively [2].
The performance of anaerobic digestion also depends on the bioaugmentation dosage.
Thus, the optimal dosage was determined to be 0.27 g VSbioaugmentation seed /g VSchicken manure ,
which could be adopted for rapid start-up or improving a continuous digester for treating
chicken manure. Higher bioaugmentation doses (0.34 g VSBS /g VSCM ) did not exhibit a
significantly improved bioaugmentation efficiency [70].
The effects of single and routine bioaugmentation with Methanosarcina thermophila
combined with the addition of biochar in the anaerobic digestion of food waste were
compared by [71]. Specifically, 10% v/v of the microbes grown on biochar (1 g/L) were
added during the setup of the reactors, which is in contrast to a routine bioaugmentation
wherein the same amount of supplements were added over ten feeding cycles. The best
routine reactor showed 37% more yield, while the best single reactor presented 32% [71].
The effect of bioaugmentation with exogenous activated sludge on the nitritation–
anammox process in a sequencing batch reactor was studied by [44]. Two bioaugmentation
strategies were tested: the exogenous sludge was added either immediately after the
inoculation with the anammox activated sludge or when a stable mode of nitrogen removal
was achieved. The authors reported a positive effect of bioaugmentation when carried out
either at the launching of a bioreactor (a 15% increase in nitrogen RE) or after its long-term
operation (a 21–35% increase in nitrogen RE); it had a short-term effect and should be used
carefully [44].
Regarding the activation of the biodegradation process by nutrients/stimulants, [9]
recently reviewed the emerging trends for the enhancement of co-metabolism for pollu-
tant degradation efficiency [9]. In the case of Fenton oxidation and bioremediation of oil
lubricant-contaminated soils, the addition of citrate or citric acid led to the further oxidation
of the oil pollutant by forming chelation and preventing ferric oxide precipitate production.
On the other hand, ammonium chloride and monosodium glutamate served as biostim-
ulants that fostered the growth of indigenous petroleum or hydrocarbon degraders [72].
The addition of methylated β-cyclodextrin for bioavailability enhancement was previously
proposed by [25] in a study of the degradation of chlorpyrifos from soil. The biostimulation
Microorganisms 2023, 11, 710 9 of 13

effect of neat biodiesel was recently reviewed by [59]. It was also shown that rhamnolipids
enhance pyrene bioaugmentation as a carbon source and as a biosurfactant, stimulating
more active pyrene degraders and reconstructing microbial communities [73].
Biostimulation can result in an even higher RE than bioaugmentation. Thus, over a
90-day experiment (7.5 L bioreactors), the biodegradation of 35 mg/kg benzo(a)pyrene
(BaP) and 28 mg/kg dichlorodiphenyltrichloroethane (DDT) was more efficient in the set
employing biostimulation only, as compared to the one using bioaugmentation. However,
bioaugmentation resulted in a toxicity drop of 90%, while this value was only 48% for the
biostimulation set [74].
The addition of natural sorbents, e.g., minerals (zeolite, kaolinite, vermiculite, di-
atomite), organics (peat), carbonaceous (biochar) materials, and mixed sorbent (consisting
of granular activated carbon and diatomite), to soils contaminated with crude oil resulted
in a reduction in soil toxicity, decrease in soil hydrophobicity, optimization of soil pH and
of the water–air regime, thus considerably stimulating the oil degradation [75].
Gibert et al. [76] studied the efficiency of nano zero-valent injection pulses for the
removal of nitrate and pesticides (dieldrin and lindane) in continuous-flow packed columns
promoted by the addition of acetate and/or an inoculum rich in denitrifiers. The combina-
tion of heterotrophic denitrifiers and abiotic chemical nitrate reduction promoted by the
Microorganisms 2023, 11, x FOR PEER REVIEW 11 of 15
pulse injection of zerovalent iron nanoparticles (nZVI) resulted in up to 99% removal of
NO3 - . The removal of the target pesticides occurred not due to biodegradation, but via
adsorption onto the soil or chemical degradation by nZVI [76].
The biomonitoring
biomonitoring parameters
parameters used
used for for assessing
assessing the performance
the performance of a
of a bioremediation
bioremediation process are summarised
process are summarised in Figure 1. in Figure 1.

Figure 1. Biomonitoring
1. Biomonitoring parameters
parameters for assessing
for assessing the performance
the performance of a bioremediation
of a bioremediation process
[16,54,74,77,77,78].
process [16,54,74,77,77,78].

7. Conclusions
7. Conclusions
In order to improve bioaugmentation performance, researchers mainly need to solve
In order to improve bioaugmentation performance, researchers mainly need to solve
the problems of long-term residence of special functional bacteria and maintenance of
the problems of long-term residence of special functional bacteria and maintenance of
multi-bacteria interactions [2]. If the total breakdown of the remaining contaminants is
multi-bacteria interactions [2]. If the total breakdown of the remaining contaminants is
not achievable, immobilising and reducing the bioavailability of organic pollutants in soils
not achievable, immobilising and reducing the bioavailability of organic pollutants in soils
is critical. Monitoring microbial activity on a regular basis by using methodologies for
is critical. Monitoring microbial activity on a regular basis by using methodologies for
controlling problematic volatile organic compounds, ecotoxicity, pollutant leaching, etc.,
is necessary [79]. Multi-compound pollution, new findings in inoculum conservation for
commercial uses, and the further development of process monitoring will be the main
topics for further research in the field of bioaugmentation-assisted bioremediation.
Microorganisms 2023, 11, 710 10 of 13

controlling problematic volatile organic compounds, ecotoxicity, pollutant leaching, etc.,

is necessary [79]. Multi-compound pollution, new findings in inoculum conservation for
commercial uses, and the further development of process monitoring will be the main
topics for further research in the field of bioaugmentation-assisted bioremediation.

Funding: This research was funded by the “State research project in the field of biomedicine, medical
technologies and pharmacy” VPP-EM-BIOMEDICĪNA-2022/1-001 (Y3-VPP32f-ZR-N-090).
Data Availability Statement: Not applicable.
Conflicts of Interest: The author declares no conflict of interest.

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