processes

Review
Synthetic Biology: A New Era in Hydrocarbon Bioremediation
Valentina Jiménez-Díaz 1 , Aura Marina Pedroza-Rodríguez 2 , Oswaldo Ramos-Monroy 3
and Laura C. Castillo-Carvajal 1, *

1 Centro de Investigación en Ciencias de la Salud, Facultad de Ciencias de la Salud, Universidad Anáhuac

Mexico Campus Norte, Huixquilucan 52786, Mexico; [Link]@[Link]
2 Laboratorio de Microbiología Ambiental y de Suelos, Unidad de Investigaciones Agropecuarias (UNIDIA),
Departamento de Microbiología, Facultad de Ciencias, Pontificia Universidad Javeriana,
Bogota 11023, Colombia; apedroza@[Link]
3 Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, Prolongación de Carpio y Plan de
Ayala S/N, Colonia Santo Tomás, Ciudad de Mexico 11340, Mexico; ibq_os@[Link]
* Correspondence: [Link]@[Link]

Abstract: Crude oil is a viscous dark liquid resource composed by a mix of hydrocarbons which, after
refining, is used for the elaboration of distinct products. A major concern is that many petroleum
components are highly toxic due to their teratogenic, hemotoxic, and carcinogenic effects, becoming
an environmental concern on a global scale, which must be solved through innovative, efficient, and
sustainable techniques. One of the most widely used procedures to totally degrade contaminants are
biological methods such as bioremediation. Synthetic biology is a scientific field based on biology
and engineering principles, with the purpose of redesigning and restructuring microorganisms to
optimize or create new biological systems with enhanced features. The use of this discipline offers
improvement of bioremediation processes. This article will review some of the techniques that use
synthetic biology as a platform to be used in the area of hydrocarbon bioremediation.






Keywords: synthetic biology; bioremediation; hydrocarbons; biosensors; consortium; genetically
Citation: Jiménez-Díaz, V.;
engineered microorganisms
Pedroza-Rodríguez, A.M.;
Ramos-Monroy, O.; Castillo-Carvajal,
L.C. Synthetic Biology: A New Era in
Hydrocarbon Bioremediation.
1. Introduction
Processes 2022, 10, 712. https://
[Link]/10.3390/pr10040712 Crude oil is a viscous dark liquid resource composed by a mix of hydrocarbons.
Primarily it is composed of carbon and hydrogen, along with minor elements such as
Academic Editor: Tao Sun
nitrogen, oxygen, sulfur, and metals. Petroleum components can be divided into two
Received: 21 February 2022 main groups: hydrocarbons and heterocompounds. Crude oil is separated by different
Accepted: 24 March 2022 distillation processes for the elaboration of distinct products. Petroleum in crude state has
Published: 6 April 2022 minimal applications. However, by refining, different useful products are obtained such as
fuels, solvents, lubricants, plastics, oils, and asphaltic products, among others [1–3].
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
Due to the accelerated growth of industry and the high demand for oil, environmen-
published maps and institutional affil-
tal problems and contamination from leaks or spills has become an important issue to
iations.
be considered [4]. This can be caused by accidental leaks from reservoirs, refineries or
transportation pipelines (not including petrochemicals products) [5], which lead to con-
tamination of soil, groundwater, and oceans [2]. Environmental pollution causes direct
damages on the ecological properties of many species, in addition to those that directly
Copyright: © 2022 by the authors. affect humans [5,6]. A major concern is that many petroleum components, such as benzene,
Licensee MDPI, Basel, Switzerland. toluene, ethylbenzene, xylenes (BTEX), and polycyclic aromatic hydrocarbons (PAH), are
This article is an open access article highly toxic due to their teratogenic, hemotoxic, and carcinogenic effects [2]. Considering
distributed under the terms and this, along with the permanent damage to ecosystems, petroleum derivatives are one of the
conditions of the Creative Commons most persistent organic pollutants in the world. For this reason, oil pollution has become
Attribution (CC BY) license (https:// an environmental concern on a global scale, which must be solved through innovative,
[Link]/licenses/by/
efficient and sustainable techniques [5].
4.0/).

Processes 2022, 10, 712. [Link] [Link]

Processes 2022, 10, 712 2 of 14

Some of the most widely used procedures to totally degrade contaminants are biologi-
cal methods such as bioremediation [5,7]. Here, the pollutants are used as carbon sources,
which allows for elimination without altering the environment [2,7]. Microorganisms
involved in bioremediation processes use multiple metabolic pathways where enzymes are
the key actors for degradation [8]. Discovery of new techniques that consider the different
forms of genetic and metabolic expression in order to choose the right microorganism
and obtain better results is a new objective in the area [8,9]. The design, construction,
and fine-tuning of a wholly-engineered organism for the monitoring and degradation of
pollutants is a strategy provided by synthetic biology, creating a new era in bioremediation
and making it the best option in terms of contamination removal [6,9].

1.1. Petroleum Composition: Crude Oil

Within the two groups of the petroleum components there are four fractions: saturated
(aliphatic hydrocarbons), aromatic (cyclic hydrocarbons), resins, and asphaltenes. The
main components of petroleum are hydrocarbons: hydrogen and carbon compounds with
different molecular structures. The range of compounds extends from methane (natural
gas), liquids that are refined into gasoline, to solid waxes [1–3].
Saturated or aliphatic hydrocarbons represent the higher percentage of oil compounds.
Some of the common alkanes are methane, ethane, propane, and butane, and among the
common cycloalkanes are naphthenes [1,3]. On the other hand, aromatic compounds
are divided into (a) monocyclic aromatics, such as BTEX; (b) asphaltic compounds; and
(c) polycyclic aromatic hydrocarbons [2,3,10]. Asphaltic compounds include resins and as-
phaltenes [1,3]. Aromatic derivatives are the most toxic components of crude oil, associated
with chronic and carcinogenic effects [2,3,10].

1.2. Traditional Hydrocarbon Bioremediation Techniques

In order to choose the appropriate oil clean-up or remediation technique, time, cost
and efficiency must be considered, as well as the type of hydrocarbon and geographic
location [7]. Traditional physicochemical methods are effective to remove oil. However, they
produce different immunotoxic and carcinogenic compounds that remain dangerous [7].
This problem, along with the increasing cost and limitation of physicochemical methods,
led to the development of technologies based on the degradative capabilities of plants and
microorganisms, an option that offers great benefits [2].
Bioremediation techniques can be classified according to the site of application, namely
either ex situ and in situ (Table 1) [11,12]. Ex situ techniques involve the transportation
of contaminants to another site for treatment. Performing treatment away from the con-
tamination site could be costly and can disrupt natural sites. On the contrary, in situ
techniques perform the treatment at the site of contamination to eliminate the toxic com-
pounds. Therefore, this technique is less expensive as it does not require excavation
nor transport, although certain environmental factors such as soil porosity, temperature,
nutrient availability, humidity and pH must be evaluated [11,12].

Table 1. Examples of hydrocarbon bioremediation methods.

Bioremediation Technique Treatment % of Degradation Reference

Biostimulation: compounds such as nutrients,
oxygen, biopolymers, biosurfactants or Mineral fertilizer with Total petroleum hydrocarbon (TPH)
[13]
fertilizers are added, in order to enhance dolomite flour decreased by 47% in 15 months.
microbial activity [13–15].

Bioaugmentation: addition of autochthonous, Microbial inoculum with TPH was reduced by 41% in
[16]
exogenous or genetically engineered Alcanivorax as the dominant genus. 63 days.
microorganisms with catabolic activity to the TPH was removed by 69.17% in
Acinetobacter sp. SCYY-5 [17]
contamination site [14–16]. 10 days.
Processes 2022, 10, 712 3 of 14

Table 1. Cont.

Bioremediation Technique Treatment % of Degradation Reference

Biopile: contaminated soil is piled followed by
Biopile system with crude oil,
the addition of nutrients and oxygen, which
nutrients and Amnite P-300 TPH were reduced by 77% in
enhance degradation. A water system can be [20]
(consortium of 10 strains mainly 156 days.
added or organic materials that acts as bulking
belonging to the Pseudomonas genus)
agents [14,18,19].
Phytoremediation: process based on plants
and their associated microorganisms to TPH by 42.2% and PAH by 49.9% in
Melilotus officinalis [21]
degrade, remove or immobilize toxic 6 months.
compounds from the environment [14,15].

Microorganisms used in the bioremediation process can be isolated from soil, water,
or air. In many occasions, these are isolated directly from the contaminated sites, as it has
been found that they already have naturally developed the capacity to use or degrade these
pollutants as an adaptation in response to the selective pressure that the contaminants in
the environment exerted. Many microorganisms use hydrocarbons as an alternative carbon
source, since they are compounds that naturally can provide energy [2,22]. Considering
this, bioremediation techniques can be used to eliminate them [23].

2. Synthetic Biology and Bioremediation

Although bioremediation techniques offer great advantages, in many cases they are
becoming traditional methods since new tools are emerging. Thanks to multi-omics analysis
and advances in genetic engineering, it is possible to obtain the information necessary to
choose the best microorganism for the remediation process [9].
For example, in order to identify catabolic genes, novel pathways or proteins involved
in the biodegradation process, genomic, proteomics and metabolomic tools can be used.
Transcriptomics also gives important information, such as how the cell responds after the
exposure to toxic compounds and how it affects its metabolic state. On the other hand, in
silico tools can give insight into the biochemical reactions that take place in the degradation
of contaminants [24]
Synthetic biology is a scientific field based on biology and engineering principles,
with the purpose of redesigning and restructuring organisms to optimize or create new
biological systems with enhanced features [25–27]. This field uses molecular tools, systems
biology and the reprogramming of the genetic framework, thus constructing synthetic
pathways to obtain microorganisms with alternative functions [26,27]. The use of this
discipline brings an improvement of bioremediation processes (Table 2).

Table 2. Examples of engineered microorganism in hydrocarbon bioremediation.

Genetically Engineered Microorganisms Results Reference

almA gene was inserted into two plasmids and Total crude oil biodegradation was up to 32% and
[28]
transformed into Escherichia coli. 50% in 72 h.
Degradation of light crude oil by 88.65% [15.23%
Enzyme consortium of three mutant alkane more than the wild type (WT)], viscous crude oil
hydroxylases (alkMa, alkMb, and almA) belonging was degraded by 90.05% (21.65% more than WT), [29]
to Acinetobacter venetianus strain RAG-1. and high waxy crude oil by 60.52% (13.06% more
than WT), in 10 days.
1,2,3-Trichloropropane was degraded by 97% in 48
An improved dehalogenase gene (dhaA31) was
days. The enzyme showed 36-foldhigher activity
cloned behind the constitutive dhlA promoter into [30]
and 26-foldhigher catalytic efficiency than the
Pseudomonas putida MC4.
WT enzyme.
A recombinant strain was constructed from the
The biodegradation rate of the oil concentrations
integration of catechol 2,3-dioxygenase in [31]
was 80% in 28 days.
Acinetobacter sp. BS3.
Processes 2022, 10, x FOR PEER REVIEW 4 of 13
Processes 2022, 10, 712 4 of 14

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3. bioremediation.
Genetically Engineered Microorganisms
Many microorganisms have the metabolic potential to use toxic compounds as a carbon
3. Genetically
source, Engineered
and can even Microorganisms
degrade some of these contaminants [38]. Thanks to advancements in
Many microorganisms have the metabolic potential to use toxic compounds as a car
bon source, and can even degrade some of these contaminants [38]. Thanks to advance
ments in genetic and metabolic engineering, these properties can be enhanced to create
new, efficient, and safe techniques that could overcome physicochemical ones [38–40].
One example is modifying or inserting the necessary metabolic machinery that wil
Processes 2022, 10, 712 5 of 14

genetic and metabolic engineering, these properties can be enhanced to create new, efficient,
and safe techniques that could overcome physicochemical ones [38–40].
One example is modifying or inserting the necessary metabolic machinery that will
allow microorganisms to degrade specific contaminants [41,42]. Recombinant DNA technol-
ogy allows the insertion of a gene of interest through a vector, so the microorganism will be
capable of expressing the desired gene and gaining a new function [43]. There are different
strategies to construct genetically engineered microorganism (GEMs) in bioremediation,
although, there are two main techniques: (a) incorporation of the necessary degradation
machinery, creating new metabolic pathways; and (b) genetic and metabolic optimization,
to enhance affinity, specificity, and efficiency of the enzymes involved in degradation pro-
cesses, as well as to improve substrate utilization, and increase bioavailability and genetic
stability [40,42,44,45].
To create new metabolic pathways, it is necessary to identify degradative enzymes,
their respective genes, and strains with catabolic capacity. Then, the chosen genetic se-
quence is inserted into a vector to subsequently transform the host. Specific experiments
need to be performed to confirm the sequence integration into the microorganisms, as well
as the efficiency of the biodegradation gene [40,45].
A very important part in creating new metabolic pathways is selecting a plasmid.
Plasmids can carry degradative genes that encodes enzymes needed for the degradation
of different contaminants, such as aliphatic, aromatic and polycyclic aromatic hydrocar-
bons [46,47]. Bacterial plasmids can be transferred through horizontal gene transfer (HGT)
which may occur by different mechanisms: transformation, transduction, and bacterial
conjugation. Transformation is the most used method to insert genes of interest into a
microorganism, by using different vectors [47–49]. Some of the most commonly used
plasmids in microbial engineering for hydrocarbon bioremediation are PHE, TOL, NAH,
and OCT [46].
The PHE plasmid contains genetic information necessary for phenol metabolism to
take place, the TOL plasmid encodes xyl genes that are necessary for the degradation of
toluene and xylene. Naphthalene catabolic genes are part of the NAH plasmid and OCT
plasmid is responsible for the octane degradation by alk genes [46,47,50].
Wang et al. [51] constructed an E. coli strain capable of converting phenol to a carbon
source thanks to the integration of synthetic modules. First, phenol degrading genes were
selected, isolated and modified. Then, two metabolic modules were constructed using
two phenol hydroxylase genes and seven catechol-degrading genes, both of which were
integrated into a vector to transform E. coli cells. Engineered strains degraded phenol
rapidly in crude-contaminated wastewater: 5 mM in 7 h [51]. The study demonstrates
the successful construction of a novel metabolic pathway capable of degrading phenol in
E. coli. This is a great advantage considering that the bacteria do not naturally degrade
phenol, making this synthetically modified strain one of the first to utilize phenol as a
carbon source [51].
In contrast, to improve the genetic and metabolic performance of enzymes involved in
the degradation process, fine-tuning can be performed at different stages of gene expression,
such as transcription, translation and post-translation. Genetic optimization is carried out
with the purpose of obtaining the best genetic platform to maximize metabolic efficiency.
Some of these methods consist in the search of homologous genes or the optimization
of codons, in order to match the host or to find the most appropriate degradation genes
for the chosen microorganisms. It is also possible to regulate the number of copies of
the plasmid and mRNA expression, or to modify the translation rate by modifying the
ribosome binding site (RBS). All of these modifications could have different effects, such as
minimizing bottlenecks, avoiding overexpression of an enzyme, or increasing the affinity
or binding of the ribosomes to the transcripts [40,52]. Gene expression can be regulated by
many factors, so fine-tuning allows to achieve a proper balance in pathways to maximize
metabolic efficiency.
Processes 2022, 10, 712 6 of 14

Research conducted by Jain et al. [53] focused on a in silico analysis of a protein

involved in hydrocarbon degradation, alkane monooxygenase (ladA). An orthologous gene
for this enzyme was found in Burkholderia thailandensis MSMB121. Homology modeling
was performed in its structure and several amino-acids of the ladA protein were substi-
tuted. This achieved an improvement in the binding energy to chains of different alkanes
which increases accessibility to the substrate, creating the basis for further validation of
hydrocarbon degradation pathways in B. thailandensis [53]. This study is a great example of
how genetic optimization is a tool to achieve metabolic improvements for degradation of
various hydrocarbons.
Another approach of metabolic optimization is when metabolic pathways can be
improved, following the theory that there is proximity between enzymes of the same
pathway, allowing products to be immediately used, which generates a substrate tunneling
effect. For this purpose, a synthetic protein scaffold can be designed, containing binding
domains along with enzymes involved in the pathway. Therefore, scaffold aligns enzymes
very closely, mimicking the substrate tunneling effect [40,52].
One example of a synthetic scaffold was made by Dueber et al. [54] with the aim of in-
creasing the effective concentration of each component of a metabolic pathway. To achieve
this, a scaffold of synthetic proteins that spatially recruited enzymes was constructed. The
scaffold contained PDZ-SH3-GBD domains, which together with their binding peptides,
was attached to each heterologous enzyme involved in a synthetic pathway, which mim-
icked the substrate tunneling effect. The use of this synthetic scaffold increased the efficacy
of the pathway by 77-fold [40,54]
Most hydrocarbons are difficult to eliminate, and although there are microorganisms
that have degradative capacities, it is not enough to achieve efficient biodegradation. For
this reason, and thanks to genetic engineering advances, it has been possible to construct
GEMs, which through genetic refinement or construction of new pathways offer several
advantages, such as: metabolic optimization, integration of catabolic modules, increased
enzyme expression and biodegradation rate, among others. Synthetic biology has given
efficient results in the degradation of pollutants, allowing to create a new alternative in
bioremediation areas.

4. Biosensors
Due to the harmful effects that oil contamination has on the environment, animal
life and human health, it is very important to evaluate the risks this kind of pollutant
may have [55,56]. In order to reduce the impact and contain the contamination as early as
possible, the development of a detection unit for these compounds is necessary [56–58].
However, this represents a great challenge due to high costs, time spent and complex
procedures. Establishing new techniques with high effectiveness and sensitivity, and rapid
detection is necessary [55,58].
A biosensor is an integral and analytical device, which through biochemical reactions,
can detect a signal to provide quantitative and precise information. Biosensors are com-
posed of three main elements: a biological recognition element, a transducer, and a system
that processes the signal (detector) [4,59,60].
The biological recognition elements (receptor) can be enzymes, antibodies, antigens,
nucleic acids and even whole cells. The detected biochemical signals could be those derived
from metabolic processes, gene expression, cellular toxicity, or enzyme activity. Finally,
the transducer can be classified according to its physicochemical nature to detect the
electrochemical, optical, calorimetric, or thermal signals [59,60].
When the biological sample comes in contact with the receptor, the transducer will
convert it into a quantifiable electrical signal [4,56,59,60]. This means that the biological
recognition element selectively identifies the analyte by generating a specific signal. The
type of signal generated depends on the kind of transducer used. Then, the signal is
quantified by the detector [56].
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There are biological recognition elements that have the ability to detect multiple ana-
lytes which are useful when monitoring multiple toxic compounds. One of the recognition
elements that have the ability to monitor multiple samples simultaneously, selectively and
with fast response, are antibodies [56].
In the oil industry it is of great importance to perform environmental monitoring to
ensure the safety of the processes and reduce possible contaminations [61]. Biosensors can
be used to monitor oil spills along the process of bioremediation [62]. In this way, they help
detecting toxic compounds on time, so potential risks can be eliminated [57].
There are physicochemical methods that evaluate oil contamination [61]. Although
they are effective, they are also highly expensive, time-consuming and require solvents for
extraction and a large sample volume [55,61]. Molecular tools offer an alternative that will
overcome these limitations [61].
Biosensors can be constructed by isolating biological components or using whole
microorganisms. These sensors have great advantages. For example, they are fast, easy-to-
use and cost-effective tools that require less sample volume, no need for solvent extraction,
are robust and have good compatibility for real-time application [56,61,62]. They can also
be cultivated on a large scale, and even be engineered to resist harsh conditions such as
extreme pH and temperatures, and environmental contamination [4,59].
Synthetic biology offers the possibility of constructing whole cell biosensors (WCB).
WCBs have been used to monitor environmental pollutants such as heavy metals, pesticides,
pharmaceutical residues, chemicals, and organic pollutants such as oil derivatives [10].
The use of microorganisms to monitor different pollutants is a result of the development
of genetic engineering techniques which have made it possible to modify or design the
necessary elements for the detection and processing of signals [63]. This new alternative has
diverse bioreceptors that can be used along with different genetic mechanisms to overcome
traditional sensors and be used in situ [61].
When building WCBs it is important to consider the interaction between promoters
and reporter genes. Promoters can be inducible by external factors or constitutively ex-
pressed, and reporter proteins must produce quantifiable signals with high sensitivity [63].
Commonly used reporter genes are: (1) lacZ gene from Escherichia coli, this gene
encodes β-galactosidase. The enzyme degrades specific substrates producing colored
compounds which are measured by colorimetry; (2) GFP gene from Aequorea victoria,
encodes a green fluorescent protein (GFP). This protein absorbs light and emits it in a
different wavelength, which can be easily measured; (3) lux operon, works via a quorum-
sensing mechanism. The most used enzyme in WCB is luciferase (Luc), and it uses two
types of configuration: LuxCDABE, from Vibrio fischeri (where the same cell synthesizes the
necessary substrate for the luciferase) and LuxAB, from Vibrio harveyi (where the substrate
needs to be added) [55,63,64].
The first genetically modified microorganism to be used as a WCB for the monitoring
of the bioremediation process of contaminated soil was Pseudomonas fluorescens HK44
strain, which contained the pUTK21 plasmid. This plasmid holds the nahG gene (which
controls the degradation of salicylate) fused with the LuxCDABE gene cluster. Another
example of a WCB genetic construct is the fusion between the Luc gene and the Pu promoter,
controlled by XylR activator protein to monitor toluene in the environment. This activator
protein binds to toluene or its derivatives and activates Pu promoter; thus, creating a
bioluminescent sensor [64].
Furthermore, the study of Patel et al. [65] developed two biosensing strains to detect
hydrocarbons. Two vectors were designed with a promoter-operator fusion with fluores-
cent protein genes: tbuT-gfp (capable of detecting BTEX compounds) and phnR-cfp (capable
of detecting naphthalene, phenanthrene, and related PAH compounds). Designed vectors
were then transformed into E. coli DH5α. Both recombinant strains were capable of de-
tecting mono and polyaromatic hydrocarbons, creating a method to measure contaminant
levels [65].
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To create an adaptable WCB capable of being tuned for a particular monoaromatic

contaminant, Roy et al. [66] combined synthetic biology with a complementary design
to construct a genetically rewired and selective biosensor, ideal to detect pollutants in
contaminated water sources [66]. This study based its design on the phenol catabolism
pathway, using the MopR protein, triggering the transcription of the gene cluster when
binding to phenol. In this case, the difference was that to achieve sensitive detection, the
catabolism gene cluster was replaced with a reporter gene (Luc); creating the MopRLuc
biosensor, capable of detecting low and high concentrations of phenol in water. Given the
efficiency of the constructed biosensor, Roy et al. [66] decided to use it as a model to create
an array of WCBs for other pollutants. They discovered that changes in sensor profile were
due to mutations in the variable region, so they could generate biosensors to detect xylenol,
ethylbenzene and xylene. Not only this study was able to obtain successful results, but also
was able to create a biosensor template that can be engineered for the detection of different
aromatic pollutants [66].
There are still some areas in biosensor development that need to be studied to over-
come limitations, such as sample interferences, cell stability, bioavailability of hydrocar-
bons and even legal considerations on the release of engineered microorganisms into
nature [61,62]. However, synthetic biology brought new possibilities in the creation of
biosensors to help achieve early detection of toxic compounds [57].
When it comes to choosing an appropriate bioremediation technique, it is important to
know the conditions of the contamination site as well as the type of pollutant involved. Lack
of information can cause setbacks in the elimination process or even affect the establishment
of necessary control measures. Therefore, correct detection of contaminants is of great
importance. Using microorganisms offers great advantages, given the fact that many of
them are able to withstand high concentrations of contaminants, their adaptation is possible
and makes them perfect candidates for monitoring. Considering many hydrocarbons are
highly toxic, synthetic biology creates the possibility of engineering microorganisms with
the necessary machinery to detect different pollutants. Studies carried out in this area
provide data to position biosensors as new effective systems for detecting hydrocarbons in
the environment.

5. Construction of Synthetic Consortia

A microbial consortium is a set of two or more microbial species that work synergisti-
cally to create a balanced community where mutual benefit exists [67,68]. The life cycle of a
microbial community depends directly on the relationships existing among them, creat-
ing cooperative or competitive dynamics [67,69], as well as neutral, positive or negative
effects [70]. These relationships are classified in: (a) symbiotic, where organisms obtain
mutual benefit (e.g., mutualisms); and (b) antagonistic, where one species can be harmed
and the other benefited (e.g., parasitism) [71].
A consortium tends to be more effective than a single microorganism due to the
synergistic relationships resulting from complementary activities and metabolic capacity of
each species [72]. Since metabolites of one microbe can be used by another one, microbial
communities can use these mutual interactions to completely degrade contaminants [29].
On that account, when designing a consortium it is necessary to establish parameters that
guarantee coexistence and stable interactions between all of the species [24,73].
Each environment has autochthonous microorganisms with different degradation
capacities. However, the degradation of hydrocarbons by these individual microorganisms
can be low. Consortia are capable of achieving complete mineralization of contaminants
thanks to a sequential degradation due to the synergistic and metabolic activities they
possess as a group [74]. Therefore, an alternative in hydrocarbon bioremediation is the
addition and combination of allochthonous microorganisms for the creation of consortia,
which is more effective and sustainable than traditional methods [75,76].
Molecular tools, along with systems biology, enable the analysis of genetic information
and cell to cell interactions [8]. Omics tools can provide information on genes, proteins
Processes 2022, 10, 712 9 of 14

and metabolic pathways that will help to understand microbial behavior to enhance the
biodegradation processes. For example, they can be used to evaluate the structure and
dynamics of microbial consortia in different environmental scenarios [24]. Thanks to this,
synthetic biology offers the possibility of creating engineered microbial communities to
make strong cellular functions and improve its microbial capabilities and cooperation [8].
Assembling synthetic consortia will improve efficacy in bioremediation. These modifica-
tions can be made by manipulating environmental conditions, communication networks,
syntrophic interactions or the genetic framework and new genetic modules [8,24,34].
Synthetic biology tools can be used in microbial consortia to facilitate the interaction
among microorganisms. Some of these tools are: (a) syntrophic interactions, to create a
metabolic network where metabolites produced by one organism can be used by another
one; (b) exogenous molecules, adding external inputs to control cell communities and gene
expression; and (c) intercellular signaling, to control communication between cells and
gene expression (e.g., quorum sensing) [34,77].
One example of a synthetic community is the study of a consortium consisting of two
bacterial strains that were modified for phenanthrene degradation, by Jia et al. [78]. The
used strains were: (a) E. coli HY, with two terminal dioxygenase modules and an electron
transfer chain; and (b) Pseudomonas aeruginosa PH2, with a catechol 1,2-dioxygenase module.
A gas chromatography-mass spectrometry (GC-MS) was performed to identify metabolites.
The initial oxidation steps were made by E. coli HY1 (phenanthrene into 9,10-dihydroxy
phenanthrene or 1,2-dihydroxy phenanthrene), and then ring cleavage was performed
by P. aeruginosa PH2 to produce catechol. Further conversion between intermediates
was through the tricarboxylic acid cycle (TCA). The modified consortium was able to
degrade 71% of phenanthrene in nine days, while the wild type (P. aeruginosa PH1) only
degraded 45% [78]. This confirmed the improved removal capacity of a constructed
consortium compared to unmodified strains, confirming once again a new alternative for
bioremediation of PAHs.
Engineered consortia offer great advantages due to their efficient and improved levels
of degradation in comparison to individual strains. This means that synthetic communities
are a useful and a valid platform for bioremediation of hydrocarbons, even with efficient
results. Analysis of each microbial community, separately and as a whole, is needed when
designing and constructing one. It is important to remember synergistic relationships are
the key to the consortium’s success since metabolic capabilities and characteristics of each
species can be integrated, enhancing biodegradation. Developing a microbial consortium
with specific parameters to bioremediate hydrocarbon-contaminated areas is one of the
most promising benefits that synthetic biology offers.

6. Risk Assessment of Synthetic Biology

Considering the bioremediation process will take place at the site of contamination,
GEMs will be leaving the laboratory and entering a natural environment, which may entail
risks or difficulties since it stops being a controlled environment [8,42]. The environmental
risks relate to gene contamination, toxicity and competition with native species. The problems
of gene contamination are related to horizontal gene transfer which leads to the delivery of
the recombinant genes [79], modifying autochthonous microorganisms and altering their
natural genetics [42]. One of the major risks is posed by plasmids containing antibiotics
as resistance genes, as they can lead to the formation of antibiotic-resistant superbugs in
nature [80]. It is also important to consider the release of compounds toxic to the environment
or related to human health due to the change in microbial metabolism [42,80]. In regards
to the competition with native species, as noted by de Lorenzo [81], the risk of altering
the microbial composition by introducing GEMs into natural ecosystems is not as high as
commonly thought. Due to the homeostasis of biological ecosystems and resistance to colo-
nization, engineered microorganism have difficulties at establishing in a new environment,
meaning that it is unlikely that the modified microorganisms could displace the indigenous
community [80,81].
Processes 2022, 10, 712 10 of 14

To minimize potential risks, strategies include using non-antibiotic selection mark-

ers and avoiding gene transfer to indigenous organisms [82]. To resolve this problem,
biocontainment techniques based on cellular circuits, inducible systems or auxotrophy
have been developed [83]. The most promising technique for biocontainment of genes in a
natural environment is through toxin-antitoxin systems. This protection system will secure
the genetic material from horizontal gene transfer [84]. A toxin is encoded in a plasmid,
while the antitoxin is encoded into the genome. Therefore, if a gene transfer occurs, the
new host would die as it incorporated the plasmid with the toxin but does not possess
the antitoxin [83]. Another strategy that can be used to protect the inserted genes is the
use of conditional replication origins. The origin needs an initiator protein to replicate the
plasmid, which is inserted into the chromosome of the host. In this way, the replication of
the plasmid is blocked if it is transferred to another cell [84].
It must be taken into consideration that legal regulations play an important role when
implementing these new alternatives, as well as the possible environmental risks it may
involve. The good news is genetic engineering and synthetic biology offer to mitigate these
biological risks through different biocontainment mechanisms. Therefore, based on the
different bioinformatics studies and laboratory results obtained, it is important to carry out
more in situ tests to analyze the behavior of GEMs in an uncontrolled environment, as well
as their proper biomonitoring, in order to establish this alternative in bioremediation as
one of the most effective and safe.

7. Conclusions
Petroleum-derived pollutants are highly toxic, creating serious and harmful conse-
quences in any environment. Developing innovative, fast, safe, and cost-effective tech-
niques for their elimination is of great importance. Bioremediation as a contaminant
removal technique has been very successful, and although several microorganisms possess
degradative capacities, optimizing these techniques is necessary due to the pollutant’s
persistence. Over the years, advances in different areas of science have led to improvements
in various degradation techniques. Knowledge in systems biology, molecular tools, and
multiomics are the basis of synthetic biology, which creates a new era in bioremediation.
Analysis, design, construction and fine-tuning of genetically and metabolically optimized
microorganisms maximize toxic compounds degradation.
Creating biosensors to detect and monitor contaminants, understanding microbial
dynamics to construct synthetic consortia, as well as creating new metabolic pathways or
enzymatic enhancement, are some of the possibilities offered by synthetic biology. It is
still necessary to carry out more in situ experiments to support different results obtained
in laboratories, as well as establishing the necessary safety parameters for an engineered
microorganism to enter the environment. The most important thing is that now it is
possible to create ideal techniques to degrade persistent and harmful pollutants such as
hydrocarbons. Even though some areas need further research, synthetic biology puts
science on the right track. With these new tools at hand, bioremediation positions itself as
one of the best and most effective pollutant removal process available today.

Author Contributions: Conceptualization, V.J.-D. and L.C.C.-C.; investigation, V.J.-D.; writing—

original draft preparation, V.J.-D. and L.C.C.-C.; writing—review and editing, V.J.-D., L.C.C.-C.,
O.R.-M. and A.M.P.-R.; supervision, L.C.C.-C.; funding acquisition, L.C.C.-C. and O.R.-M. All authors
have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Acknowledgments: The authors acknowledge to Universidad Anáhuac Mexico for the support given
to publish this work.
Processes 2022, 10, 712 11 of 14

Conflicts of Interest: The authors declare no conflict of interest.

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