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Citation: Yotinov, I.; Belouhova, M.;
Foteva, A.; Dinova, N.; Todorova, Y.;
Schneider, I.; Daskalova, E.; Topalova,
Y. Application of Nanodiamonds in
Modelled Bioremediation of Phenol
Pollution in River Sediments.
Processes 2022,10, 602. https://
doi.org/10.3390/pr10030602
Academic Editor: Maria Jose Martin
de Vidales
Received: 31 January 2022
Accepted: 17 March 2022
Published: 19 March 2022
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processes
Article
Application of Nanodiamonds in Modelled Bioremediation of
Phenol Pollution in River Sediments
Ivaylo Yotinov 1,2,*, Mihaela Belouhova 1,2 , Anna Foteva 1, Nora Dinova 1,2, Yovana Todorova 1,2 ,
Irina Schneider 1,2, Elmira Daskalova 1and Yana Topalova 1,2
1Department of General and Applied Hydrobiology, Faculty of Biology, Sofia University “St. Kliment
Ohridski”, 8, Dragan Tzankov Blvd., 1164 Sofia, Bulgaria; mihaela.kirilova@uni-sofia.bg (M.B.);
anna.foteva@gmail.com (A.F.); norakdbg@yahoo.com (N.D.); yovanatodorova@biofac.uni-sofia.bg (Y.T.);
i.schneider@abv.bg (I.S.); baba_emi@abv.bg (E.D.); yanatop@abv.bg (Y.T.)
2Center of Competence “Clean Technologies for Sustainable Environment—Water, Waste,
Energy for Circular Economy”, 1000 Sofia, Bulgaria
*Correspondence: ivaylo_yotinov@uni-sofia.bg; Tel.: +359-2-8167-205
Abstract:
The pollution of aquatic ecosystems is a big problem that has its impact on river sediments.
In recent decades, an effective solution to this problem has been the application of bioremediation
technologies. Nanoremediation is an innovative part of these technologies. We still know little
about the efficiency of nanoparticles, especially nanodiamonds, in modelled conditions. The aim of
the present study is to investigate the effect of nanodiamonds on the key parameters of modelled
bioremediation of river sediments that are polluted with phenol, as well their effect on the structures
and functions of microbial communities. An important indicative mechanism that was used is the
application of fluorescent in situ hybridization for sediment microbial communities. The results
of this study revealed the positive role of nanodiamonds that is associated with their intoxication
with high concentrations of phenol. Readaptation was also found, in which the xenobiotic biodegra-
dation potential evolved by increasing the relative proportions of non-culturable bacteria, namely
Acinetobacter (at the 144th hour) and Pseudomonas (at the 214th hour). The results can help to find
an effective solution to the question of how information from such precise molecular methods and
the application of nanodiamonds can be translated into the accessible language of management and
bioremediation technologies.
Keywords: biodetoxification; bioremediation; FISH; nanodiamonds; river sediments; phenol
1. Introduction
Sediments play an important role in the assessment of the condition of water sys-
tems [
1
–
5
]. Sediments consist of particles of different sizes that can be transported by a fluid
stream and that precipitate as a layer of solid particles over the bed or the bottom of water
basins [
6
]. The upper layers of the sediments in lake and river ecosystems have a higher
content of organic compounds, which is a precondition for the presence of a large number of
microorganisms. This is mostly due to the higher concentrations of carbohydrates, proteins,
and amino acids that are in the interstitial water [
7
–
9
]. The microorganisms have a main role
in the performance of the bio-transforming processes of organic compounds and nutrients.
This also defines the key significance of the microbial communities for the realization of the
self-purifying potential of water ecosystems. Therefore, an understanding of the functional
structure of the microbial community would contribute to the better understanding of this
potential. The study of the groups of microorganisms that are present, through separation
that is based on a specific physiological process, would contribute to the study of the details
of the bio-transformation processes [10,11].
An example in which the self-purification biotransformation processes that occur in
river sediments can be studied in detail is the case of the cascade “Middle Iskar”, which
Processes 2022,10, 602. https://doi.org/10.3390/pr10030602 https://www.mdpi.com/journal/processes
Processes 2022,10, 602 2 of 20
includes five small hydro power plants (SHPPs) on the middle stream of the Iskar River
in the region of the villages Rebrovo and Gabrovnitsa in Bulgaria. The river in this region
is strongly anthropogenically impacted, mostly by the villages that are near it and by
different productions that do not have built or well-functioning treatment plants. As a
result, into the waters and the sediments of the river fall a large quantity of trivial and
xenobiotic pollutants. Of the different xenobiotics polluting the river, the most critical
ones are the heavy metals which have been accumulating for decades in the sediments
of the river [
12
]. The SHPPs play the role of a filter, holding the river sediments and the
domestic, industrial and naturally originated pollutants that have entered into them. These
pollutants hinder the self-purification processes. The SHPP which was first built in the
cascade was SHPP Lakatnik, which has functioned since 2008. In its storage are found the
largest accumulations of river sediments, as well as an accumulation of large quantities of
hardly degradable pollutants within the sediments [13].
An example of a typical pollutant that accumulates in these sediments is phenol—a
toxic compound that is found in the water media exceptionally often due to pollution from
different industrial processes from petrol refineries, the chemical industry, the production
of dyes, wood conservation, paper production, etc. [
14
,
15
]. Phenol is a comparatively
easily degradable xenobiotic for the microbial degraders. A higher concentration could
inhibit the growth of the microorganisms and microbial communities. On the other hand,
phenol is toxic to water organisms and to people [
16
] as it provokes carcinogenicity and
neurotoxicity [
17
]. As a consequence of this, it is reasonable to propose that the living
organisms that are in the river water and river sediments in the cascade could be at risk.
Therefore, the elimination of phenol is vitally important for the good ecological status of
the river. All this makes phenol a very convenient model xenobiotic for the study and
management of the bioremediation processes in sediments.
In the last few decades, together with the improvement of biotechnologies, bioremedi-
ation has transformed into one of the fastest-developing fields in environment protection.
Bioremediation is an application of biological processes for the purification of underground
waters, soils, sediments, and sludges that are polluted with dangerous chemicals [
18
]. This
approach is a more effective alternative to the physicochemical methods that have higher
expenses and produce other toxic final products [
17
]. This technology, when applied in
situ, causes the least disruption to the environment and, compared to other technologies, is
relatively cheap. It can use autochthonous microflora, selected and/or adapted microbial
cultures or microbial communities [
18
]. The application of the bioremediation process
requires an entire study of the essence and the range of the pollution, the characteristics of
the place, the legal restrictions, and the long-term management of the specific project as well
as the juxtapositions between the value and the benefits of the specific technology [
18
,
19
].
The main aims of the prevention of the pollution of sediments are a decrease in the regional
pollutions within the water basins and an increase in the essential self-purifying processes
and bioremediation technologies for the recovery of the sediments’ quality.
One of the main challenges in the use of the bioremediation technologies is the identi-
fication of adequate microorganisms with a high enough level of metabolic activity that
would be able to effectively eliminate the pollutants [
14
]. The bioremediation of phenol
compounds could be performed by many microorganisms from different genera such as
Pseudomonas, Vibrio, Serratia, Bacillus, Achromobacter, and Acinetobacter [15,20]. The identifi-
cation of the microorganisms could be performed with standard culture methods [
21
,
22
].
However, by means of these methods, just a small subset of the microorganisms—not
more than 3%—can be characterized. More complete information about the quantity, the
biodiversity, the localization, and the distribution of the microorganisms in the commu-
nities allow molecular methods such as denaturing gradient gel electrophoresis (DGGE),
polymorphism with a limited length of the fragment (T-RFLP), and fluorescence in situ
(FISH) [23–28].
One of the most often applied molecular methods is fluorescence in situ hybridization
analysis (FISH). The application of this method allows the analysis of the distribution of the
Processes 2022,10, 602 3 of 20
target microorganisms in situ in the biological system [
29
–
31
]. FISH combines the precision
of molecular genetics with the visual information of microscopic analysis, this combination
allows the identification of key microbial cells in their essential microhabitat as well as
the study of the population dynamics and interspecies interactions of the separate cell
levels [
32
–
36
]. The FISH method gives an opportunity for a study of the physiological
marks of selected microbial populations and responds to the question, “who does what
in this microbial community?”. However, to the present date, studies of the microbial
communities in the sediments of freshwater basins with FISH are not numerous [
37
–
40
].
Although FISH analysis is more often applied to underground waters [
41
–
43
], it has been
proven that more than 90% of the microbial biomass within water can be found attached
to precipitated particles [
44
,
45
] and that this biomass manifest a much higher level of
activity than the plankton microbial community [
46
–
49
]. Therefore, detailed studies of
sediments from water bodies are ecologically more convenient than studies of samples
from underground and surface water [50].
The intensive development of nanotechnologies accelerates the studies and applica-
tions of nanodiamonds (ND) due to their mechanical, chemical and optic properties [
51
].
All of these characteristics contribute to the more effective application of ND in different
biomedical and ecological applications and other biotechnologies, in comparison to other
carbon nanoparticles. The application of nanodiamonds has expanded dramatically in
recent years. This is the result of increasingly successful laboratory results and patents. The
commercial production of nanodiamonds makes their application in industrial technologies
more realistic. This could also lead to the production of widely manufactured products
containing nanodiamonds. From an economic point of view, the increased production of
ND leads to a decrease in their price and, therefore, their scope of application increases [
52
].
An example of such a new production, which is significantly more economically profitable,
is described by Dolmatov et al. [
53
]. In previous experiments with nanodiamonds with
hydrophobic groups, their positive effect on the biodegradation of phenol at concentrations
of 250 mg/L has already been proven. Because of the hydrophobic properties of ND, they
form consortia together with bacterial cells [
54
]. These properties are the base for the
capability of ND to actively to adsorb viruses and microorganisms. An example is the at-
tachment of the bio-compatible ND to enzymes or different medications that have a proven
effectiveness against pathogenic bacteria [
55
] and cancer cells [
56
]. Regarding environment
protection, ND could be used in different aspects. A main application of ND, which is
examined in detail in the present paper, is their use for environment remediation [
57
]. The
aim of the study is to investigate the effect of nanodiamonds on the key parameters of the
model bioremediation of river sediments that are polluted with phenol, as well as their
effect on the structure and functions of microbial communities. Kinetic and microbiological
parameters, as well as fluorescent in situ hybridization analysis, were key to the process.
2. Materials and Methods
2.1. Experimental Design
The design of this analogous modeling included the making of 2 bioremediation sites
simulating the processes in the sediments of the dams of the Middle Iskar cascade. Each
bioremediation site had a volume of 1 dm
3
, contained 500 g of sediments from Lakatnik
dam and 500 g of filler in the form of quartz sand, with a particle size of 0.8–1.6 mm. The
duration of functioning was 214 h in a semi-continuous mode of action. The experiment
aimed to follow the processes that were occurring in two variant conditions: (1) the control
variant (Ph), containing sediments, quartz sand, and a model xenobiotic phenol and (2) the
model variant (PhND), in which nanodiamonds were added to the sediments with quartz
sand and phenol (ND) as nanomodulators (Figure 1).
Processes 2022,10, 602 4 of 20
Processes2022,10,xFORPEERREVIEW4of21
and(2)themodelvariant(PhND),inwhichnanodiamondswereaddedtothesediments
withquartzsandandphenol(ND)asnanomodulators(Figure1).
Figure1.Experimentaldesign.
ThesedimentswerecollectedfromMHPPLakatnik,theoldeststorageintheMiddle
Iskarcascade.Inthispartoftheriver,largequantitiesofpollutantshaveaccumulatedin
thesediments.Inthecourseofthemodelbioremediation,thebiodetoxifyingandadaptive
processesofthemicrobialcommunityinthevariantwithphenol(Ph)andinthevariant
withthephenolandnanodiamonds(PhND)wereinvestigated.Duringtheprocess,the
increaseintheconcentrationofphenoloccurredaccordingtoaspecializedalgorithm:2nd
hour—250mg/kg;49thhour—500mg/kg;and72ndhour—500mg/kg[30].Thefollowing
processesandsituationsweresimulated:
Pollutionwithacomparativelylowconcentrationofphenol:fromthe2nd–48th
hours;
Simulationofacriticalsituationwiththeenteringofahighconcentrationofphenol:
atthe49thand72ndhours;
Studyofthesystemafteralongadaptationandenteringintothephaseofsystem
recoveryaftertheexplosivephenolloading:fromthe144th–214thhours.
Theparametersoftheseprocessedweremeasuredatcriticalcontrolpoints—CCP0,
CCP2,CCP48,CCP144,andCCP214.
Thisselectionofphenolasthemodelxenobioticwasmadeduetothefactthatitis
easilybiodegradable,hasahighlevelofsolubilityinwater,hasahighleveloftoxicity
andisfoundinlargequantitiesinessentialwaterecosystems,sediments,andindustrial
wastewaters[15,58].
2.2.AnalyticalMethodsandIndicators
Theresidualphenolwasmeasuredbytheuseofthespectrophotometricmethodwith
4‐aminoantipyrineafterdistillation.Afterthepreliminarydistillation,thesampleswere
analyzedaccordingtothespecificationsthataredescribedintheBDSandEPAmethods
[59,60].TheabsorptionwasmeasuredspectrophotometricallyatSpectrophotometer—
PharmaciaBiotechUltrospec3000.Allofthechemicalreagentsthatwereusedinthe
measurementoftheresidualphenolweresuppliedbyMerck,Inc,USA.
Thefollowingformulasforthecalculationofbiodegradationindicatorswereused.
Effectivenessofphenolbiodegradation(Eff):
Figure 1. Experimental design.
The sediments were collected from MHPP Lakatnik, the oldest storage in the Middle
Iskar cascade. In this part of the river, large quantities of pollutants have accumulated in
the sediments. In the course of the model bioremediation, the biodetoxifying and adaptive
processes of the microbial community in the variant with phenol (Ph) and in the variant
with the phenol and nanodiamonds (PhND) were investigated. During the process, the
increase in the concentration of phenol occurred according to a specialized algorithm: 2nd
hour—250 mg/kg; 49th hour—500 mg/kg; and 72nd hour—500 mg/kg [
30
]. The following
processes and situations were simulated:
•
Pollution with a comparatively low concentration of phenol: from the 2nd–48th hours;
•
Simulation of a critical situation with the entering of a high concentration of phenol:
at the 49th and 72nd hours;
•
Study of the system after a long adaptation and entering into the phase of system
recovery after the explosive phenol loading: from the 144th–214th hours.
The parameters of these processed were measured at critical control points—CCP
0
,
CCP2, CCP48, CCP144 , and CCP214.
This selection of phenol as the model xenobiotic was made due to the fact that it is
easily biodegradable, has a high level of solubility in water, has a high level of toxicity
and is found in large quantities in essential water ecosystems, sediments, and industrial
wastewaters [15,58].
2.2. Analytical Methods and Indicators
The residual phenol was measured by the use of the spectrophotometric method with
4-aminoantipyrine after distillation. After the preliminary distillation, the samples were ana-
lyzed according to the specifications that are described in the BDS and EPA methods [
59
,
60
].
The absorption was measured spectrophotometrically at Spectrophotometer—Pharmacia
Biotech Ultrospec 3000. All of the chemical reagents that were used in the measurement of
the residual phenol were supplied by Merck, Inc, Kenilworth, NJ, USA.
The following formulas for the calculation of biodegradation indicators were used.
Effectiveness of phenol biodegradation (Eff ):
E f f =Ct1−Ct2
Ct1
·100 [%],
where Ct
1
is the phenol concentration at time t
1
and Ct
2
is the phenol concentration at the
next time, t2.
Processes 2022,10, 602 5 of 20
Rate of phenol biodegradation (RB):
RB =Ct1−Ct2
t2−t1
mg/gxh,
Specific rate of phenol biodegradation (SRB):
SRB =Ct1−Ct2
(t2−t1)·Bm mg/gxh,
where Bm is the biomass in g/L dry weight.
Rate of biomass accumulation (RAB):
RAB =DW t2−DWt1
t2−t1
mg/lxh,
where DWt1is the dry weight at time t1and DWt2is the dry weight at the next time, t2.
2.3. Microbiological Analysis
The quantitative determination of the microorganisms was conducted through culture
methods by using solid nutrient media according to the rules of routine microbiological
practice [
61
]. The sediment samples were taken from both of the variants (Ph and PhND) at
the 0th hour—CCP
0
, 48th hour—CCP
48
, and 214th hour—CCP
214
. The sediment samples
were firstly treated with an ultrasound disintegrator UD-20 automatic (Techpan), in three
repetitions for 10 s for the desorption of the bacterial cells from the surface layer of the
sediment particles (1 g sediment + 9 mL mineral solution of NaCl (0.9%)). The conditions of
the cultivation are presented in Table 1. All of the results are the average values of the three
independent repetitions, represented as CFU/g dry weigh. The microbiological indicators
that were investigated in this process were physiological and taxonomic groups of bacte-
ria like aerobic heterotrophic bacteria (AeH), bacteria from genus Pseudomonas (Ps), and
bacteria from the genus Acinetobacter (Ac) (Table 1). The amount of the phenol-degrading
bacteria (Ph-degr.) were determined on the solid medium according to Furukawa [
62
], with
phenol (200 mg/L) as the sole carbon and energy source (Table 1).
Table 1. Studied groups of microorganisms, nutrient media, and cultivation conditions.
Microbiological Indicator Nutrient Media Producer Incubation Conditions
Aerobic heterotrophic bacteria
(AeH) Nutrient agar in aerobic conditions HiMedia 24 h,
37 ◦C
Genus Pseudomonas
(Ps.) Glutamate starch Pseudomonas agar HiMedia 24 h,
37 ◦C
Genus Acinetobacter
(Ac.) Sellers differential agar HiMedia 24 h,
37 ◦C
Phenol-degrading bacteria
(Ph.degr.)
Synthetic salt medium with added
phenol
according to Furukawa,
1983 2–7 days, 28 ◦C
2.4. FISH Analysis
Samples were taken from both of the variants (Ph and PhND) of the experiment at the
points CCP
0
and CCP
2
as well as at the points CCP
48
, CCP
144
, and CCP
214
of the process.
The samples were fixed with an 8% paraformaldehyde. This conducted a dehydration and
a permeabilization process, according to Nielsen (2009) [
31
]. The fluorescent signal for
the genera Pseudomonas was received with the 5
0
-labelled oligonucleotide probe 5
0
-GCT
GGC CTA GCC TTC-3
0
with the red dye Cy3 [
63
–
66
]. The fluorescent signal for the genera
Acinetobacter was received with the 5
0
-labelled oligonucleotide probe (ACA652) 5’-ATC CTC
TCC CAT ACT CTA-3’ with the yellow dye Fluos, 5’-labeled. For a control, a non-sense
Processes 2022,10, 602 6 of 20
probe coH
д
a NON338 (5
0
-ACT CCT ACG GGA GGC AGC-3
0
) was used. The hybridization
was performed with 20% formamide for the genus Pseudomonas and 35% formamide for the
genus Acinetobacter. The samples were dyed with DAPI for the visualization of all of the
microorganisms in the biofilm. The images were made with an epifluorescent microscope
(Leica Micro Systems DFC 310 FX) with a magnification of 400×.
2.5. Application of Nanodiamonds
The ND were added in a concentration of 1.667 mg/g at the start of the process. The
nanodiamonds that were applied in the present model bioremediation of river sediments
were provided by Professor Stavry Stavrev from the International Center for Nanomaterials,
Nanotechnologies and Nanomedicine in the town of Smolyan, Bulgaria [
67
]. A method
of the detonation synthesis of ND was applied in order to create the obtained ND. The
detonation of the explosive charge was performed in an aqueous medium in the presence
of active reductants [
68
]. The nanodiamonds that were used in the present experiment
were characterized by transmission electron microscopy (TEM) and X-ray measurements,
as described in the study by Prokharov et al. [
69
]. The data from both of these methods
has proven the characteristics of the nanodiamonds. In addition to these two methods,
Raman spectroscopy was performed [
69
]. The Raman spectrum contained the line for
nanodiamonds at 1332 cm
−1
, D-line of graphite at 1350 cm
−1
, and G-line of graphite at
1580 cm
−1
. The lines D and G were relatively narrow and had the same intensity, indicating
the presence of carbon nanoclusters smaller than 5 nm [
69
]. In our experiments ND with
hydrophobic properties without a specially treated surface have been used [70].
3. Results and Discussion
3.1. Residual Concentration of Phenol
In this section, the dynamics of the residual concentration of phenol, the kinetic
parameters of the phenol elimination and the effect of the nanodiamonds on them will be
discussed. The ecological situations of an explosive, risky pollution incident with phenol in
high concentrations were simulated.
In Figure 2, the phenol dynamics at residual concentrations in the different hours of the
detoxifying process are represented. Phenol was added on the 2nd hour at a concentration
of 250 mg/kg. Between the 2nd and the 49th hours, a small decrease in the phenol
concentration in both of the working variants was registered. The residual concentration
was around 200 mg/kg in both of the systems. At the 49th hour, phenol was added
into the systems at a two-times higher concentration—500 mg/kg and thus the phenol
concentrations reached the values of 527.63 mg/kg for variant Ph and 504.99 mg/kg for
variant PhND. At the 72nd hour, the third and last addition of phenol with a concentration
of 500 mg/kg was performed. The phenol then reached the concentration of 738.87 mg/kg
for variant Ph and 682.28 mg/kg for PhND; after which, at the 75th hour, there was a
new discharge of the phenol that was accumulated in the cells which led to a bigger
increase in the phenol concentration—835.06 mg/kg for variant Ph and 721.89 mg/kg
for variant PhND. The lower values of the phenol at these hours in the variant PhND
manifest the adsorbing properties of the nanoparticles which are due to the one of their
specific features—their surface-active groups. From the 75th to the 96th hours a sharp
decrease in the phenol concentration was registered. Due to the critical concentration being
reached and the significant inhibition of the microorganisms, the process of elimination
was probably accompanied by different biotic and abiotic processes, but it could hardly be
considered a real biodegradation process. Between the 75th and the 96th hours, a sharp
decrease in the phenol concentration was registered, which contradicts the biological logic
of the realization of the biodegradation process. Here, phenol elimination from the model
sediment site had occurred based on physicochemical processes—phenols flying in the
air (this is a volatile pollutant at an extremely high concentration in periodically mixed
sediments), polymerization and a permanent adsorption of the polymerized products on
the sediment particles and others like them. After the physicochemical decrease in the
Processes 2022,10, 602 7 of 20
phenol concentration after the 96th hour to around 310 mg/kg (a concentration which is
near-critical), the long phase of the readaptation of the inhibited biological system began.
This process of readaptation (from the 96th to 192nd hour) began around the 96th hour and
only at the 192nd hour did the microbial community start to recover its biodegradation
potential. Again, from the 192nd to the 214th hours, a real biodegradation process was
registered. This is the reason for the later study of the kinetic biodegrading parameters
which constituted a second real biodegradation stage from the 192nd to the 214th hours.
Processes2022,10,xFORPEERREVIEW7of21
realbiodegradationprocess.Betweenthe75thandthe96thhours,asharpdecreaseinthe
phenolconcentrationwasregistered,whichcontradictsthebiologicallogicoftherealiza‐
tionofthebiodegradationprocess.Here,phenoleliminationfromthemodelsedimentsite
hadoccurredbasedonphysicochemicalprocesses—phenolsflyingintheair(thisisavol‐
atilepollutantatanextremelyhighconcentrationinperiodicallymixedsediments),
polymerizationandapermanentadsorptionofthepolymerizedproductsonthesediment
particlesandotherslikethem.Afterthephysicochemicaldecreaseinthephenolconcen‐
trationafterthe96thhourtoaround310mg/kg(aconcentrationwhichisnear‐critical),
thelongphaseofthereadaptationoftheinhibitedbiologicalsystembegan.Thisprocess
ofreadaptation(fromthe96thto192ndhour)beganaroundthe96thhourandonlyatthe
192ndhourdidthemicrobialcommunitystarttorecoveritsbiodegradationpotential.
Again,fromthe192ndtothe214thhours,arealbiodegradationprocesswasregistered.
Thisisthereasonforthelaterstudyofthekineticbiodegradingparameterswhichconsti‐
tutedasecondrealbiodegradationstagefromthe192ndtothe214thhours.
Figure2.ResidualconcentrationofphenolinPhandPhNDvariantsinthecourseofmodelbiore‐
mediationofcontaminatedsediments.Arrowsindicatetheadditionofphenolinthemodelsys‐
tem—atthe2nd,49thand72ndhours.
3.2.KineticParametersofBiodetoxificationProcesses
Table2presentsthekineticparametersofthephenolbiodegradationprocessinthe
experimentalvariantsPhandPhNDwithinthebioremediationofthesediments.Therates
ofbiodegradationofphenolatthe48thhourwerefoundtobe1.598mg/kg∙hinvariant
Phand0.891mg/kg∙hinvariantPhND.Attheendoftheprocess(atthe214thhour)an
increaseinthisratewasfoundinthevariantwiththeaddedND—5.446mg/kg∙h,com‐
paredtothecontrolvariantPh—4.578mg/kg∙h.Thelowerrateatthe48thhourmaybe
duetotheadsorptionofphenolontothenanodiamondsatthebeginningoftheprocess.
Thisledtolowerconcentrationsoftheresidualphenol,andthusinducedtheenzymatic
activitiesthatwereresponsibleforthebiodegradationofthexenobiotic.
Table2.Kineticparametersofphenolicbiodegradationandbiomassgrowth.
ParametersVariants48thhour214thhour
Rateofbiodegradationofphenol
/RBP/mg/kgxh
Ph1.598±0.1664.578±0.295
PhND0.891±0.1175.446±0
Specificrateofbiodegradationof
phenol/SRBP/mg/kgxh
Ph0.211±0.0220.348±0.027
PhND0.100±0.0160.436±0
Rateofaccumulationofbiomass
/RAB/mg/lxh
Ph−0.082±0.0530.034±0.008
PhND−0.054±0.0280.021±0.008
Effectivenessofeliminationof
phenol/EFF./%
Ph26.15±1.97629.64±1.638
PhND16.81±2.26033.42±0.486
Figure 2.
Residual concentration of phenol in Ph and PhND variants in the course of model bioreme-
diation of contaminated sediments. Arrows indicate the addition of phenol in the model system—at
the 2nd, 49th and 72nd hours.
3.2. Kinetic Parameters of Biodetoxification Processes
Table 2presents the kinetic parameters of the phenol biodegradation process in the
experimental variants Ph and PhND within the bioremediation of the sediments. The
rates of biodegradation of phenol at the 48th hour were found to be 1.598 mg/kg
·
h in
variant Ph and 0.891 mg/kg
·
h in variant PhND. At the end of the process (at the 214th
hour) an increase in this rate was found in the variant with the added ND—5.446 mg/kg
·
h,
compared to the control variant Ph—4.578 mg/kg
·
h. The lower rate at the 48th hour may
be due to the adsorption of phenol onto the nanodiamonds at the beginning of the process.
This led to lower concentrations of the residual phenol, and thus induced the enzymatic
activities that were responsible for the biodegradation of the xenobiotic.
Table 2. Kinetic parameters of phenolic biodegradation and biomass growth.
Parameters Variants 48th Hour 214th Hour
Rate of biodegradation of phenol
/RBP/mg/kgxh
Ph 1.598 ±0.166 4.578 ±0.295
PhND 0.891 ±0.117 5.446 ±0
Specific rate of biodegradation of
phenol/SRBP/mg/kgxh
Ph 0.211 ±0.022 0.348 ±0.027
PhND 0.100 ±0.016 0.436 ±0
Rate of accumulation of biomass
/RAB/mg/lxh
Ph −0.082 ±0.053 0.034 ±0.008
PhND −0.054 ±0.028 0.021 ±0.008
Effectiveness of elimination of
phenol/EFF./%
Ph 26.15 ±1.976 29.64 ±1.638
PhND 16.81 ±2.260 33.42 ±0.486
The specific rate of the biodegradation of the phenol value at the 48th hour for the
variant Ph was 0.211 mg/kg
·
h and 0.100 mg/kg
·
h for the variant PhND. At 214th hour, the
specific rate for the variant PhND was 0.436 mg/kg
·
h versus 0.348 mg/kg
·
h for the variant
Ph. The data in Table 1show the negative modulating effect of the nanodiamonds on the
first phase of the biodegradation of the phenol and the positive modulating effect at the
end of the process.
Processes 2022,10, 602 8 of 20
Another finding is that the effectiveness of the elimination of phenol at the 48th
hour was lower in the PhND variant. This confirms the negative modulating effect of
nanodiamonds on the initial phase. At the end of the process (at the 214th hour), the
effectiveness of variant Ph was 29.64% and for PhND—33.42%, i.e., the effectiveness was
almost equal (with a difference of about 4%). These results show the low effectiveness of
the elimination of the toxic pollutant.
Figure 3presents the effect of nanodiamonds on the kinetic parameters of the biodegra-
dation of phenol, compared to the control variant Ph. It was found that, at the 48th hour,
the rate of the biodegradation of the phenol in the variant with the added ND was 44.37%
lower than that of the control variant Ph, while at the 214th hour there was an increase of
19.00% in the variant PhND. From the data that are presented in Figure 3, it was found that,
at the 48th hour, the specific rate of the biodegradation of phenol in the variant PhND was
52.38% lower than that of the variant Ph, while at the 214th hour we registered an increase
in the specific rate by 25.71% in the variant PhND. Similar to the RBP, the data showed a
higher SRBP for the 214th hour in the variant with ND. Regarding the EFF, it was found to
be 35.72% lower at the 48th hour than in the control variant Ph, while at the 214th hour it
was found to increase the EFF by 12.75%.
Processes2022,10,xFORPEERREVIEW8of21
Thespecificrateofthebiodegradationofthephenolvalueatthe48thhourforthe
variantPhwas0.211mg/kg∙hand0.100mg/kg∙hforthevariantPhND.At214thhour,the
specificrateforthevariantPhNDwas0.436mg/kg∙hversus0.348mg/kg∙hforthevariant
Ph.ThedatainTable1showthenegativemodulatingeffectofthenanodiamondsonthe
firstphaseofthebiodegradationofthephenolandthepositivemodulatingeffectatthe
endoftheprocess.
Anotherfindingisthattheeffectivenessoftheeliminationofphenolatthe48thhour
waslowerinthePhNDvariant.Thisconfirmsthenegativemodulatingeffectofnanodia‐
mondsontheinitialphase.Attheendoftheprocess(atthe214thhour),theeffectiveness
ofvariantPhwas29.64%andforPhND—33.42%,i.e.,theeffectivenesswasalmostequal
(withadifferenceofabout4%).Theseresultsshowtheloweffectivenessoftheelimination
ofthetoxicpollutant.
Figure3presentstheeffectofnanodiamondsonthekineticparametersofthebio‐
degradationofphenol,comparedtothecontrolvariantPh.Itwasfoundthat,atthe48th
hour,therateofthebiodegradationofthephenolinthevariantwiththeaddedNDwas
44.37%lowerthanthatofthecontrolvariantPh,whileatthe214thhourtherewasan
increaseof19.00%inthevariantPhND.FromthedatathatarepresentedinFigure3,it
wasfoundthat,atthe48thhour,thespecificrateofthebiodegradationofphenolinthe
variantPhNDwas52.38%lowerthanthatofthevariantPh,whileatthe214thhourwe
registeredanincreaseinthespecificrateby25.71%inthevariantPhND.Similartothe
RBP,thedatashowedahigherSRBPforthe214thhourinthevariantwithND.Regarding
theEFF,itwasfoundtobe35.72%loweratthe48thhourthaninthecontrolvariantPh,
whileatthe214thhouritwasfoundtoincreasetheEFFby12.75%.
Figure3.EffectofNDonkineticparameters.
Sofar,itisclearthattheratesandeffectivenessofphenoleliminationatthe48thhour
werelowerthanthoseatthe214thhourinbothoftheexperimentalvariants.Italsobe‐
cameclearthatthenanodiamondsreducedthekineticparametersuptothe48thhourand
increasedthesameparametersofthebiodegradationofphenoluptothe214thhour.At
thesametime,inthemomentsofphenoladdition(theexplosivepollution),thenanodia‐
mondsalmostexpresslyreducetheamountoftheresidualphenolandrelievedthesystem
byslightlyweakeningtheintoxicationshock.
Thenexttwosectionswillconsiderthemicrobialcommunity,theadaptivemecha‐
nismswithwhichitresponded,andhowitchangeditsfunctionalstructurewithinthe
sedimentduringthemodelbioremediationthroughthestudyofclassicalcultivation
Figure 3. Effect of ND on kinetic parameters.
So far, it is clear that the rates and effectiveness of phenol elimination at the 48th
hour were lower than those at the 214th hour in both of the experimental variants. It
also became clear that the nanodiamonds reduced the kinetic parameters up to the 48th
hour and increased the same parameters of the biodegradation of phenol up to the 214th
hour. At the same time, in the moments of phenol addition (the explosive pollution), the
nanodiamonds almost expressly reduce the amount of the residual phenol and relieved the
system by slightly weakening the intoxication shock.
The next two sections will consider the microbial community, the adaptive mechanisms
with which it responded, and how it changed its functional structure within the sediment
during the model bioremediation through the study of classical cultivation techniques and
fluorescent in situ hybridization analysis. Autochthonous microflora play a key role in the
transformation of naturally occurring pollutants in sediments, as well as pollutants that are
of an anthropogenic origin. Sediments accumulate high concentrations of organic matter,
ions and toxic substances that are expected to have a negative effect on microorganisms.
However, the biofilm in the sediments, through the processes of the succession and synergy
of communities, has a high level of bioremediation potential.
Processes 2022,10, 602 9 of 20
3.3. Relationship of Culturable and Non-Culturable Microorganisms during Adaptation
without ND
This section discusses the results of the culture techniques and non-cultivation meth-
ods (FISH methods) that were used for studying the functional diversity of the microorgan-
isms during of model bioremediation of phenol-contaminated sediments. The dynamics
of the key physiological and taxonomic groups of the microorganisms are presented. A
comparison was also made in the restructuring of the microbial communities during the
process with the variant without added nanodiamonds.
Figure 4a shows the quantity of aerobic heterotrophs, as well as the phenol-degrading
microorganisms that were present in the variant without added ND at the 0th, 48th and
214th hours—the three critical points characterizing the process. The number of aero-
bic heterotrophs shows a gradual increase throughout the process. The results for this
group reveal the ability of the microbial sediment community to adapt to the available
toxicant, based on the multiplication of the number of bacterial cells. The phenol-degrading
microorganisms, during of the biodegradation process, increased their quantity despite
intoxication shock (Figure 4). For them, phenol is both a substrate and an inhibitor. These
two effects of phenol on the quantity of phenol-degrading microorganisms are related.
During the adaptation process of sediment microbial communities (from the 48th–214th
hours) these microorganisms increased their number more than 2 times. All of this leads to
the intermediate conclusion that one of the adaptive reactions of the sediment microflora
is an increase in the density of the cultured phenol-degrading microbial segment. In the
microbial consortium at the 214th hour, their number was at its largest. At this time, micro-
bial restructuring processes had already taken place and processes of a specific enzymatic
response to the phenol were likely to take place in the microbial communities. So far,
the data well illustrate the ability of xenobiotic-degrading bacteria to cope with phenol
loading at near-critical concentrations (approximately 280 mg/kg) [
71
,
72
]. This result is
not uncommon, as the intensive biodegradation of 200 mg/kg phenol under anaerobic
conditions within 192 hours has been demonstrated in previous research [
73
]. It should be
noted that the cultivation methods do not reflect the real picture of the metabolic processes
in the microbial community and that these results should be interpreted more fully when
considering the data from the fluorescent in situ hybridization analysis.
Processes2022,10,xFORPEERREVIEW10of21
Figure4.Quantitiesof:(a)aerobicheterotrophicbacteriaandphenol‐degradingbacteriainthevar‐
iantwithoutaddedND;(b)bacteriafromgenusPseudomonasandgenusAcinetobacterinthevariant
withoutaddedND.
InthevariantPh,thequantityofPseudomonasandAcinetobacterbacteriaincreasedat
the48thhour(Figure4b).Thisleadsustoinfertheexclusiveroleofbothtypesofbacteria,
thequantitiesofwhichdeterminetheirmetaboliccapacitytoeliminatepollutantsofdif‐
ferentnaturesinthemicrobialcommunity.Theseresultscoincidewiththeresearchofa
numberofauthors,accordingtowhomtheadaptationofmicrobialcommunitiestothe
biodegradationoftoxicpollutantsinconcentrationsthatareclosetocriticalinvolvesthe
numberofmicroorganismsofthegenusPseudomonasandAcinetobacterincreasing;these
arethemaincomponentoftheculturablexenobiotic‐degradingcomplex[72,74,75].Inour
case,thebacteriaofthegenusAcinetobacterhadanumericaladvantage,duringthewhole
process,overthebacteriaofthegenusPseudomonas.Inbothgroupstherewasadecrease
inthenumberofculturedbacteriaafterthehighintoxicationshockatthe214thhour.
ThebacteriaofthegenusAcinetobacterandespeciallythosewithinthegroupofphe‐
nol‐degradingbacteriaremainedpresentinrelativelylargequantitiesatthe214thhour.
Incontrast,thepseudomonadslosttheirleadingroleinbiodetoxification.Thisisprobably
duetocompetitionforsubstratesandelectronicacceptors.Despitethesmallernumberof
thesebacteria,itispossiblethattheyshowedhigherbiodegradationactivity.Thequantity
ofculturedpseudomonadsmaybereducedbyincreasingtheamountandactivityofnon‐
culturablePseudomonas.Theliteratureshowsthatbothmechanismsareinforceandthat
thesetwomechanismsareusuallycombined[30,72].Thecommunityintensifiesitsbio‐
degradationactivityattheexpenseofthesimultaneousmanifestationofthefollowing
biologicalmechanisms:
(1) Intheearlyphase(atthe48thhour—inthebiodegradationprocesswhileatphenol
concentrationsthatareclosetocritical)ofadaptationtolowphenolconcentrations,
microbialcommunitiesrespondbyincreasingthequantityofmicroorganismsfrom
thekeygroupsforthebiodegradationprocess.
(2) Inthelatephase(aftertheintoxicationshockandthestartofthereadaptationpro‐
cess)afterprolongedadaptationtohighconcentrationsofxenobioticsthatareclose
toacriticallevel,theadaptiveresponseismorecomplexandhighlyspecialized.Syn‐
ergisticrelationshipsandzonesareformedinthecommunitieswithhighoxygenase
enzymeactivity,formationofmaturebiofilmwithzoneswithhighbiodegradation
activityandothersimilarmechanisms.Inthiscase,thequantityofculturablemicro‐
organismsisnottheonlyadaptiveresponse.ForthegeneraPseudomonasandAcineto‐
bacter,themechanismsthatareassociatedwithincreasedactivitytodegradethexe‐
nobioticarevalid.
Figure 4.
Quantities of: (
a
) aerobic heterotrophic bacteria and phenol-degrading bacteria in the
variant without added ND; (
b
) bacteria from genus Pseudomonas and genus Acinetobacter in the
variant without added ND.
In the variant Ph, the quantity of Pseudomonas and Acinetobacter bacteria increased
at the 48th hour (Figure 4b). This leads us to infer the exclusive role of both types of
bacteria, the quantities of which determine their metabolic capacity to eliminate pollutants
of different natures in the microbial community. These results coincide with the research of
a number of authors, according to whom the adaptation of microbial communities to the
Processes 2022,10, 602 10 of 20
biodegradation of toxic pollutants in concentrations that are close to critical involves the
number of microorganisms of the genus Pseudomonas and Acinetobacter increasing; these
are the main component of the culturable xenobiotic-degrading complex [
72
,
74
,
75
]. In our
case, the bacteria of the genus Acinetobacter had a numerical advantage, during the whole
process, over the bacteria of the genus Pseudomonas. In both groups there was a decrease in
the number of cultured bacteria after the high intoxication shock at the 214th hour.
The bacteria of the genus Acinetobacter and especially those within the group of phenol-
degrading bacteria remained present in relatively large quantities at the 214th hour. In
contrast, the pseudomonads lost their leading role in biodetoxification. This is probably
due to competition for substrates and electronic acceptors. Despite the smaller number of
these bacteria, it is possible that they showed higher biodegradation activity. The quantity
of cultured pseudomonads may be reduced by increasing the amount and activity of
non-culturable Pseudomonas. The literature shows that both mechanisms are in force and
that these two mechanisms are usually combined [
30
,
72
]. The community intensifies its
biodegradation activity at the expense of the simultaneous manifestation of the following
biological mechanisms:
(1)
In the early phase (at the 48th hour—in the biodegradation process while at phenol
concentrations that are close to critical) of adaptation to low phenol concentrations,
microbial communities respond by increasing the quantity of microorganisms from
the key groups for the biodegradation process.
(2) In the late phase (after the intoxication shock and the start of the readaptation process)
after prolonged adaptation to high concentrations of xenobiotics that are close to a crit-
ical level, the adaptive response is more complex and highly specialized. Synergistic
relationships and zones are formed in the communities with high oxygenase enzyme
activity, formation of mature biofilm with zones with high biodegradation activity
and other similar mechanisms. In this case, the quantity of culturable microorganisms
is not the only adaptive response. For the genera Pseudomonas and Acinetobacter, the
mechanisms that are associated with increased activity to degrade the xenobiotic
are valid.
The genera Pseudomonas and Acinetobacter were studied by fluorescence in situ hy-
bridization analysis. Bacteria of the genus Pseudomonas and the genus Acinetobacter have
been selected as key microorganisms due to their importance in the biodegradation of
xenobiotics. It is known that microorganisms from these two genera can use alternative
pathways for energy supply and this property gives them more opportunities to develop
resistance and reach their detoxification potential [72].
From the data in Figure 5for the percentage of non-culturable bacteria of the genus
Pseudomonas, in relation to the total number of microorganisms that were stained with
DAPI, it was found that at the beginning of the process at the 0th hour the non-culturable
Pseudomonas bacteria numbered 11.46%, while the non-culturable Acinetobacter numbered
16.61%. These results from the beginning of the process are correlated with the results for
the non-culturable bacteria of these two genera, where bacteria of the genus Acinetobacter
were dominant. At the 48th hour, the pseudomonads responded with an increase in
their percentage, reaching 22.81%, while the genus Acinetobacter decreased its presence
to 7.95%. After the explosive addition of phenol at the 72nd hour and after a certain
period of readaptation of the sedimentary microbial communities, at the 144th hour there
was a change in the dominant genus. Bacteria of the genus Acinetobacter increased their
percentage to 17.72%. Pseudomonads decreased their presence to 9.52%. At the end of the
study process at the 214th hour, the genus Acinetobacter maintained its higher percentage
by 15.57%, compared to 6.99% for the genus Pseudomonas. These results are identical to
those that were obtained for the culturable bacteria, where again the genus Acinetobacter
had a leading role in the biodegradation of the phenol.
Processes 2022,10, 602 11 of 20
Processes2022,10,xFORPEERREVIEW11of21
ThegeneraPseudomonasandAcinetobacterwerestudiedbyfluorescenceinsituhy‐
bridizationanalysis.BacteriaofthegenusPseudomonasandthegenusAcinetobacterhave
beenselectedaskeymicroorganismsduetotheirimportanceinthebiodegradationof
xenobiotics.Itisknownthatmicroorganismsfromthesetwogeneracanusealternative
pathwaysforenergysupplyandthispropertygivesthemmoreopportunitiestodevelop
resistanceandreachtheirdetoxificationpotential[72].
FromthedatainFigure5forthepercentageofnon‐culturablebacteriaofthegenus
Pseudomonas,inrelationtothetotalnumberofmicroorganismsthatwerestainedwith
DAPI,itwasfoundthatatthebeginningoftheprocessatthe0thhourthenon‐culturable
Pseudomonasbacterianumbered11.46%,whilethenon‐culturableAcinetobacternumbered
16.61%.Theseresultsfromthebeginningoftheprocessarecorrelatedwiththeresultsfor
thenon‐culturablebacteriaofthesetwogenera,wherebacteriaofthegenusAcinetobacter
weredominant.Atthe48thhour,thepseudomonadsrespondedwithanincreaseintheir
percentage,reaching22.81%,whilethegenusAcinetobacterdecreaseditspresenceto
7.95%.Aftertheexplosiveadditionofphenolatthe72ndhourandafteracertainperiod
ofreadaptationofthesedimentarymicrobialcommunities,atthe144thhourtherewasa
changeinthedominantgenus.BacteriaofthegenusAcinetobacterincreasedtheirpercent‐
ageto17.72%.Pseudomonadsdecreasedtheirpresenceto9.52%.Attheendofthestudy
processatthe214thhour,thegenusAcinetobactermaintaineditshigherpercentageby
15.57%,comparedto6.99%forthegenusPseudomonas.Theseresultsareidenticaltothose
thatwereobtainedfortheculturablebacteria,whereagainthegenusAcinetobacterhada
leadingroleinthebiodegradationofthephenol.
time, h
0 24 48 72 96 120 144 168 192 216
% / All microorga nis ms
0
10
20
30
40
50
60
70
80
90
100
phenol, mg/kg
0
200
400
600
800
Pseudomonas
Acinetobacter
Phenol
Figure5.Percentageofnon‐culturablebacteriaofthegenusPseudomonasandgenusAcinetobacter
andresidualphenolconcentrationinthevariantwithoutaddedND.
3.4.RelationshipofCulturableandNon‐CulturableMicroorganismsduringAdaptationwiththe
ParticipationofND
Thequantityofaerobicheterotrophsatthe0thand48thhourswasfoundtobehigher
inthePhNDvariant,aresultwhichwasprobablyinfluencedbytheNDthatwereadded
tothesystem(Figure6a).Thispeakinthequantityofaerobicheterotrophswasfollowed
byasharpdecreaseatthe214thhour.ThestimulatingeffectoftheNDatthe48thhour
wasduetothefactthattherewasahighconcentrationoftrivialsubstratesinthesystem.
Ontheotherhand,duetotheadsorptionofphenolontheNDandinthe“micelles”its
Figure 5.
Percentage of non-culturable bacteria of the genus Pseudomonas and genus Acinetobacter
and residual phenol concentration in the variant without added ND.
3.4. Relationship of Culturable and Non-Culturable Microorganisms during Adaptation with the
Participation of ND
The quantity of aerobic heterotrophs at the 0th and 48th hours was found to be higher
in the PhND variant, a result which was probably influenced by the ND that were added
to the system (Figure 6a). This peak in the quantity of aerobic heterotrophs was followed
by a sharp decrease at the 214th hour. The stimulating effect of the ND at the 48th hour
was due to the fact that there was a high concentration of trivial substrates in the system.
On the other hand, due to the adsorption of phenol on the ND and in the “micelles” its
toxic effect on the other microorganisms, in this case the complex of aerobic heterotrophs,
was reduced.
Processes2022,10,xFORPEERREVIEW12of21
toxiceffectontheothermicroorganisms,inthiscasethecomplexofaerobicheterotrophs,
wasreduced.
Figure6.Quantitiesof:(a)aerobicheterotrophicbacteriaandphenol‐degradingbacteriainthevar‐
iantwithoutaddedND;(b)culturablebacteriaofthegenusPseudomonasandthegenusAcinetobacter
inthevariantwithaddedND.
Thephenol‐degradingmicroorganisms,duringthebiodegradationprocess,in‐
creasedtheirquantity,despitetheintoxicationshock(Figure6a).Inthemicrobialconsor‐
tiumatthe214thhour,theirnumberwasthelargestinbothvariants.Atthistime,micro‐
bialrestructuringprocesseshadalreadytakenplaceandprocessesofaspecificenzymatic
responsetophenolwerelikelytotakeplaceinthemicrobialcommunities.Inthecontrol
variantPh,thequantityofculturedphenol‐degradingmicroorganismsexceededthat
whichwaspresentinthevariantwithNDatthe48thand214thhours.Itshouldbenoted
thattheculturemethodsdonotreflecttherealsituationofthemetabolicprocessesinthe
microbialcommunityandthattheseresultsshouldbeinterpretedmorefullyinthesection
ontheapplicationoffluorescentinsituhybridizationanalysis.
ThedatathatarepresentedinFigure6balsoestablishtheinfluenceofNDontwo
importanttaxonomicgroupsofbacteria—PseudomonasandAcinetobacter.Nanodiamonds,
atthe48thhour,hadapositiveeffectonthedevelopmentofbacteriaofthegenusAcineto‐
bacter,butatthe214thhourtheirquantitywasnegativelyaffectedinthecontrolvariant
Ph.Atthe48thhourtherewasapeakinthequantityofAcinetobacterandadecreaseinthe
numberofbacteriaatthe214thhour.Thispeakwasprobablyduetotheincreasedbioa‐
vailabilityofpollutantsinthesystem,bothtrivialandxenobiotic.Inthiscase,NDledto
thestrongstimulationofthebacteriaofthegenusAcinetobacteratthe48thhourandtheir
inhibitionatthe214thhour,whilethenanoparticlescausedadecreaseinthebacteriaof
thegenusPseudomonasatthe48thhourandhadalmostnoeffectontheiramountatthe
214thhour.Thechangeinthedominantroleofthesetwobacterialgeneraintermsofthe
characteristicsofthedetoxificationprocesswasagainconfirmed.Here,mostlikely,the
specificmechanismsthatarearesponsetocommunityadaptationhavebeenactivated.
However,herewehavetoemphasize—theseareonlyculturablebacteriafrombothgen‐
era.
Asabriefsummary,whenmonitoringthequantityofthebacteriafromthexenobi‐
otic‐degradingcomplex(genusPseudomonasandgenusAcinetobacteraswellasphenol‐
degradingmicroorganisms)itwasfoundthat,aftertheexplosiveloadinthe48thhour,
thebacterialsegmentchangedasfollows:
Figure 6.
Quantities of: (
a
) aerobic heterotrophic bacteria and phenol-degrading bacteria in the variant
without added ND; (
b
) culturable bacteria of the genus Pseudomonas and the genus Acinetobacter in
the variant with added ND.
The phenol-degrading microorganisms, during the biodegradation process, increased
their quantity, despite the intoxication shock (Figure 6a). In the microbial consortium at
the 214th hour, their number was the largest in both variants. At this time, microbial
Processes 2022,10, 602 12 of 20
restructuring processes had already taken place and processes of a specific enzymatic
response to phenol were likely to take place in the microbial communities. In the control
variant Ph, the quantity of cultured phenol-degrading microorganisms exceeded that which
was present in the variant with ND at the 48th and 214th hours. It should be noted that the
culture methods do not reflect the real situation of the metabolic processes in the microbial
community and that these results should be interpreted more fully in the section on the
application of fluorescent in situ hybridization analysis.
The data that are presented in Figure 6b also establish the influence of ND on two
important taxonomic groups of bacteria—Pseudomonas and Acinetobacter. Nanodiamonds, at
the 48th hour, had a positive effect on the development of bacteria of the genus Acinetobacter,
but at the 214th hour their quantity was negatively affected in the control variant Ph. At the
48th hour there was a peak in the quantity of Acinetobacter and a decrease in the number
of bacteria at the 214th hour. This peak was probably due to the increased bioavailability
of pollutants in the system, both trivial and xenobiotic. In this case, ND led to the strong
stimulation of the bacteria of the genus Acinetobacter at the 48th hour and their inhibition
at the 214th hour, while the nanoparticles caused a decrease in the bacteria of the genus
Pseudomonas at the 48th hour and had almost no effect on their amount at the 214th hour. The
change in the dominant role of these two bacterial genera in terms of the characteristics of
the detoxification process was again confirmed. Here, most likely, the specific mechanisms
that are a response to community adaptation have been activated. However, here we have
to emphasize—these are only culturable bacteria from both genera.
As a brief summary, when monitoring the quantity of the bacteria from the xenobiotic-
degrading complex (genus Pseudomonas and genus Acinetobacter as well as phenol-degrading
microorganisms) it was found that, after the explosive load in the 48th hour, the bacterial
segment changed as follows:
•
The genus Pseudomonas reduced their quantity. In this standard method of studying
microbial cenosis, ND inhibited the growth and adaptation of culturable bacteria of
this genus within 48 h.
•
At the 48th hour, the other two physiological and taxonomic groups increased their
populations of culturable bacteria, namely Acinetobacter and the phenol-degrading
microorganisms. Bacteria of the genus Acinetobacter played a leading role in the
bioremediation of the phenol-contaminated sediments. These bacteria have occupied
this ecological niche and there was an increase in their population compared to the
observed decline in the quantity of bacteria of the genus Pseudomonas.
From the data that are presented in Figure 7, the percentage of non-culturable bacteria
from Acinetobacter was found to be 16.61% at the 0th hour, while for Pseudomonas it was
11.47%. These results for the start of the process are correlated with the results for culturable
bacteria of these two genera, where the dominant bacteria were Acinetobacter. At the 48th
hour, the pseudomonads responded by reducing their percentage to 9.40%, while the
Acinetobacter decreased to 15.80%. After the explosive addition of phenol at the 72th hour
and after a certain period of readaptation of the sediment microbial communities at the
144th hour, the genus Acinetobacter’s tendency to predominate was preserved. The bacteria
of the genus Acinetobacter increased their percentage to 24.68% at the 144th hour. The
pseudomonads also increased their percentage to 11.21% by the 144th hour. At the end
of the process at the 214th hour, genus Pseudomonas played a dominant role at 20.69%
compared to 11.66% for the genus Acinetobacter. These results are in contrast to those
of the culturable bacteria, where the genus Acinetobacter played a leading role in the
biodegradation of the phenol at the end of the process.
Processes 2022,10, 602 13 of 20
Processes2022,10,xFORPEERREVIEW13of21
ThegenusPseudomonasreducedtheirquantity.Inthisstandardmethodofstudying
microbialcenosis,NDinhibitedthegrowthandadaptationofculturablebacteriaof
thisgenuswithin48hours.
Atthe48thhour,theothertwophysiologicalandtaxonomicgroupsincreasedtheir
populationsofculturablebacteria,namelyAcinetobacterandthephenol‐degrading
microorganisms.BacteriaofthegenusAcinetobacterplayedaleadingroleinthebio‐
remediationofthephenol‐contaminatedsediments.Thesebacteriahaveoccupied
thisecologicalnicheandtherewasanincreaseintheirpopulationcomparedtothe
observeddeclineinthequantityofbacteriaofthegenusPseudomonas.
FromthedatathatarepresentedinFigure7,thepercentageofnon‐culturablebacte‐
riafromAcinetobacterwasfoundtobe16.61%atthe0thhour,whileforPseudomonasitwas
11.47%.Theseresultsforthestartoftheprocessarecorrelatedwiththeresultsforcultura‐
blebacteriaofthesetwogenera,wherethedominantbacteriawereAcinetobacter.Atthe
48thhour,thepseudomonadsrespondedbyreducingtheirpercentageto9.40%,whilethe
Acinetobacterdecreasedto15.80%.Aftertheexplosiveadditionofphenolatthe72thhour
andafteracertainperiodofreadaptationofthesedimentmicrobialcommunitiesatthe
144thhour,thegenusAcinetobacterʹstendencytopredominatewaspreserved.Thebacte‐
riaofthegenusAcinetobacterincreasedtheirpercentageto24.68%atthe144thhour.The
pseudomonadsalsoincreasedtheirpercentageto11.21%bythe144thhour.Attheendof
theprocessatthe214thhour,genusPseudomonasplayedadominantroleat20.69%com‐
paredto11.66%forthegenusAcinetobacter.Theseresultsareincontrasttothoseofthe
culturablebacteria,wherethegenusAcinetobacterplayedaleadingroleinthebiodegra‐
dationofthephenolattheendoftheprocess.
time, h
0 24 48 72 96 120 144 168 192 216
% / All microorganisms
0
10
20
30
40
50
60
70
80
90
100
phenol, mg/kg
0
200
400
600
800
Pseudomo nas
Acinetobacter
Phenol
Figure7.Percentageofnon‐culturablebacteriaofthegenusPseudomonasandgenusAcinetobacter
andresidualphenolconcentrationinthevariantwithaddedND.
3.5.EffectofNDontheRestructuringoftheMicrobialCommunity
Table3presentstheeffectofNDontheamountsofthekeygroupsofmicroorganisms
thatwerepresentatthe48thhourofthebiodegradationprocesscomparedtothosethat
werepresentatthesametimeinthecontrolvariantPh.Inthisphase,thenanomodulator,
aswehavealreadyestablished,hadthestrongestinfluenceontheinitialadaptationtothe
degradationofpollutantsandhadapositiveeffectonthequantitativegrowthoftheaer‐
obicheterotrophsandbacteriaofthegenusAcinetobacter.Theaerobicheterotrophsand
Figure 7.
Percentage of non-culturable bacteria of the genus Pseudomonas and genus Acinetobacter
and residual phenol concentration in the variant with added ND.
3.5. Effect of ND on the Restructuring of the Microbial Community
Table 3presents the effect of ND on the amounts of the key groups of microorganisms
that were present at the 48th hour of the biodegradation process compared to those that
were present at the same time in the control variant Ph. In this phase, the nanomodulator,
as we have already established, had the strongest influence on the initial adaptation to
the degradation of pollutants and had a positive effect on the quantitative growth of the
aerobic heterotrophs and bacteria of the genus Acinetobacter. The aerobic heterotrophs
and bacteria of the genus Acinetobacter had the largest percentage increases, 33.87% and
74.65%, respectively.
Table 3.
Effect of ND on the amount of culturable key groups of microorganisms at 48 h and 214 h of
the bioremediation process (expressed in %).
Groups of M.O. Change (%) versus Variant
Ph at 48 h
Change (%)
versus Variant Ph
at 214 h
AeH +33.87% −19.45%
Ps. −76.30% +71.83%
Ac. +74.65% −37.58%
Ph.degr. −60.61% −75.35%
The largest decrease was registered in the percentage of the bacteria of the genus
Pseudomonas (
−
76.30%) and the phenol-degrading microorganisms (
−
60.61%) (Table 3).
Phenol-degrading microorganisms and especially bacteria of the genus Pseudomonas have a
plastic metabolism for the degradation of trivial organics and they play a key role in the
degradation of xenobiotics [
72
,
76
,
77
]. In this case, in microbial restructuring, ND activate
the degradation of organic matter by heterotrophic microorganisms and ND have positively
stimulated bacteria of the genus Acinetobacter from the xenobiotic-degrading complex.
Under the modulating effect of the ND, the largest percentage negative change in the
214th hour was registered by the phenol-degrading bacteria (
−
75.35%) (Table 3). At the
same time, the aerobic heterotrophs decreased by
−
19.45% compared to the control variant
Ph (Table 3).
At the 214th hour, the ND stimulated the Pseudomonas bacteria by 71.83%. At the same
time (the 214th hour), the phenol-degrading microorganisms and bacteria of the genus
Processes 2022,10, 602 14 of 20
Acinetobacter decreased by 75.35% and 37.58%, respectively. Bacteria of the genus Pseu-
domonas are also involved in the processes of organic transformation, but here they probably
played a significant role in the biodegradation of phenol when the trivial substrates were
depleted (Table 3).
During the experiment, dynamic changes in the development and restructuring of
the microbial community were found in both of the variants. The effect of the ND, as
compared to the control variant, was registered in the 48th hour within bacteria of the
genus Acinetobacter (+74.65%) and the aerobic heterotrophic bacteria increased by about 30%.
At the 214th hour, the bacteria of the genus Pseudomonas increased by 71.83%, compared to
the control variant. These changes are related to microheterogeneity, due to nanoparticles,
oxygen access, the number of bacteria and the influence of the toxic agent.
The discussed microbiological dependences are reflected and associated with the
changes in the kinetic parameters of the process of biodegradation of phenol and the
bioremediation of the sediments and also with the changes in the enzymatic profile; the
role of non-culturable microorganisms in sedimentary communities [
72
]. Comprehensive
interpretation of this information is a key tool for use in future algorithms for managing
risk situations stemming from the explosive and accumulative loading of sediments with
toxic pollutants.
From the data that are presented in Figure 8, it could be concluded that the nanodia-
monds had a positive effect on the density of the microorganisms of the genus Pseudomonas
at the 144th and 214th hour (+17.71% and +195.54%, respectively). At the end of the
biodegradation process, a peak in the density of bacteria of the genus Pseudomonas was
reached, which means that the system had already adapted and could degrade the phenol
that was present in the sediments. The ND helped the microbial system to stay active for
longer, i.e., there was an increase in the readiness of the pseudomonads to degrade the
toxicant. In the control variant, the density was highest at the 48th hour, but the distribution
of the microorganisms was uneven, without the clear formation of microbial structures.
These structures and clusters are the most likely places (zones), or micro-niches, where
phenolic biodegradation is enhanced at the expense of the synergetic relationships that
are formed there. These assumptions are supported by the kinetic parameters of phenolic
biodegradation, enzyme activity and other data from researchers working with amaranth,
pentachlorophenol and other toxic pollutants [72,74,75,78].
Processes2022,10,xFORPEERREVIEW15of21
distributionofthemicroorganismswasuneven,withouttheclearformationofmicrobial
structures.Thesestructuresandclustersarethemostlikelyplaces(zones),ormicro‐
niches,wherephenolicbiodegradationisenhancedattheexpenseofthesynergeticrela‐
tionshipsthatareformedthere.Theseassumptionsaresupportedbythekineticparame‐
tersofphenolicbiodegradation,enzymeactivityandotherdatafromresearchersworking
withamaranth,pentachlorophenolandothertoxicpollutants[72,74,75,78].
Figure8.EffectofNDonnon‐culturablebacteriaofthegenusPseudomonasandthegenusAcineto‐
bacter.
FromthedatathatarepresentedinFigure8,itcouldbeconcludedthattheNDhad
apositiveeffectonthedensityofAcinetobactermicroorganismsatthe48thand144th
hours.ComparingtheresultsthatarepresentedinFigure8,itcanbeconcludedthata
clearsynergismwasregisteredinthehoursofthestrongestadaptationofthebacteriaof
thetwotargetgeneraintheNDvariant.TheNDhadtheirstrongesteffectonthegenus
Acinetobacteratthe48thhour,whileforbacteriaofthegenusPseudomonastheeffectwas
thestrongestatthe214thhourTheseresultscanbeconsideredasaresponsetothemicro‐
bialsystem,whichthroughitssmoothrestructuringadaptstotoxicshock.Thedevelop‐
mentofthissynergismandmetabolicsuccessionintheactivityofthetwotargetgenera
canbeconsideredasasuccessfuladaptationofthemicrobialcommunitytoeffectively
copewiththetoxiceffectsofhighconcentrationsofphenolanditsadaptationtosubse‐
quentbiodegradation.
JudgingbythephotographicmaterialdetailingthebacteriaofthegenusPseudomonas
atthe48thhourinthePhNDvariant,accumulationsofasmallsizeandratherdispersed
locationsofthebacteriaofthetargetgenuswerereported(Table4).Atthe144thhour,the
genusPseudomonasshowedstrongerfluorescence(comparedtothe48thhour),corre‐
spondingtoitsincreasedmetabolicactivity.Clustersofincreasedsizewerefound,which
isanindicationofthedevelopmentofcooperativeandsynergeticrelationships.These
relationshipsareconnectedwithimprovedandincreasedtargetactivity.Inthiscasethis
activitywastheincreaseddetoxificationofphenol.Atthe214thhour,well‐formedaccu‐
mulationsofmicroorganismswereestablished—anindicationofestablishedsynergetic
relationshipsinthebiodegradationcommunity.Attheendoftheexperiment,high‐inten‐
sityfluorescencewasdetectedinthePhNDvariant,whichindicateshighlevelsofdensity
andactivityofPseudomonasspp.inthemicrobialcommunity.Atthe214thhour,aninverse
relationshipwasfoundwhencomparingtheculturedandnon‐culturedmicroorganisms
(Figure9).Pseudomonasspp.predominatedamongthenon‐culturableones,whileAcineto‐
bacterspp.predominatedamongtheculturableones(Figure9).
Figure 8. Effect of ND on non-culturable bacteria of the genus Pseudomonas and the genus Acinetobacter.
From the data that are presented in Figure 8, it could be concluded that the ND had a
positive effect on the density of Acinetobacter microorganisms at the 48th and 144th hours.
Comparing the results that are presented in Figure 8, it can be concluded that a clear
synergism was registered in the hours of the strongest adaptation of the bacteria of the two
target genera in the ND variant. The ND had their strongest effect on the genus Acinetobacter
Processes 2022,10, 602 15 of 20
at the 48th hour, while for bacteria of the genus Pseudomonas the effect was the strongest at
the 214th hour These results can be considered as a response to the microbial system, which
through its smooth restructuring adapts to toxic shock. The development of this synergism
and metabolic succession in the activity of the two target genera can be considered as a
successful adaptation of the microbial community to effectively cope with the toxic effects
of high concentrations of phenol and its adaptation to subsequent biodegradation.
Judging by the photographic material detailing the bacteria of the genus Pseudomonas
at the 48th hour in the PhND variant, accumulations of a small size and rather dispersed
locations of the bacteria of the target genus were reported (Table 4). At the 144th hour, the
genus Pseudomonas showed stronger fluorescence (compared to the 48th hour), correspond-
ing to its increased metabolic activity. Clusters of increased size were found, which is an
indication of the development of cooperative and synergetic relationships. These relation-
ships are connected with improved and increased target activity. In this case this activity
was the increased detoxification of phenol. At the 214th hour, well-formed accumulations
of microorganisms were established—an indication of established synergetic relationships
in the biodegradation community. At the end of the experiment, high-intensity fluorescence
was detected in the PhND variant, which indicates high levels of density and activity of
Pseudomonas spp. in the microbial community. At the 214th hour, an inverse relationship
was found when comparing the cultured and non-cultured microorganisms (Figure 9).
Pseudomonas spp. predominated among the non-culturable ones, while Acinetobacter spp.
predominated among the culturable ones (Figure 9).
Table 4.
Fluorescent images of the microbial community for the variant PhND (400
×
). Subjects were
represented by hybridization with an oligonucleotide probe specific to the genus Pseudomonas (red)
and the genus Acinetobacter (yellow).
2nd hour 48th hour 144th hour 214th hour
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Table4.FluorescentimagesofthemicrobialcommunityforthevariantPhND(400X).Subjectswere
representedbyhybridizationwithanoligonucleotideprobespecifictothegenusPseudomonas(red)
andthegenusAcinetobacter(yellow).
2ndhour48thhour144thhour214thhour
Ps./FISH/‐2ndhour
Ps./FISH/‐48thhour
Ps./FISH/‐144thhour
Ps./FISH/‐214thhour
Ac./FISH/‐2ndhour
Ac./FISH/‐48thhour
Ac./FISH/‐144thhour
Ac./FISH/‐214thhour
Figure9.Comparisonbetweenculturableandnon‐culturablebacteriaofthegenusPseudomonasand
thegenusAcinetobacterinthevariantPhND.
ThecorrespondingphotographicmaterialforthegenusAcinetobacterrecordedlow
fluorescenceatthe2ndhour,correspondingtolowmetabolicactivityofthetargetgroup
(Table4).Atthe48thhour,arelativelystrongfluorescenceandstrongclusteringofthe
genusAcinetobacterwasrecorded,showingtheactivationofthepopulationofthese
microorganismsinthebiofilm.Anincreaseinthequantityofthetargetmicroorganisms
wasobservedatthe144thhour,buttheclusteringwasnotascompactasitwasatthe48th
hour.ItcanbeassumedthatatthistimebacteriaofthegenusAcinetobacterhadhigh
activitywithrespecttothebiodegradationofthexenobiotic,butthesynergistic
relationshipsinthisgroupwerenotsowellestablished.Anexplanationcanbefoundin
Ps. /FISH/- 2nd hour
Processes2022,10,xFORPEERREVIEW16of21
Table4.FluorescentimagesofthemicrobialcommunityforthevariantPhND(400X).Subjectswere
representedbyhybridizationwithanoligonucleotideprobespecifictothegenusPseudomonas(red)
andthegenusAcinetobacter(yellow).
2ndhour48thhour144thhour214thhour
Ps./FISH/‐2ndhour
Ps./FISH/‐48thhour
Ps./FISH/‐144thhour
Ps./FISH/‐214thhour
Ac./FISH/‐2ndhour
Ac./FISH/‐48thhour
Ac./FISH/‐144thhour
Ac./FISH/‐214thhour
Figure9.Comparisonbetweenculturableandnon‐culturablebacteriaofthegenusPseudomonasand
thegenusAcinetobacterinthevariantPhND.
ThecorrespondingphotographicmaterialforthegenusAcinetobacterrecordedlow
fluorescenceatthe2ndhour,correspondingtolowmetabolicactivityofthetargetgroup
(Table4).Atthe48thhour,arelativelystrongfluorescenceandstrongclusteringofthe
genusAcinetobacterwasrecorded,showingtheactivationofthepopulationofthese
microorganismsinthebiofilm.Anincreaseinthequantityofthetargetmicroorganisms
wasobservedatthe144thhour,buttheclusteringwasnotascompactasitwasatthe48th
hour.ItcanbeassumedthatatthistimebacteriaofthegenusAcinetobacterhadhigh
activitywithrespecttothebiodegradationofthexenobiotic,butthesynergistic
relationshipsinthisgroupwerenotsowellestablished.Anexplanationcanbefoundin
Ps. /FISH/-48th hour
Processes2022,10,xFORPEERREVIEW16of21
Table4.FluorescentimagesofthemicrobialcommunityforthevariantPhND(400X).Subjectswere
representedbyhybridizationwithanoligonucleotideprobespecifictothegenusPseudomonas(red)
andthegenusAcinetobacter(yellow).
2ndhour48thhour144thhour214thhour
Ps./FISH/‐2ndhour
Ps./FISH/‐48thhour
Ps./FISH/‐144thhour
Ps./FISH/‐214thhour
Ac./FISH/‐2ndhour
Ac./FISH/‐48thhour
Ac./FISH/‐144thhour
Ac./FISH/‐214thhour
Figure9.Comparisonbetweenculturableandnon‐culturablebacteriaofthegenusPseudomonasand
thegenusAcinetobacterinthevariantPhND.
ThecorrespondingphotographicmaterialforthegenusAcinetobacterrecordedlow
fluorescenceatthe2ndhour,correspondingtolowmetabolicactivityofthetargetgroup
(Table4).Atthe48thhour,arelativelystrongfluorescenceandstrongclusteringofthe
genusAcinetobacterwasrecorded,showingtheactivationofthepopulationofthese
microorganismsinthebiofilm.Anincreaseinthequantityofthetargetmicroorganisms
wasobservedatthe144thhour,buttheclusteringwasnotascompactasitwasatthe48th
hour.ItcanbeassumedthatatthistimebacteriaofthegenusAcinetobacterhadhigh
activitywithrespecttothebiodegradationofthexenobiotic,butthesynergistic
relationshipsinthisgroupwerenotsowellestablished.Anexplanationcanbefoundin
Ps. /FISH/-144th hour
Processes2022,10,xFORPEERREVIEW16of21
Table4.FluorescentimagesofthemicrobialcommunityforthevariantPhND(400X).Subjectswere
representedbyhybridizationwithanoligonucleotideprobespecifictothegenusPseudomonas(red)
andthegenusAcinetobacter(yellow).
2ndhour48thhour144thhour214thhour
Ps./FISH/‐2ndhour
Ps./FISH/‐48thhour
Ps./FISH/‐144thhour
Ps./FISH/‐214thhour
Ac./FISH/‐2ndhour
Ac./FISH/‐48thhour
Ac./FISH/‐144thhour
Ac./FISH/‐214thhour
Figure9.Comparisonbetweenculturableandnon‐culturablebacteriaofthegenusPseudomonasand
thegenusAcinetobacterinthevariantPhND.
ThecorrespondingphotographicmaterialforthegenusAcinetobacterrecordedlow
fluorescenceatthe2ndhour,correspondingtolowmetabolicactivityofthetargetgroup
(Table4).Atthe48thhour,arelativelystrongfluorescenceandstrongclusteringofthe
genusAcinetobacterwasrecorded,showingtheactivationofthepopulationofthese
microorganismsinthebiofilm.Anincreaseinthequantityofthetargetmicroorganisms
wasobservedatthe144thhour,buttheclusteringwasnotascompactasitwasatthe48th
hour.ItcanbeassumedthatatthistimebacteriaofthegenusAcinetobacterhadhigh
activitywithrespecttothebiodegradationofthexenobiotic,butthesynergistic
relationshipsinthisgroupwerenotsowellestablished.Anexplanationcanbefoundin
Ps. /FISH/-214th hour
Processes2022,10,xFORPEERREVIEW16of21
Table4.FluorescentimagesofthemicrobialcommunityforthevariantPhND(400X).Subjectswere
representedbyhybridizationwithanoligonucleotideprobespecifictothegenusPseudomonas(red)
andthegenusAcinetobacter(yellow).
2ndhour48thhour144thhour214thhour
Ps./FISH/‐2ndhour
Ps./FISH/‐48thhour
Ps./FISH/‐144thhour
Ps./FISH/‐214thhour
Ac./FISH/‐2ndhour
Ac./FISH/‐48thhour
Ac./FISH/‐144thhour
Ac./FISH/‐214thhour
Figure9.Comparisonbetweenculturableandnon‐culturablebacteriaofthegenusPseudomonasand
thegenusAcinetobacterinthevariantPhND.
ThecorrespondingphotographicmaterialforthegenusAcinetobacterrecordedlow
fluorescenceatthe2ndhour,correspondingtolowmetabolicactivityofthetargetgroup
(Table4).Atthe48thhour,arelativelystrongfluorescenceandstrongclusteringofthe
genusAcinetobacterwasrecorded,showingtheactivationofthepopulationofthese
microorganismsinthebiofilm.Anincreaseinthequantityofthetargetmicroorganisms
wasobservedatthe144thhour,buttheclusteringwasnotascompactasitwasatthe48th
hour.ItcanbeassumedthatatthistimebacteriaofthegenusAcinetobacterhadhigh
activitywithrespecttothebiodegradationofthexenobiotic,butthesynergistic
relationshipsinthisgroupwerenotsowellestablished.Anexplanationcanbefoundin
Ac. /FISH/-2nd hour
Processes2022,10,xFORPEERREVIEW16of21
Table4.FluorescentimagesofthemicrobialcommunityforthevariantPhND(400X).Subjectswere
representedbyhybridizationwithanoligonucleotideprobespecifictothegenusPseudomonas(red)
andthegenusAcinetobacter(yellow).
2ndhour48thhour144thhour214thhour
Ps./FISH/‐2ndhour
Ps./FISH/‐48thhour
Ps./FISH/‐144thhour
Ps./FISH/‐214thhour
Ac./FISH/‐2ndhour
Ac./FISH/‐48thhour
Ac./FISH/‐144thhour
Ac./FISH/‐214thhour
Figure9.Comparisonbetweenculturableandnon‐culturablebacteriaofthegenusPseudomonasand
thegenusAcinetobacterinthevariantPhND.
ThecorrespondingphotographicmaterialforthegenusAcinetobacterrecordedlow
fluorescenceatthe2ndhour,correspondingtolowmetabolicactivityofthetargetgroup
(Table4).Atthe48thhour,arelativelystrongfluorescenceandstrongclusteringofthe
genusAcinetobacterwasrecorded,showingtheactivationofthepopulationofthese
microorganismsinthebiofilm.Anincreaseinthequantityofthetargetmicroorganisms
wasobservedatthe144thhour,buttheclusteringwasnotascompactasitwasatthe48th
hour.ItcanbeassumedthatatthistimebacteriaofthegenusAcinetobacterhadhigh
activitywithrespecttothebiodegradationofthexenobiotic,butthesynergistic
relationshipsinthisgroupwerenotsowellestablished.Anexplanationcanbefoundin
Ac. /FISH/-48th hour
Processes2022,10,xFORPEERREVIEW16of21
Table4.FluorescentimagesofthemicrobialcommunityforthevariantPhND(400X).Subjectswere
representedbyhybridizationwithanoligonucleotideprobespecifictothegenusPseudomonas(red)
andthegenusAcinetobacter(yellow).
2ndhour48thhour144thhour214thhour
Ps./FISH/‐2ndhour
Ps./FISH/‐48thhour
Ps./FISH/‐144thhour
Ps./FISH/‐214thhour
Ac./FISH/‐2ndhour
Ac./FISH/‐48thhour
Ac./FISH/‐144thhour
Ac./FISH/‐214thhour
Figure9.Comparisonbetweenculturableandnon‐culturablebacteriaofthegenusPseudomonasand
thegenusAcinetobacterinthevariantPhND.
ThecorrespondingphotographicmaterialforthegenusAcinetobacterrecordedlow
fluorescenceatthe2ndhour,correspondingtolowmetabolicactivityofthetargetgroup
(Table4).Atthe48thhour,arelativelystrongfluorescenceandstrongclusteringofthe
genusAcinetobacterwasrecorded,showingtheactivationofthepopulationofthese
microorganismsinthebiofilm.Anincreaseinthequantityofthetargetmicroorganisms
wasobservedatthe144thhour,buttheclusteringwasnotascompactasitwasatthe48th
hour.ItcanbeassumedthatatthistimebacteriaofthegenusAcinetobacterhadhigh
activitywithrespecttothebiodegradationofthexenobiotic,butthesynergistic
relationshipsinthisgroupwerenotsowellestablished.Anexplanationcanbefoundin
Ac. /FISH/-144th hour
Processes2022,10,xFORPEERREVIEW16of21
Table4.FluorescentimagesofthemicrobialcommunityforthevariantPhND(400X).Subjectswere
representedbyhybridizationwithanoligonucleotideprobespecifictothegenusPseudomonas(red)
andthegenusAcinetobacter(yellow).
2ndhour48thhour144thhour214thhour
Ps./FISH/‐2ndhour
Ps./FISH/‐48thhour
Ps./FISH/‐144thhour
Ps./FISH/‐214thhour
Ac./FISH/‐2ndhour
Ac./FISH/‐48thhour
Ac./FISH/‐144thhour
Ac./FISH/‐214thhour
Figure9.Comparisonbetweenculturableandnon‐culturablebacteriaofthegenusPseudomonasand
thegenusAcinetobacterinthevariantPhND.
ThecorrespondingphotographicmaterialforthegenusAcinetobacterrecordedlow
fluorescenceatthe2ndhour,correspondingtolowmetabolicactivityofthetargetgroup
(Table4).Atthe48thhour,arelativelystrongfluorescenceandstrongclusteringofthe
genusAcinetobacterwasrecorded,showingtheactivationofthepopulationofthese
microorganismsinthebiofilm.Anincreaseinthequantityofthetargetmicroorganisms
wasobservedatthe144thhour,buttheclusteringwasnotascompactasitwasatthe48th
hour.ItcanbeassumedthatatthistimebacteriaofthegenusAcinetobacterhadhigh
activitywithrespecttothebiodegradationofthexenobiotic,butthesynergistic
relationshipsinthisgroupwerenotsowellestablished.Anexplanationcanbefoundin
Ac. /FISH/-214th hour
The corresponding photographic material for the genus Acinetobacter recorded low
fluorescence at the 2nd hour, corresponding to low metabolic activity of the target group
(Table 4). At the 48th hour, a relatively strong fluorescence and strong clustering of the
genus Acinetobacter was recorded, showing the activation of the population of these mi-
croorganisms in the biofilm. An increase in the quantity of the target microorganisms was
observed at the 144th hour, but the clustering was not as compact as it was at the 48th hour.
It can be assumed that at this time bacteria of the genus Acinetobacter had high activity
with respect to the biodegradation of the xenobiotic, but the synergistic relationships in
this group were not so well established. An explanation can be found in the assumption
Processes 2022,10, 602 16 of 20
that ND have a positive stimulation on the development of the adaptive potential of the
genus Acinetobacter. Accumulations of the microorganisms were registered at the 214th
hour, but the clusters were reduced in size and their fluorescence was reduced in intensity
compared to that which was observed at the 144th hour. The addition of ND helped to
overcome the early toxic shock by increasing the amount of non-culturable bacteria of
the genus Acinetobacter, especially at the 48th and 144th hours (Figure 9). This led to an
increase in the number of synergistic relationships and the ability of the sediment microbial
communities to reactivate their biodegradation detoxification potential. At the same time,
Pseudomonas spp. played the largest role in the late adaptation. The clear proof of this
was the significant clustering that was observed at the 214th hour. At the 214th hour, the
Pseudomonas spp. prevailed as culturable and non-culturable bacteria, probably with high
detoxification activity.
Processes2022,10,xFORPEERREVIEW16of21
Table4.FluorescentimagesofthemicrobialcommunityforthevariantPhND(400X).Subjectswere
representedbyhybridizationwithanoligonucleotideprobespecifictothegenusPseudomonas(red)
andthegenusAcinetobacter(yellow).
2ndhour48thhour144thhour214thhour
Ps./FISH/‐2ndhour
Ps./FISH/‐48thhour
Ps./FISH/‐144thhour
Ps./FISH/‐214thhour
Ac./FISH/‐2ndhour
Ac./FISH/‐48thhour
Ac./FISH/‐144thhour
Ac./FISH/‐214thhour
Figure9.Comparisonbetweenculturableandnon‐culturablebacteriaofthegenusPseudomonasand
thegenusAcinetobacterinthevariantPhND.
ThecorrespondingphotographicmaterialforthegenusAcinetobacterrecordedlow
fluorescenceatthe2ndhour,correspondingtolowmetabolicactivityofthetargetgroup
(Table4).Atthe48thhour,arelativelystrongfluorescenceandstrongclusteringofthe
genusAcinetobacterwasrecorded,showingtheactivationofthepopulationofthese
microorganismsinthebiofilm.Anincreaseinthequantityofthetargetmicroorganisms
wasobservedatthe144thhour,buttheclusteringwasnotascompactasitwasatthe48th
hour.ItcanbeassumedthatatthistimebacteriaofthegenusAcinetobacterhadhigh
activitywithrespecttothebiodegradationofthexenobiotic,butthesynergistic
relationshipsinthisgroupwerenotsowellestablished.Anexplanationcanbefoundin
Figure 9.
Comparison between culturable and non-culturable bacteria of the genus Pseudomonas and
the genus Acinetobacter in the variant PhND.
4. Conclusions
The adaptive response of microbial communities to the biodegradation of phenol at
low concentrations (close to critical, about 250 mg/kg) manifests itself as the multiplica-
tion of culturable bacteria and an increase in AeH (by 33.87%) and Ac. (by 74.65%) as
well as a relatively well-developed non-culturable microbial segment of Pseudomonas and
Acinetobacter in the presence of the ND.
In the case of the modelled explosive or accumulative contamination of sediments
with phenol in concentrations many times higher than that which is considered critical
(in the case of phenol up to 835 mg/kg), the sediment communities fell into intoxication
shock. This requires a technological, physicochemical or bioremediation reduction of the
concentration of the toxicant and the chosen approach to this is critical for the community.
ND as an augmentation factor improved the parameters of the adaptation to and biodegra-
dation of the toxic pollutants: they increased RB by 19%, Eff by 13%, and activated the
increase in the amount of Ps. (culturable) by 72% after the intoxication shock. In long-term
adaptation processes, the presence of ND when associated with high phenol intoxication
and readaptation led to the evolution of the xenobiotic biodegradation potential. This was
done by increasing the relative part of non-culturable Ac. (FISH) (at the 48th and 144th
hours) and Ps (FISH) (at the 144th and 214th hours). In parallel, ND presence increased
the cometabolic and synergistic relationships in the xenobiotic-degrading consortium. ND
increased the relative part of Ac. (FISH) by 98.98% at the 48th hour and by 39.94% at the
144th hour, it also increased the relative part of Ps. (FISH) at the 144th and 214th hours (by
Processes 2022,10, 602 17 of 20
+17.71% and +195.54%, respectively). The reasons for these results are complex—including
the deeper anaerobic conditions in clusters of ND and the greater ability of Acinetobacter
and especially Pseudomonas to use alternative energy sources to overcome intoxication.
In conclusion, it was found that ND accelerated the detoxification processes and their
effect was higher in risky situations that involve explosive pollution with extremely high
concentrations of xenobiotics. This effect from nanoparticles is extremely important for
the effective implementation of bioremediation in river sediments. The results that were
obtained could be used as a reference for similar studies for lake and dam sediments, as
well as for dewatered sludge from wastewater treatment plants. Nanodiamonds could also
be used as nanomodulators in future experiments on the biodegradation of xenobiotics
such as nitrophenols and pentachlorophenol.
Author Contributions:
Conceptualization, Y.T. (Yana Topalova); methodology, I.Y., M.B. and E.D.;
data collection—I.Y.; A.F. and I.S.; software, I.Y.; kinetic parameters, I.Y., N.D. and I.S.; microbiological
analysis, I.Y., E.D. and Y.T. (Yovana Todorova); FISH analyses—M.B., I.Y., N.D. and A.F.; writing—
original draft preparation, I.Y.; writing—review and editing, Y.T. (Yana Topalova), Y.T. (Yovana
Todorova) and M.B.; visualization, I.Y.; project administration, Y.T. (Yana Topalova). All authors have
read and agreed to the published version of the manuscript.
Funding:
This paper was supported by the Grant
№
BG05M2OP001-1.002-0019: “Clean Technologies
for Sustainable Environment—Waters, Waste, Energy for a Circular Economy”, financed by the
Science and Education for Smart Growth Operational Program (2014–2020) and co-financed by the
EU through the ESIF by means of financing the labor.
Data Availability Statement:
The data that are presented in this study are available on request from
the corresponding author.
Acknowledgments:
This work has been carried out in the framework of the National Science Pro-
gram “Environmental Protection and Reduction of Risks of Adverse Events and Natural Disasters”,
approved by the Resolution of the Council of Ministers
№
577/17.08.2018, and supported by the
Ministry of Education and Science (MES) of Bulgaria (Agreement №D01-322/18.12.2019).
Conflicts of Interest: The authors declare no conflict of interest.
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