Elimination of Oil Pollution

A. I. Dzhangarov

1 a

, N. V. Potapova

and R. U. Selimov

Kadyrov Chechen State University, 32 Sheripova Street, Grozny, Russia

Kuban State University, Krasnodar, Russia

Keywords: Industry, oil refining, petrochemistry, nature pollution, biological cleaning methods, oil decomposition.

Abstract: In recent years, there has been an increase in the industry in the field of oil refining and petrochemistry, which

contributes to an increase in the volume of pollution entering the natural environment; the produced petroleum

products, as well as, in the first place, the crude oil itself, are dangerous pollution. Biological methods are

considered to be the most promising methods of cleaning from oil pollution. They are based on the natural

mechanisms of oil decomposition. This is what will be discussed in this scientific work.

1 INTRODUCTION

When it comes to cleaning up oil pollution, oil-

oxidizing bacteria play a key role in this process.

Coryneform bacteria are the most effective group of

microorganisms used in the destruction of oilfield

waste and the elimination of the consequences of

spills and oil pollution. They do not lose their

potential even at high concentrations of contaminants.

Also, coryneforms are widely represented in various

fields of biotechnology (Bendinger, 1992).

At present, bioremediation of soil and water from

oil pollution, based on the use of microorganisms-

destructors of oil hydrocarbons, shows the best

results. Thus, the high biotechnological and

biodegradative potential indicates the relevance of

research in the study of this genus of bacteria.

In the past, bacterial cultures such as micrococci,

pseudomonads, bacilli, etc. were used. However,

these groups of microorganisms are not able to

survive in an anhydrous environment containing only

oil hydrocarbons.

In this connection, their biodegradative activity is

manifested only superficially. To the number of

disadvantages already listed, there is also a limited

range of hydrocarbons that these bacteria are able to

assimilate, which is generally expressed in the lower

efficiency of cleaning measures carried out with their

use in comparison with coryneform bacteria.

Coryneform bacteria are able to develop directly

in the oil column, which gives them an advantage

https://orcid.org/0000-0001-6962-9593

over other oil-oxidizing bacteria. They play an

important role in cleaning up oil spills into the

environment: participating in the initial stage, they

create favorable conditions for the growth of oil-

oxidizing microorganisms of other genera.

These bacteria are of great ecological importance,

but they are also used as biological agents in other

areas: biotechnology, agriculture, and medicine (Lin,

2022).

Bacteria of the genus Arthrobacter are typical

inhabitants of the soil microflora, but are also found

in other sources: in water bodies, peat, inside or on

the surface of other organisms as a symbiont. Thus,

some species of this genus are symbionts of the

chiromonid larva Polypedilum vanderplanki.

Ammonium salts and some organic substances

containing nitrogen are used as a source of nitrogen.

Individual representatives of the genus Arthrobacter

are associative nitrogen fixers.

Artrobacteria can utilize phenol and 2,4-

dichlorophenol, which can bring tangible benefits in

the treatment of wastewater from plywood, pulp and

paper industries. Also, they noted the ability to

oxidize a wide range of oil components, including

mono- and polycyclic aromatic hydrocarbons.

Bacteria A. globiformis destroy 2,4-

dichlorophenoxyacetic and 2,4,5-

trichlorophenoxyacetic acids, surpassing other

microorganisms in this; breaking down such

compounds as chlorine derivatives of benzoic acid,

halogen derivatives of polycyclic aromatic

Dzhangarov, A., Potapova, N. and Selimov, R.

Elimination of Oil Pollution.

DOI: 10.5220/0011570000003524

In Proceedings of the 1st International Conference on Methods, Models, Technologies for Sustainable Development (MMTGE 2022) - Agroclimatic Projects and Carbon Neutrality, pages

263-267

ISBN: 978-989-758-608-8

 2023 by SCITEPRESS – Science and Technology Publications, Lda. Under CC license (CC BY-NC-ND 4.0)

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hydrocarbons, acridine orange, are able to metabolize

carbohydrates, alcohols, carboxylic acids, and other

organic substances.

The genus Arthrobacter is extremely valuable

from the point of view of biotechnology since its

representatives are used as producers for the synthesis

of such important groups of organic substances as

enzymes, amino acids, and peptides, for the

production of porphyrins, vitamin B12.

In agriculture, the Mizorin P preparation based on

A. mysorens is widely used. Seed treatment with its

use has a positive effect on soybean seedlings. Some

strains of this genus are resistant to cadmium, which

opens the way to new promising ways to protect

plants from soil pollution by heavy metals, in

particular cadmium. Artrobacteria is able to

accelerate plant growth by synthesizing growth

substances.

Gordonia is another genus of coryneform

microorganisms used for bioremediation due to their

ability to metabolize hydrocarbons, a wide range of

environmental pollutants, various xenobiotics, and

difficult to decompose substances.

Members of the genus Gordonia play an important

role in biofiltration and wastewater treatment. Some

other species of Gordonia are able to decompose or

convert aliphatic and aromatic hydrocarbons,

halogenated aromatic compounds, benzothiophene,

nitrile, polyisoprene, xylene, etc. The presence of

long-chain mycolic acids in the composition of the

cell wall makes it waxy and highly hydrophobic,

which plays an important role in the destruction of

also hydrophobic pollutants. An example of this is the

growth of some strains of bacteria of the genus

Gordonia on the surface of the rubber and the

metabolization of hydrophobic hydrocarbons by

some species of this genus.

2 MATERIALS AND METHODS

In the process of studying an object, difficulties often

arise: the inaccessibility of the original or the

inappropriateness of its use, or the involvement of the

original requires the use of a large number of

resources. All these problems can be solved with the

help of simulation. The model can serve as an almost

complete or complete replacement of the object under

study. With the help of mathematical models, it is

possible to evaluate the behavior of microorganisms

under certain conditions, based on the principle that

the reaction of microorganisms to environmental

factors is reproducible. The ability to predict the

growth of microorganisms and their survival is of

great importance for all branches of microbiology

(Pepper, 1995).

Before introducing a mathematical model into

production or starting to use it in a laboratory, it is

necessary to verify its ability to adequately reflect the

features of the life processes of microorganisms.

There are many equations and models in the literature

that are used as growth functions. The main

difference between them is the complexity and

number of parameters of the equation. Some authors

compared the behavior of growth models from

different points of view: mathematical indicators of

the quality of fit and other statistical criteria. Quality

of fit scores for comparing models in these studies are

calculated by calculating bias (Bf) and precision (Af)

indices as done by Ross, coefficient of determination

(R2), standard error, or by performing an F-test. Other

authors have focused on direct comparisons of

individual growth parameters. All these studies led

the authors to different conclusions, which means that

there is no clear consensus in the literature about

which model is the most optimal for predicting

bacterial growth (Buchanan, 1997).

A group of scientists led by Osipenko M.A. built

a probabilistic mathematical model of the

morphogenetic development of bacteria of the genus

Rhodococcus. Within the framework of this study,

theoretical images simulated on the basis of

mathematical formulas were created and compared

with real microphotographs obtained during the

experiment. A comparison of them showed that, in

general, the model is correct, both in a qualitative and

quantitative aspect, but it has a drawback in the form

of somewhat increased sizes of cells of great length

(Dalgaard, 2001).

Vodopyanov V. in his research work, analyzing

the progress made in the field of modeling the

decomposition of pollutants in the soil, notes that they

cannot properly take into account the change in the

number of bacteria. Subsequently, the mathematical

model he created made it possible to establish that the

only way that can increase the efficiency of

biodegradation in the soil is to stimulate the native

microflora by introducing appropriate additives or

carbon-assimilating associations of bacteria.

Biosurfactants released by some bacteria have a

very promising future in pharmacology, the food

industry, and bioremediation. Therefore, a

mathematical model of biomass accumulation and

production of biosurfactants by Nocardia amarae

bacteria was created. The model presented by them

gave an adequate picture of the growth process. At the

same time, the average error in the production of

surfactant during the fermentation process was 5.74,

MMTGE 2022 - I International Conference "Methods, models, technologies for sustainable development: agroclimatic projects and carbon

neutrality", Kadyrov Chechen State University Chechen Republic, Grozny, st. Sher

264

and the error in the concentration of microorganisms

was 10.172%. Another group of scientists studied and

modeled the kinetics of surfactant formation by

various strains of Bacillus sp. As a result, the

productivity of biosurfactant formation and the

optimal fermentation conditions, in particular the

ratio of carbon and nitrogen, as well as the surface

tension force, were established (Hesty, 2017).

Understanding how a complex set of phenotypic

traits depends on individual molecules and their

various interactions is a top priority in bioinformatics.

Carr and colleagues developed a comprehensive

computer model of the cell. Its greatest importance

among similar works is great in connection with the

ability to predict the phenotype based on the

genotype. The phenotype of a particular

microorganism in the model is made up of its

molecular components, as well as the processes

occurring inside the cells.

3 RESULTS AND DISCUSSION

Of great interest to applied science is a mathematical

model simulating nitrogen regulation in the bacterium

Corynebacterium glutamicum. This species is widely

used for the synthesis of lysine. It is based on

piecewise linear differential equations and, due to the

high degree of knowledge of the simulated processes,

even with a small amount of input data, allows

obtaining adequate information about the state of the

biological system.

Fractal analysis and modeling of the growth of

colonies of biological objects were studied in detail

by Sletkov D.V. He created a computer software

package for visualizing a model in which the shape of

cells is closely related to their so-called fractal

dimension. In this study, he managed to obtain a

qualitative correspondence between the model and

natural experiment, which allowed him to conclude

that the model adequately describes various growth

processes and the dynamics of their course.

To date, there is a problem of increasing antibiotic

resistance of bacteria. This is happening against the

background of the lack of new developments in the

field of industrial production of antibiotics and the

irrational use of the latter in the treatment of people

and in agriculture. With this formulation of the

question, it is important to understand the mechanism

of the adaptation of bacteria to the action of

antibiotics. To this end, Serovaisky S. Ya. and

colleagues resorted to the creation of a mathematical

model for reducing sensitivity to antibiotics for

microorganisms such as Chlamydia pneumoniae,

Haemophilus influenzae, Mycoplasma pneumoniae,

Streptococcus pneumoniae. The created model allows

you to dynamically track the sensitivity and resistance

of the studied bacteria, which to a certain extent helps

to correct the tactics of treating patients with

pneumonia.

Predicting the concentration or number of

microorganisms, taking into account growth

conditions, using mathematical models of growth rate

and temperature dependence is an effective tool that

finds very wide applications in many areas of

biotechnology (in particular, bioremediation) and the

food industry. In the work of Zwietering M. H. and

co-authors, the suitability of various mathematical

models existing at the time of the study was

considered. The models were compared using the F-

test. Modified forms of the Ratkowski model were

chosen as the most suitable growth rate models. An

important condition for growth is the presence of

biogenic elements in the environment. Thus, a model

is proposed that describes the growth of

microorganisms in the soil, taking into account the

exchange of carbon and nitrogen. The approach

combines modified classical equations for microbial

growth by introducing a new state variable (r) that

determines microbial activity. The activity factor, in

turn, controls microbial growth and mortality rates, as

well as the rate of decomposition of insoluble organic

matter. The model was tested for conditions of

periodic and continuous application of the substrate

at various amounts of biomass and the content of

carbon and nitrogen in the soil. The proposed

structure of the mathematical model makes it possible

to reproduce such features of the vital activity of

microorganisms in the soil as the transition of the

microbial population to a dormant state in the case of

a lack of carbon or nitrogen, the primary effect on the

decomposition of soil organic matter, and a decrease

in the efficiency of microbial biosynthesis in the case

of nitrogen deficiency (Blagodatsky, 1998).

Evaluating the effect of temperature on microbial

growth was the goal of the work of scientists led by

Lihan Huang. They developed a new model based on

the Eyring and Arrhenius equations and compared the

results of its calculations with experimental data

published in the literature for such microorganisms as

Pseudomonas spp., Listeria monocytogenes,

Salmonella spp., Clostridium perfringens, and

Escherichia coli. Another team of scientists attempted

to develop mathematical equations to calculate the

maximum increase in biomass as a function of

temperature for a sigmoid empirical growth model.

Here we compared the performance of two models

based on empirical parameters and two more based on

Elimination of Oil Pollution

265

biological parameters. The considered models were

adapted to the experimental data for Lactobacillus

Plantarum under six isothermal conditions.

The role of software in microbiology.

Computer software, based on mathematical

formulas, is able to visually visualize and even predict

many important aspects of the dynamics of growth

and development of bacterial microorganisms and

their communities, both those that actually exist in

wildlife and artificially created ones (Parish, 1979;

Shi, 2020).

For example, a software system such as

"AgentCell" models biochemical processes, as well

as the movement of cells in a three-dimensional

environment. It is designed to study stochastic

fluctuations. This software was tested on the example

of the chemotaxis response of E. coli cells in relation

to a chemoattractant gradient.

The software complex "Haploid Evolutionary

Constructor" allows you to simulate genetic

mutations, transfer, and loss of genes, as well as fix

these genetic changes. Using this software, it is

possible to simulate phage infection and the

functioning of gene networks.

The AQUASIM computer simulation system

(Wanner and Morgenroth, 2004) creates a model of

bacterial biofilms in aquatic ecosystems, which, in

turn, makes it possible to analyze the sensitivity of the

model and evaluate its parameters.

In his scientific work, Davydov A. A. developed

mathematical models and a set of programs called

"Biomod", which serve to simulate the dynamics of

the interaction of microorganisms used in the

production of raw smoked sausages in order to ensure

their stable quality and biological safety.

The software "Biodestructor", which is used for

simulation modeling, allows you to simulate the

dynamics of the spread of oil pollution in the sea,

taking into account several phenomena at once:

diffusion, convective transfer, oil fractionation,

biological decay, and bacterial population.

Material and research methods.

This work on the study of oil pollution was carried

out on the basis of the Department of Genetics,

Microbiology, and Biochemistry of the Faculty of

Biology of Kuban State University. As part of the

study, data were also obtained on the dynamics of the

process of biological treatment of oil-contaminated

soil at the site of the Tikhoretskaya oil depot from the

Biotechnology Research Center of the Kuban State

University.

Object of study.

The objects of study in this work were the oil-

contaminated soil of the Tikhoretskaya tank farm and

bacterial strains of Arthrobacter globiformis AC1112,

Gordonia alkanivorans K9. A. globiformis culture

cells are gram-variable (gram-positive in the

stationary phase; gram-negative, in the exponential

growth phase). obligate aerobes. Motionless, non-

sporing. During growth, the colony changes color

from yellow to white. During growth, a bacillus-

coccus cycle is observed.

G. alkanivorans culture cells, Gram-positive.

Aerobes. Non-spore-forming. Colonies are orange.

The growth cycle is two-stage: bacillus-coccus.

To examine the morphological features of the

cells of the studied cultures, a phase-contrast

microscope CX41 (Olympus, Japan) with a 1000x

magnification was used. First, the pre-prepared

preparation is placed on the object stage, then the

microscope is adjusted, focused, and then aimed at

different fields of view, using a standard and

micrometric eyepiece.

To determine the increase in cell biomass (optical

density) of microorganisms in culture, a KFK-2MP

photoelectrocolorimeter (Zagorsk Optical and

Mechanical Plant, Russia) was used.

When working with a photoelectric colorimeter,

first of all, calibration is carried out. This happens by

establishing the OD of the BCH liquid medium, into

which no biomass was introduced, as a reference.

Then, a small amount of culture content is added to

another cuvette and its OD is measured; the resulting

value is recorded in the logbook.

In the framework of this work, a special

preparation consisting of living cells was used to

examine the cell morphology. The procedure for its

preparation: a small drop of water is placed on a

sterile glass slide, then a small amount of live

bacterial mass is introduced into it using a loop; The

resulting suspension is covered with a coverslip.

The pre-prepared preparation is microscoped

using a phase-contrast microscope with an immersion

objective. Look through 10 fields of view. For each

of them, the total number of cells and the number of

individual cell forms are fixed, then for each group of

cells, a percentage of the total is calculated. The final

indicators of the determined value are the arithmetic

mean of the indicators obtained for each field of view.

In the work, we used nutrient media produced by

the FBSI SRC PMB (Federal Budgetary Institution of

Science "State Scientific Center for Applied

Microbiology and Biotechnology") - the standard

nutrient medium of MPA, as well as MPB.

The composition of the MPA medium (per 1000

ml): pancreatic hydrolyzate of fish meal (12.0 g);

enzymatic peptone (12.0 g); sodium chloride (6.0 g);

microbiological agar (10.0 g); distilled water.

MMTGE 2022 - I International Conference "Methods, models, technologies for sustainable development: agroclimatic projects and carbon

neutrality", Kadyrov Chechen State University Chechen Republic, Grozny, st. Sher

266

The composition of the MPB medium (per 1000

ml): dry enzymatic peptone (10.0 g), meat extract

(11.0±1.0 g), sodium chloride (5.0 g), and distilled

water.

The MPA medium was used to obtain isolated

colonies, which subsequently served for the

preparation of "crushed drop" preparations. Studying

the preparations using a phase-contrast microscope,

the morphological changes in the cells of the studied

microorganisms were recorded, as well as the sizes

(length and width or diameter, depending on the

shape of the cells) of the cells. In this case, special

attention was paid to the ratio of cell shapes in a

variety of fields of view of the preparation. These

manipulations were carried out with a frequency of 3

hours, during the morphogenetic cycle of the

development of microorganisms. In parallel, in order

to assess the growth of bacteria and build growth

curves, cultivation was carried out in a liquid medium

of the MPB. OD was measured with the same

frequency; the value was entered into the journal.

Thus, serious work has been done to prepare the

results of the study, with the help of which it is

possible to build objective mathematical and

biological models. This solution will significantly

automate the research process and increase its

practical significance.

4 CONCLUSIONS

Summing up, we can conclude that the construction

of models and the use of software systems in the field

of microbiology plays a very significant role, being

the basis of various methods of observation and

knowledge of the processes of development of

microorganisms and phenomena. The issue of

pollution of natural resources is more relevant than

ever. And only the combination of traditional

methods of studying and solving global problems

with advanced information tools - can answer many

questions and allow you to develop a whole apparatus

for responding to changing environmental conditions.

Therefore, it is so important to develop research in

this direction, to develop biological and mathematical

models and special software that could track the

cycles and phases of the development of

microorganisms capable of cleaning up oil pollution.

The results obtained can play a fundamental role in

solving this type of problem.

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