JOURNAL LA LIFESCI

VOL. 01, ISSUE 01 ( 018-023), 2020

Microbial Isolation
Rhezqy Furwati Jufri

Biology Education Department,

University of Muhammadiyah Makassar, Indonesia
Corresponding Author: Rhezqy Furwati Jufri
Email: rhezqy28@gmail.com

Article Info Abstract

Article history:
Received 07 January 2020
Received in revised form 14
January 2020
Accepted 24 January 2020
Keywords:
Microbe, isolation, bacteria
The importance of isolating a microbe from the environment, such as
food (solid substrate), drinks (liquid substrate), and yourself because
of the many microbes that are difficult to observe or distinguish
directly using the five senses. A sample can contain bacteria or fungi.
By isolating, the shape of the colonies and the contents in a sample can
be observed. Bacteria from the air and normal flora form colonies with
lobate-shaped edges, whereas bacteria found in well water samples
form colonies with irregular edges and there are also fungi found in
the well water samples.

Introduction
Specific skills are needed in learning microbiology so that learning objectives can be
achieved. In addition to learning in the classroom, practicum activities in the laboratory also
need to be done because the object of learning microbiology is very difficult to observe
directly in daily life. Related to this, observations need to be made in the laboratory.
Observation of microorganisms can only be done if the microorganisms to be observed are
isolated in certain places so that they are easily observed under a microscope.
Microorganisms live freely in the environment, spread in the air, soil, water, food, even those
of microorganisms that live in the human body. Observations on certain microorganisms can
only be made if microorganisms are separated from the environment and other
microorganisms. This can be done with isolation techniques. Several methods are often used
in microbial isolation. The methods used are also adapted to the type of microbes to be
observed. Observation activities in microbiological practice cannot be separated from
microbial isolation, so this practicum needs to be done.
Microbial isolation technique is an attempt to grow microorganisms outside of their natural
environment. Separation of microorganisms outside the environment aims to obtain bacterial
cultures that are no longer mixed with other bacteria called pure cultures. The principle of
microbial isolation is to separate one type of microbe with other microbes derived from a
mixture of various microbes. This can be done by growing it in solid media, microbial cells
will form a colony of cells that remain in place (Lestari, 2017).
Providing samples as a source of microbial isolates can be obtained in a variety of ways there
are samples obtained from soil, livestock manure, livestock rumen fluid. From the plant part.
There are samples from food products such as milk, meat, fish, shrimp paste and others.
There is a sampling by doing the fermentation process to obtain indigenous microorganisms
(MOI) which aim to spur the process of decomposition by microbes. Microbes that have
grown and developed from MOI media are used as isolate sources for isolation purposes.

18
Copyright © 2020, Journal La Lifesci, Under the license CC BY-SA 4.0
  
 

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  Mikrobiologi Keperawatan Gigi 

Gambar 1.8 Bentuk

Bentuk-bentuk Koloni Bakteri
Sumber; https://www.sciencebuddies.org diunduh 7 Agustus 2017

b. Morfologi Mikroskopis
Morfologi mikroskopik adalah karakteristik bakteri yang dilihat melalui pengamatan
dibawah mikroskop. Bentuk bakteri sangat bervariasi, tetapi secara umum aada 3 tipe, yaitu :
bentuk bulat/kokus, bentuk batang/basil dan bentuk spiral/
spiral/spirilium.

Bentuk bulat
Bentuk kokus (coccus = sferis / tidak bulat betul) dapat di bedakan lagi menjadi beberapa
formasi, yaitu :
1. Micrococcus : berbentuk bulat, satu
satu-satu. Contohnya Monococcus gonorrhoe.
2. Diplococcus
cus : berbentuk bulat, bergandengan dua dua-dua.
dua. Contohnya Diplococcus
pneumoniae
3. Staphylococcus : berbentuk bulat, tersusun seperti untaian buah anggur. Contohnya
Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprofiticus.
4. Streptococccus : berbentuk bulat, bergandenbergandengan
gan seperti rantai, sebagai hasil
pembelahan sel kesatu atau dua arah dalam satu garis. Contohnya Streptococcus
faecalis, Streptococcus lactis, dan lain
lain-lain.
5. Sarcina : berbentuk bulat, terdiri dari 8 sel yang tersusun da
dalam
lam bentuk kubus sebagai
hasil pembelahan
embelahan sel ke 3 arah. Contohnya : Thiosarcina rosea
6. Tetracoccus / gaffkya : berbentuk bulat tersusun dari 4 sel berbentuk bujur sangkar,
sebagai hasil pembelahan sel kedua arah. Contohnya Pediococcus

18
  Mikrobiologi Keperawatan Gigi 

perkembangan mikroorganisme dalam makanan sangat erat hubungannya dengan

kehidupan manusia.
Mikroorganisme merupakan makhluk hidup yang sangat banyak, baik di tanah, air
maupun udara. Untuk itu perlunya isolasi maupun pemurnian untuk mendapatkan
mikroorganisme tersebut. Populasi yang besar dan kompleks dengan berbagai mikroba
terdapat dalam tubuh manusia termasuk di gigi dan mulut, saluran pencernaan dan kulit.
Isolasi adalah cara untuk memisahkan atau memindahkan mikroba tertentu dari
lingkungannya, sehingga diperoleh kultur murni atau biakan murni. Kultur murni ialah kultur
yang sel-sel mikrobianya berasal dari pembelahan dari satu sel tunggal. Kultur murni atau
biakan murni diperlukan karena semua metode mikrobiologis yang digunakan untuk
menelaah dan mengidentifikasi mikroorganisme, termasuk penelaahan ciri-ciri kultural,
morfologis, fisiologis, maupun serologis, memerlukan suatu populasi yang terdiri dari satu
macam mikroorganisme saja.
Dikenal beberapa cara atau metode untuk memperoleh biakan murni dari suatu biakan
campuran. Dua diantaranya yang paling sering digunakan adalah metode cawan gores dan
metode cawan tuang. Yang didasarkan pada prinsip pengenceran dengan maksud untuk
memperoleh spesies individu. Dengan anggapan bahwa setiap koloni dapat terpisah dari
satu jenis sel yang dapat diamati (Afrianto, 2004).
Biakan murni diperlukan dalam berbagai metode mikrobiologis, antara lain digunakan
dalam mengidentifikasi mikroba. Untuk mengamati ciri-ciri kultural morfologi, fisiologi, dan
serologi dibutuhkan mikroba yang berasal dari satu spesies (Dwijoseputro, 2005). Beberapa
faktor yang perlu diperhatikan dalam melakukan isolasi mikroba yaitu antara lain:
1. Sifat setiap jenis mikroba yang akan diisolasi.
2. Tempat hidup atau asal mikroba tersebut.
3. Medium pertumbuhan yang sesuai.
4. Cara menginkubasi mikroba.
5. Cara menginokulasi mikroba.
6. Cara menguji bahwa mikroba yang diisolasi telah berupa kultur murni dan sesuai
dengan yang dimaksud.
7. Cara memelihara agar mikroba yang telah diisolasi tetap merupakan kultur murni.
8. Untuk mengisolasi mikroorganisme, hal yang tidak kalah pentingnya untuk
diperhatikan pula adalah teknik pengambilan sampel. Mikroorganisme terdapat
dimana-mana di dalam lingkungan kita mereka ada pada tubuh kita, dan di sekeliling
kita. Mereka merupakan komponen penting dalam ekosistem. Di habitat alamiahnya,
mereka hidup dalam suatu komunitas yang terdiri dari berbagai jenis mokroorganisme,
bersama spesies-spesies biologi lainnya. Didalam komunitas ini, satu spesies mikroba
dapat mempengaruhi spesies lain dengan berbagai cara-cara beberapa bersifat
menguntungkan beberapa merugikan ( Pelezar, 198)
9. Dalam suatu analisis mikrobiologi, pengambilan sampel merupakan salah satu kunci
utama yang sangat mendukung keberhasilan suatu analisa, yaitu memindahkan sampel
atau kultur bakterial dari satu tempat ke tempat yang lain secara aseptis (terhindar
dari kontaminasi).

268
Advances in Tropical Biodiversity and Environmental Sciences 4(2): 35-38, September 2020 e-ISSN: 2622-0628
DOI: 10.24843/ATBES.2020.v04.i02.p02 Available online at: https://ojs.unud.ac.id/index.php/ATBES/article/view/59571
35

Identification and Analysis of Diversity of Soil

Microorganism Colonies in Limestone Reclamation Land

Supiana Dian Nurtjahyani1*, Dwi Oktafitria2, Sriwulan1, Ahmad Zaenal Arifin3, Eko Purnomo4

1
Biology Education Study Program, Faculty of Teacher and Educational Science, PGRI Ronggolawe University
2
Department of Biology, Faculty of Mathematic and Natural Science, PGRI Ronggolawe University
3
Departement of Mathematic, Faculty of Mathematic and Natural Science, PGRI Ronggolawe University
Jl. Manunggal No. 61 Tuban-62381, East Java, Indonesia
4
PT. Semen Indonesia Tbk Gresik, Semen Indonesia Group
Jl. Veteran No.93, Gapurosukolilo, Gresik-61122, East Java, Indonesia
*
Corresponding author: diananin39@gmail.com

Abstract. Reclamation of land used for mining is very important because it is related to microbial activity in the soil which
has an impact on soil fertility. The purpose of this study is to identify and analyze the diversity of bacterial colonies in the
reclaimed land of the former Limestone Mining. The method used in this research was a laboratory experimental method.
The results of the study showed that 6 bacterial isolates were found and the highest total plate count in GPS land (GPS
system) is 1.97 X 106 cfu, the dominant colony characteristics were irregular shapes, flat edges and milky white color.

Keywords: bacterial colony; diversity; isolate; reclamation land; soil

microbial colonies in groups of bacteria in the reclaimed

I. INTRODUCTION
Reclamation of a former limestone quarry is an
obligation that must be carried out by companies or
holders as stipulated in the Minister of Energy and
Mineral Resources decree No. 7 of 2014[1]. The success
of a reclamation is largely determined by many things,
including aspects of land use, fertility of the planting
media, technical planting and maintenance of plants.
Reclamation has been carried out by PT Semen
Indonesia (Persero) Tbk since 2010 until now. Along
with the increase in years, modifications in planting are
always done, not only by doing conventional systems, but
also Groove Planting Systems (GPS). Since the initial
reclamation in 2010, 2014 and 2016, the method used is
the conventional method. This conventional method is
carried out by distributing topsoil evenly. The
implementation of this reclamation activity is closely
related to the biodiversity in the reclamation land,
including the biodiversity of soil microorganisms.
Biodiversity of soil microorganisms can be used as an
indicator of land quality. This is because, the presence of
soil microorganisms can provide a number of benefits for
plant growth, including helping provide nutrient
availability, helping to mobilize nutrients, helping to
produce Plant Growt Regulator (PGR), and as a pathogen
biocontrol agent[2]. In addition to bacteria there are also
mycorrhizae that can help soil fertility [3]. Therefore this
study aims to identify and analyze the diversity of soil
land of limestone mining.
II. METHODS
This research is an exploratory study, the method used
is a laboratory experimental method carried out in the
biology laboratory of the University of PGRI
Ronggolawe, Tuban. Research samples from the former
reclaimed land of limestone mining PT Semen Indonesia
Tbk.
Data Collection and Analysis of Soil Microorganisms
Analysis of soil microorganism diversity in this study
was limited to soil bacterial groups. This analysis process
is carried out through several stages, which include
isolation, purification, and characterization of bacterial
isolates obtained.
Bacterial isolation was carried out by taking soil
samples from the post-limestone quarry land that was
reclaimed with conventional systems and grooves. Soil
samples were taken at 5 points from each location. Then a
serial dilution was carried out on the soil sample that has
been taken. Dilutions were done at levels 10-4, 10-5, and
10-6. The diluted samples were then inoculated on NA
(Nutrient Agar) media and incubated at room temperature
for 2 x 24 hours.The isolates obtained from the isolation
process were then purified. Purification was carried out
by the quadrant method [4] [5]. After obtaining a single
colony, the isolates were characterized. Then identified
Advances in Tropical Biodiversity and Environmental Sciences 4(2): 35-38, September 2020 e-ISSN: 2622-0628
DOI: 10.24843/ATBES.2020.v04.i02.p02 Available online at: https://ojs.unud.ac.id/index.php/ATBES/article/view/59571
37

TABLE II
AVERAGE OF TOTAL PLATE COUNT OF BACTERIA FROM SOIL ON CRETACEOUS MINE

Rate of Dilution
No Sample Origin TPC value (cfu)
10-4 10-5 10-6

1 Conventional 116 263 525 4.9 x 105

Groove Planting
2 567 231 140 1.97 x 105
System (GPS)

Discussion characteristics of the bacterial colonies found in the

former limestone quarry had different diversity.
In Table 1 it can be seen that from the isolation there
Bacterial colonies have special properties in solid
were 6 isolates obtained. The six isolates consisted of
media, where the shape of the colony can be described as
Isolate 1 which was only found in soil samples taken
dot, round or circular, filamentous and irregular. The
from conventional systems. Isolate 2, Isolate 3, Isolate 4,
surface of the colony can be flat, arise flat, curved,
and Isolate 5 were found from both Groove Planting
bulging, hilly and similar to the crater, while the edges of
System (GPS) and conventional soil samples. Whereas
the colony can be intact or whole, split or lobate, swim or
Isolate 6 was only found from soil samples taken on GPS
filamentus and curled. In color, bacterial colonies are
systems.
mostly whitish or yellowish in color [11].
In Table 2 it can be seen that the total bacterial plate
Naturally in polluted soils there are biosurfactant-
count from land with a GPS system (1.97 X 10 6 cfu) is
producing bacteria. Biosurfactants are compounds
higher than conventional land (4.9 X 10 5 cfu). These
produced by microorganisms that function to reduce the
results indicate that the number of bacterial colonies
surface tension of the liquid so that the bioremediation
found on GPS land is higher than conventional land. With
process can take place properly. With the presence of
more and more bacteria this Soil fertility depends on the
biosurfactant-producing bacteria, the process of
physical, chemical, and biological soil conditions. Soil
degradation of the polluted soil environment will proceed
physical conditions include effective depth, texture,
well [12] [13].
structure, humidity, and soil air condition. Soil chemical
The reduced microbial activity in the soil will also
conditions include: soil reaction (soil pH), base
affect soil density, because it can dissolve nutrients in the
saturation, and organic matter, abundance of nutrients,
soil. Microorganisms in the soil can also use the results of
nutrient reserves, and availability of nutrients for plants.
pollutants for metabolism so that the number increases
Biological conditions of the soil include: microbial
and can help soil fertility [14] [15] [16]. The results of
activity of organic material remodeling in the process of
this study indicate there are 6 isolates of which 5 isolates
humification, and binding of nitrogen free from the air.
were obtained from the GPS environment and
Evaluation of soil fertility can be done in several ways,
conventional, while the other 1 from the conventional
namely through visual observation of plant deficiency
environment. From these data, the combination of
symptoms [7] [8] [9].
conventional environment and GPS environment provides
The number of bacterial colonies in the GPS
more environmental support for bacterial growth because
environment is higher because of the GPS environment
the remnants of the mining area still contain nutrients and
because the carrying capacity of the environment in the
pollutants that can be used for bacterial metabolism.
form of nutrients both macro and micro is better than
conventional ones. This is in line with Erfandi's research,
IV.CONCLUSION
2017[10] which states that bacteria have an important role
in the process of decomposition of organic matter, There are 6 bacterial isolates found in conventional
especially at high humidity levels. Characteristics of land and GPS land in the former limestone quarry. The
bacterial colonies found are the convex surface dominant total bacterial plate count from land with GPS systems
with milky white color. This diverse bacterial colony (1.97 X 106 cfu) is higher than conventional land (4.9 X
shows the diversity of soil microbes. The edges of the 105 cfu). Bacterial colonies on limestone quarry have the
colonies that were found all had flat edges, while the characteristics of irregular shape, flat edges and the color
dominant colony was irregularly rounded, the of milky white colonies.
AISTSSE 2020 IOP Publishing
Journal of Physics: Conference Series 1819 (2021) 012044 doi:10.1088/1742-6596/1819/1/012044

Isolation and Characterization of Bacteria from River Origin

in Mandailing Natal North Sumatera

Mhd. Yusuf Nasution, Ahmad Shafwan S. Pulungan, Eko Prasetya, Salwa Rezeqi
Biology Department, Universitas Negeri Medan

Yusufnasution1963@gmail.com

Abstract. The indicator of water pollution according to microbiological standards is the

presence of microbiological pollutants. To see the presence of bacteria in the waters, it is
necessary to study the isolation and characterization of these bacteria. The existence of the
river in the Mandailing Natal area has experienced a change in character due to gold mining
activities along the river flow. The purpose of this study was to determine the types of bacteria
in the river in the Mandailing Natal area of North Sumatra. This research was conducted at the
Laboratory of Microbiology, Department of Biology, Faculty of Mathematics and Natural
Sciences, State University of Medan. The research period was from June to October 2020. The
population in this study was the water of the Batang Gadis Mandailing Natal river, North
Sumatra. The sample in this study is the Batang Gadis river water around the Mandailing Natal
gold mine, North Sumatra. The research was carried out by taking samples and isolating
bacteria which included planting the samples on media enriching the planting of the samples
on various growing media, purifying bacterial colonies and observing the morphology of
bacterial colonies. The results showed that there were 6 bacterial isolates living in river waters
in Mandailing Natal, North Sumatra.

1. Introduction
The mining sector, especially gold mining, needs special attention by the public because it has a high
risk of environmental damage. One of the problems that is still a problem is the large number of illegal
mining activities (without permits).The main problem in gold mining without a permit is the use of
hazardous materials and substances in its processing. The waste produced generally still contains
mercury [1]. Mercury which is often used in gold ore mining is elemental mercury, which is mercury
in its original form [2].
Metallic mineral content (especially gold) has long been stored in the Mandailing Natal Regency
area. The reserves of gold mining in Mandailing Natal (Madina) Regency, North Sumatra Province are
quite large and reach 1.5 million ounces (Au) with a content of 2.2 grams tonnes of Au.
Gold mining in Madina has been around since 2008. The rise of illegal mining using hazardous
materials has resulted in various diseases caused by the dumping of hazardous heavy metal waste into
the environment. One of the alternatives for handling mercury-polluted environment is bioremediation
technique, which is one of the uses of microorganisms to improve the polluted environment.
The role of microbes in wastewater treatment has provided many encouraging results [3]. Organic
compounds found in wastewater are a source of nutrition for microbes. Microbes will break down
these compounds into simpler and more stable forms so that the levels of pollutants contained in the
waste decrease. Enzymatic reactions by bacteria are the key to implementing a gradual transformation

Content from this work may be used under the terms of the Creative Commons Attribution 3.0 licence. Any further distribution
of this work must maintain attribution to the author(s) and the title of the work, journal citation and DOI.
Published under licence by IOP Publishing Ltd 1
AISTSSE 2020 IOP Publishing
Journal of Physics: Conference Series 1819 (2021) 012044 doi:10.1088/1742-6596/1819/1/012044

process in wastewater management from substrates, which are generally organic materials with
complex molecular arrangements, into simple elements.
Several types of bacteria are known to have the ability to reduce or absorb heavy metals. Bacteria
that are resistant to mercury stress are called mercury resistant bacteria (BRM). One of the
mechanisms contained in BRM to reduce mercury is by converting Hg2 + to Hg0 with the enzyme
mercury reductase encoded by the merA gene [4].
The potential possessed by bacteria in reducing heavy metals (mercury) is an environmentally
friendly technique in improving the environment [5-6]. As an initial effort to determine the types of
bacteria that are capable of reducing mercury, it is necessary to conduct a preliminary study to
determine the types of bacteria that are resistant to mercury content in the gold mining area in
Mandailing Natal, North Sumatra.

2. Materials and Method

This research was conducted in the microbiology laboratory, Department of Biology, Faculty of
Mathematics and Natural Sciences, State University of Medan. The time of this research is planned to
start from June to October 2020. The population in this study are all bacteria obtained from soil and
water from the Mandailing Natal gold mine, North Sumatra. The sample in this study was a total
sample, that is, all bacteria that are resistant to mercury obtained from the Mandailing Natal gold
mine, North Sumatra.
The main materials used in this study were soil and waste water samples obtained from the
Mandailing Natal gold mine, North Sumatra, tissue, umbrella paper, rubber, cotton, plastic wrap,
aluminum foil, masks, gloves, markers, standard solutions. mercury, distilled water, spirits, 70%
alcohol, thick adhesive plastic, HCl, NaOH, HNO3. In addition, the media needed are BHI (Brain
Heart Infusion) Media, LDS (Lactose Double Streng), EMBA (Eosin Methylen Blue Agar), Cled
Agar, MSA (Manitol Salt Agar), MCA (Mac Conkey Agar), Blood Agar , NaCl, HgCl2 produced by
Merck Germany, as well as reagents for Gram positive and negative tests.
The tools used in the research included Laminar Air Flow with a local brand, autoclave with the
Hirayama HVE50 brand, an oven with the Memmert brand, Petri dishes with the pyrex brand, test
tubes with the pyrex brand, incubators with the Memmert Churt brand, measuring pipettes with the
pyrex brand, cupboards ice with the LG brand, ose needle, digital balance with the sartorius brand,
Beaker glass with the pyrex brand, spatula, Erlenmeyer with the pyrex brand, measuring cup with the
pyrex brand, measuring flask with the pyrex brand, micropipette with the Accumax pro brand.

2.1. Sampling
Samples were taken from the Mandailing Natal gold mine, North Sumatra. 250 ml of samples from
each shelter were then put into dark reagent bottles and carried using transport media. Next, 50 ml of
samples from each bottle were taken and mixed into bottles, each containing 450 ml of Nutrient Broth.
The mixture is then propagated at room temperature for 5 days. Propagation aims to increase bacterial
culture.

2.2. Isolation of Bacteria

- Planting samples on enriched media
The samples were planted on enriched media
BHI and LDS.
- Planting samples on various growing media
Samples were grown on EMBA, Blood Agar, Cled Agar, MCA and MSA media.
- Purification of Bacterial Colonies
Bacterial colonies that grew on the growing media of Blood Agar, EMBA, Cled Agar and MCA
were observed to determine the differences in the characteristics of the growing bacterial colonies.
Furthermore, each colony with different characteristics was replanted in the appropriate planting
medium to obtain a pure colony, then incubated at 37oC for 24 hours.

2
 BUSZEWSKI ET AL.: JOURNAL OF
SPECIAL GUEST EDITOR SECTION
Identification of Microorganisms by Modern Analytical
Techniques
BOGUSŁAW BUSZEWSKI, AGNIESZKA ROGOWSKA, PAWEŁ POMASTOWSKI, MICHAŁ ZŁOCH, and
VIORICA RAILEAN-PLUGARU
Nicolaus Copernicus University, Faculty of Chemistry, Department of Environmental Chemistry and Bioanalytics, Gagarina 7, 87-
100 Torun, Poland; Nicolaus Copernicus University, Interdisciplinary Centre of Modern Technology, Wileńska 4, 87-100 Torun,
Poland
Rapid detection and identification of
microorganisms is a challenging and important
aspect in a wide range of fields, from medical to
industrial, affecting human lives. Unfortunately,
classical methods of microorganism identification
are based on time-consuming and labor-intensive
approaches. Screening techniques require the
rapid and cheap grouping of bacterial isolates;
however, modern bioanalytics demand
comprehensive bacterial studies at a molecular
level. Modern approaches for the rapid
identification of bacteria use molecular techniques,
such as 16S ribosomal RNA gene sequencing
based on polymerase chain reaction or
electromigration, especially capillary zone
electrophoresis and capillary isoelectric focusing.
However, there are still several challenges with the
analysis of microbial complexes using
electromigration technology, such as uncontrolled
aggregation and/or adhesion to the capillary
surface. Thus, an approach using capillary
electrophoresis of microbial aggregates with UV
and matrix-assisted laser desorption ionization
time-of-flight MS detection is presented.
M
icroorganisms are well known for both positive and
negative properties. They are used on a wide scale in
the food industry, biotechnology, and modern genetic
engineering. However, some microorganisms can cause food
spoilage or serious disease. Because of these two negative
effects, the unequivocal and rapid identification of
microorganisms in real samples represents a very important
area of focus (1, 2). This is particularly important in medical
diagnostics. Microbial infections can lead to dangerous diseases,
such as sepsis (3), diabetic foot infections (4), or meningitis (5).
Rapid and unambiguous identification of the pathogen causing
the infection is a necessary factor for the implementation of an
appropriate therapy to save patient lives (6, 7).
Guest edited as a special report on “Chromatography – Theory and
Applications. In memoriam of Prof. Dr. hab. Edward Soczewiński” by
Tomasz Tuzimski.
Corresponding author’s e-mail: bbusz@chem.umk.pl
This work was supported by Maestro 6 No. 2014/14/A/ST4/00641
(2015–2017) and Opus 11 No. 2016/21/B/ST4/02130 (2016–2019) from
the National Science Centre of Poland.
DOI: 10.5740/jaoacint.17-0207
AOAC INTERNATIONAL VOL. 100, NO. 6, 2017 1607
Downloaded from https://academic.oup.com/jaoac/article/100/6/1607/5654264 by guest on 11 November 2023
Conventional techniques currently used for the identification
of microorganisms are based on the culture of microorganisms
and the determination of the phenotypic characteristics thereof.
However, these methods are labor-intensive, time-consuming
(taking up to 3 days), and often inadequate for the differentiation
of phenotypically similar species (8–12). These limits are
especially important from a medical diagnostics point of view
because the rapid and proper identification of pathogens is an
essential factor in the implementation of the appropriate therapy
(10, 11). In addition, screening identification of microorganisms
is also a key issue in areas such as the pharmaceutical industry, food
QC, and environmental research (1, 11, 13). Therefore, much
emphasis has been placed on the development of alternative
methods for the rapid and unambiguous identification of
microorganisms directly from tested material (8–11).
Nowadays, in the taxonomy of microorganisms, molecular
biology methods—such as 16S ribosomal RNA (rRNA) gene
sequencing (8, 14), polymerase chain reaction (PCR; 15), and
other related PCR-based methods (16–18)—are very popular.
These methods are characterized by high sensitivity and
reproducibility. 16S rRNA gene sequencing is considered the
most accurate method and deemed the gold standard for the
identification of microorganisms at the species level (19).
Unfortunately, due to the high cost of this analysis, in routine
diagnostics, it is impossible to use this method. Moreover, such
studies are usually performed by external institutions, resulting
in prolonged wait times for results (14, 20).
One of the strategies to reduce the time required for the
identification of microorganisms in routine diagnostics is the
use of semiautomatic and automatic systems based on
biochemical methods. Such methods allow the attainment
of results in maximum of 24 h (21–23). However, this time
frame is still too long, and the obtained results are not always
satisfactory (24). An alternative could be MS methods, such as
matrix-assisted laser desorption ionization time-of-flight
mode (MALDI-TOF MS) or electromigration techniques
(8). These methods allow the performance of quick and
clear analyses of microorganisms directly from infected
liquid samples while maintaining unit costs low (25–30).
The most recent approach involves the use of multivariate
techniques, such as PCR electrospray ionization (ESI) MS
(31, 32), PCR-microchip capillary electrophoresis (ME; 33),
PCR-capillary electrophoresis (PCR-CE; 34), capillary isoelectric
focusing (CIEF)-MALDI-TOF MS (34), or micropreparative
solution CIEF (sCIEF)-HPLC (35). Such connections allow
the attainment of conclusive results in a very short amount
of time and have great potential for further research
(24, 31–35).
1608 BUSZEWSKI ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 100, NO. 6, 2017
Biochemical Techniques
At present, automatic, semiautomated, and miniaturized
systems are the most used biochemical methods for the
identification of microbes. Their operation is generally based
on the same principles as conventional tests, i.e., on the analysis
of the biochemical properties of microorganisms. Biochemical
techniques have numerous advantages over conventional
diagnostic methods, such as short analysis times (2–4 h) and
the ability to simultaneously determine many microorganisms,
while retaining accuracy in the results (23). The most commonly
used techniques are Vitek 2 Compact (bioMérieux; 36, 37), BD
Phoenix (BD Diagnostics; 38, 39) and Analytical Profile Index
(API) 20 E (bioMérieux; 40, 41). These systems allow for both
rapid identification of microorganisms (ID) and the determination
of drug susceptibility [antimicrobial susceptibility testing
(AST); Table 1]. Such devices are equipped with different
types of panels for the identification of different groups of
microorganisms (38, 42, 43).
The BD Phoenix device determines the growth of
microorganisms by turbidimetry and colorimetry methods
(44). The simultaneous analysis of 99 panels can be
performed, monitoring each panel every 20 min. The analysis
lasts 8–12 h. Available panels enable the identification of Gram-
positive and -negative bacteria, Streptococcus pneumoniae, and
b-hemolytic streptococci that belong to the viridans group (23, 44).
Beuving et al. (39) used this system for the rapid identification
and AST of positive blood cultures. They were able to correctly
identify 71.4% of Pseudomonas aeruginosa strains and 95.2% of
all tested Enterobacteriaceae. Moreover, AST results for all the
tested antibiotics, except trimethoprim-sulfamethoxazole and
erythromycin, can be obtained 24 h earlier compared with
standard methods (39).
The Vitek 2 Compact system turbidimetrically monitors
bacterial growth during incubation (44). Depending on the
model, the simultaneous analysis of 30 or 60 microorganisms
in 1–10 h can be carried out. By using the appropriate card with
this technique, aerobic Gram-positive and -negative bacteria,
anaerobic bacteria, Streptococcus, Neisseria sp., Haemophilus
sp., Corynebacterium sp., and yeasts can be identified. The
device periodically scans each card and determines the
kinetics of microbial growth in each well, subsequently
identifying the unknown culture (21, 23, 44–46). Although
this method requires the use of expensive equipment and
Table 1. Review of the biochemical methods for the identification of different groups of microorganisms
Microorganisms Source of isolation
Anaerobic bacteria Clinical isolates
Blood cultures Blood
Enterococcus Clinical isolates
Streptococcus pneumoniae Clinical isolates
Gram-negative bacteria Collection
Lactobacillus paracasei Fermented milk
Yeast Food
Candida spp. Clinical isolates
Psychrotrophic bacteria Raw bovine milk
Staphylococcus spp. Goat’s milk
Coagulase-negative staphylococci Goat’s milk
machining of a large number of samples, it does reduce the
analysis cost per unit (22, 37).
Several studies have tested the corresponding cards for the
correct identification of various microorganisms and their drug
resistance (Table 1). Lee et al. (36) evaluated the Vitek 2
anaerobe and Corynebacterium card specific to 301 anaerobic
isolates. As a result of these studies, the card managed to
correctly identify 239 (79.4%) clinical isolates at the genus
level, including 100 species that were not in the database.
Gherardi et al. (38) compared the Vitek 2 Compact and
Phoenix systems for identification and AST of positive blood
cultures (38). They correctly identified 75% and 43.75% of
Gram-positive isolates and 100% and 92.3% of Gram-
Downloaded from https://academic.oup.com/jaoac/article/100/6/1607/5654264 by guest on 11 November 2023
negative isolates by Vitek 2 and Phoenix, respectively (38).
The semiautomatic API system is a system of miniaturized
microtubes with 20 small wells to carry out 23 standard
biochemical tests (21, 47). In these studies, catabolic reactions
between microorganisms and components of the substrate are
used. Results are typically available after 18–24 h of incubation
at 35–37°C. These kinds of tests are usually used to identify
Enterobacteriaceae, but application of similar formats can lead
to the identification of Gram-positive and -negative bacteria,
Streptococci, Enterococci, Listeria, and anaerobic bacteria
(Table 1). A significant advantage of this method is the
availability of an extensive database; unfortunately, the major
disadvantage of this method is that it is time consuming (21, 22).
Molecular Biology Methods
Rapid progress in the field of molecular biology has enabled
the development of new methods for the identification of
microorganisms. These techniques have proven to be particularly
useful in the identification of microorganisms that are difficult to
detect by phenotypic methods. Molecular biology methods can be
used to confirm the presence of a specific microorganism in the
tested material, as well as screen research. The most commonly
used molecular methods include amplification of species-specific
PCR products (15), PCR-based methods, and 16S rRNA/16S
ribosomal DNA (rDNA) gene sequencing (currently considered
the gold standard in identifying microorganisms; 48–52).
The PCR technique was developed by Mullis and Faloona
(53) in the early 1980s. PCR is based on the amplification of
DNA chains by repeatedly heating and cooling the sample in the
presence of a polymerase and primers (i.e., short DNA fragments
Method Refs.
Vitek 2 ANC (36, 45)
Vitek 2 GNI/GPI BD Phoenix (37, 38) (38, 39)
Vitek 2 API 20 STREP (42)
Vitek 2 BD Phoenix (43)
Vitek 2 GN (46)
API 50 CHL (40)
API 32 C Phoenix (144)
Vitek 2 (41)
API (112)
API 20 STAPH (24)
API Staph (145)
Microorganisms for Sustainability 27
Series Editor: Naveen Kumar Arora

Charles Oluwaseun Adetunji

Deepak G. Panpatte
Yogeshvari K. Jhala Editors

Microbial
Rejuvenation
of Polluted
Environment
Volume 3
118 A. Inobeme

of iron and manganese naturally in soil is relatively high, and their pollution effects
are not usually insignificant. Heavy metals in soil are divided into two categories
from the biochemical characteristics: one is harmful to crops and humans and
animals, such as Pb, Cd, and Hg; the other is beneficial when present in the desired
quantity, but when present in excess would have lethal effects. Examples are Cu, Zn,
Mn, and so on (Xie et al. 2016).
Metals when present in soil exist either as separate entity or combined with other
components. These components may include exchangeable forms adsorbed on the
surfaces of ionic solids, non-exchangeable forms and insoluble metal compounds
such as calcium and magnesium carbonates and phosphates, soluble metal com-
pound such as chlorides, metal complex of organic materials, and metals binding to
silicate ores (Chibuike and Obior 2014).
Some heavy metals play an extremely important role in biochemical reactions,
which are significant for the growth and development of microorganisms, plants, and
animals (Kavamura and Esposito 2010). For example, heavy metals such as copper
and zinc are vital components of physiological processes in all living organisms,
including plants, animals, and microorganisms. Zinc (Zn), copper (Cu), etc. are vital
micronutrient for growth and enzymatic activities of heterotrophic bacteria. How-
ever, excess load of the same metals show toxicity and inhibition to microbial
processes (John 2017).
Although heavy metals are naturally present in the soil, geologic and anthropo-
genic activities increase the concentration of these elements to amounts that are
harmful to both plants and animals. Some of these activities include mining and
smelting of metals, burning of fossil fuels, use of fertilizers and pesticides in
agriculture, production of batteries and other metal products in industries, sewage
sludge, and municipal waste disposal (Chibuike and Obior 2014). Soil also gets
contaminated with metals from a variety of anthropogenic inputs, most especially
irrigation with wastewater. The use of contaminated water for irrigation contributes
to the accumulation of chemical and biological contaminants in soils and alters the
physicochemical and biological properties of soils (Kouchou et al. 2018).
Pollution of soil by heavy metals refers to the release of metals such as cadmium,
lead, mercury, chromium, and other elements that are biotoxically significant in the
soil, bringing about amounts that are higher than the permissible limit. Some heavy
metals have been identified as priority pollutants, which include argon, arsenic,
copper, mercury, nickel, lead, zinc, selenium, thallium, beryllium, and silver.
These metals and metalloids are 13 in all (Sparks 2005). They are found naturally
in rocks, minerals, and various anthropogenic inputs such as mining, sewage sludge,
industrial waste, agricultural activities, electronics, and energy production among
others (Gilmour and Riedel 2009). Heavy metals are found naturally in soils
resulting from weathering of underlying bedrock or through mineral processing
processes (Shakoor et al. 2015) and their contents in soils depend on the nature of
the parent material, where it is found and its age. They represent less than 1% of the
composition of the earth crust due to alteration of rocks and natural incidents.
More recently, the release of a large amount of these metals from industries and
mining which finally goes into the soil has led to a rise in the contents of heavy
120 A. Inobeme

375 ppm of lead was added to it, the lowest concentration used, whereas a clay soil
required 1500 ppm Pb to give the same inhibitory effect. Peat soils showed no effects
even when the concentration of lead was highest (7500 ppm).

6.4.4 Growth Studies

This method was employed by Nair et al. (1992) in assessing the effect of different
salt concentrations on microorganisms. Growth studies of bacteria such as Bacillus
subtilis, Escherichia coli, and Klebsiella pneumoniae can be carried out in nutrient
broth medium supplemented with peptone and yeast extract, etc. 10 μl of culture is
inoculated into the nutrient broth medium supplemented with copper and nickel
metal; then it is analyzed for growth studies. This can be done in a broad or narrow
range:
Broad range: using this technique, different salt concentrations of metals are used in
this technique to test the effect on the different isolates. The isolates are inocu-
lated in a broth containing heavy metals at varying concentrations and incubated
for 3 h. Then readings in UV spectrophotometer are taken at different time
intervals. A plot is then drawn using the obtained readings.
Narrow range: The isolates are tested for metal tolerance using about three different
concentrations of the metals. Copper chloride and ammonium sulfate salts of
nickel are used in varying concentrations such as 10, 15, and 20 mM. The isolates
are inoculated in a broth containing heavy metals at different concentrations and
are incubated for 24, 48, and 72 h. Then readings in UV spectrophotometer are
taken at different time intervals. By using the readings, graphs were plotted
(Joonu and Divya 2017).

6.5 Role of Soil Microorganisms

Soil contains a variety of microorganisms including bacteria, fungi, and many others
which together constitute the natural ecosystem. Microorganisms play a vital role in
nutritional chains that are an important part of the biological balance in nature, where
bacteria are essential for maintaining some of the natural cycles such as nitrogen,
carbon, phosphorus, and sulfur (Kummerer 2004).
Microorganisms are also known to play important roles in plant nutrient
recycling, soil structure conservation, poisonous chemical detoxification, and plant
pest control and management (Filip 2002). In their geoactive roles in the biosphere,
they are also essential, especially in the areas of biotransformation elements and
biogeochemical cycling, metal and mineral transformations, decomposition,
bioweathering, and soil and sediment formation. All sorts of microbes, including
prokaryotes and eukaryotes and their symbiotic interactions with each other and
6 Effect of Heavy Metals on Activities of Soil Microorganism 129

example, the non-living brown algae (Ascophyllum nodosum) exchanges the original
cell wall adsorption of K+, Ca2+, and Mg2+ to adsorb Co2+ (Freitas et al. 2006).
Brady and Duncan showed that yeast releases approximately 70% of K+ rapidly and
60% of Mg2+ slowly in the process of adsorbing Cu2+ (Aly 2018).
Numerous microbial species, including bacteria and fungi from Bacillus, Pseu-
domonas, Streptomyces, Aspergillus, Rhizopus, and Penicillium, have significant
removal ability. At present, it has been found that a variety of bacteria can absorb soil
heavy metals. Among them, Escherichia coli K-12 can absorb the widest variety of
metal ions; the outer membrane of this stain can absorb more than 30 different kinds
of metal ions. Rhizopus can absorb Zn, Cu, Cd, Pb, and other heavy metal ions, and
Thiobacillus can absorb heavy metal ions as well as inorganic ions, such as S, which
combines with the metal ions to form a precipitate that can be separated from the soil.
Mechanisms involved in the detoxification and transformation of metals, include
the mechanism that restricts entry into the cell and intracellular detoxification or
organellar compartmentation, the latter occurring in some eukaryotes, e.g., algae and
fungi. Operation of a number of mechanisms is possible depending on the organism
and the cellular environment; mechanisms may be dependent on and/or independent
of metabolism. A variety of mechanisms may be involved in transport phenomena
contributing to decreased uptake and/or efflux. A variety of specific or nonspecific
mechanisms may also affect redox transformations, intracellular chelation, and
intracellular precipitation. Biomineral formation (biomineralization) may be biolog-
ically induced, i.e., caused by physicochemical environmental changes mediated by
the microbes, or biologically controlled (solid rectangles) (Gadd 2009).

6.9 How Microorganism Are Used to Effect the Process

of Metal Remediation

The utilization of organisms, primarily microbes, to clean up contaminated soils,

aquifers, sludges, residues, and air, known as “bioremediation,” is a rapidly chang-
ing and expanding area of environmental biotechnology that offers a potentially
more effective and economical clean-up technique than conventional physicochem-
ical methods (Garbisu and Alkorta 2003). The use of microbes to change the
concentration of heavy metals in soil and improve the ability of plants to deal with
elevated metals concentrations has significant economic and ecological benefits (Jin
et al. 2018).
Microbes are known for enhancement of plant growth and survival under heavy
metal stress condition as they have the capability of consuming waste and converting
the complex waste into simple nontoxic by-products/compounds. This is feasible
because microorganisms have developed many resistance mechanisms for survival
in the presence of toxic heavy metals in their environment (Mustapha and Halimoon
2015).
130 A. Inobeme

These microbes interact with metals and minerals in natural and synthetic envi-
ronments, altering their physical and chemical state, with metals and minerals also
able to affect microbial growth, activity, and survival (Gadd 2010).
The use of microbial remediation has become more common, and it is generally
considered promising owing to its many advantages, including retention of soil
structure, and the fact that the pollutants and microbes can be almost completely
removed from the soils, and secondary pollution can be avoided (Singh and Prasad
2015). Microbial remediation therefore presents new techniques for addressing the
problem of heavy metal pollution in soil, and it has become a focus of new research
and development in bioremediation technology (Jin et al. 2018).
Sodango et al. (2018) reported algae to be a more efficient biosorbent (with a
reported rate of 15.3–84.6%) compared to other microbial biosorbents (bacteria and
fungi). Microbes have a variety of properties that can effect changes in metal
speciation, toxicity, and mobility, as well as mineral formation or mineral dissolution
or deterioration.
Microbes possess transport systems for essential metals; inessential metal species
can also be taken up. Microbes are also capable of mediating metal and mineral
bioprecipitation, e.g., by metabolite production, by changing the physico-chemical
microenvironmental conditions around the biomass, and also by the indirect release
of metal-precipitating substances from other activities, e.g., phosphate from organic
decomposition or phosphate mineral solubilization.
Many different metal-containing minerals formed as a direct or indirect result of
microbial activity, e.g., various carbonates, phosphates, etc., are omitted from the
table. Microbial cell walls, outer layers, and exopolymers can sorb, bind, or entrap
many soluble and insoluble metal species as well as, e.g., clay minerals, colloids,
oxides, etc. which also have significant metal-sorption properties. Redox transfor-
mations are also widespread in microbial metabolism; some are also mediated by the
chemical activity of structural components (Gadd 2010).
Fungal cell wall contains different materials which proved to be efficient metal
biosorbents. Due to their properties, some fungal species, such as Aspergillus niger
or Mucor rouxii, have been successfully investigated for their usefulness in adsorp-
tion of heavy metals and therefore metal removal from various environments (Javaid
et al. 2011; Joshi and Sahu 2014).

6.10 Effect of Heavy Metals on Soil Microorganisms

Monitoring the structure of microbial communities and animals is a sensitive tool for
the assessment of soil quality and health. Generally, the microbial parameters in the
soil ecosystems are considered to be the best indicators reflecting changing soil
quality and properties, and thus, enabling early detection of soil degradation
(Fazekašova and Fazekaš 2019).
Heavy metals also have large effects on processes important for soil fertility by
affecting the structure and function of microbial communities (Sandaa et al. 1999).
 microorganisms
Article
Microbiological Sulfide Removal—From Microorganism
Isolation to Treatment of Industrial Effluent
Zhendong Yang 1 , Zhenghua Liu 2 , Aleksandra Sklodowska 1 , Marcin Musialowski 1 , Tomasz Bajda 3 ,
Huaqun Yin 2 and Lukasz Drewniak 1, *






Citation: Yang, Z.; Liu, Z.;
Sklodowska, A.; Musialowski, M.;
Bajda, T.; Yin, H.; Drewniak, L.
Microbiological Sulfide
Removal—From Microorganism
Isolation to Treatment of Industrial
Effluent. Microorganisms 2021, 9, 611.
https://doi.org/10.3390/
microorganisms9030611
Academic Editor: Anna H. Kaksonen
Received: 14 February 2021
Accepted: 14 March 2021
Published: 16 March 2021
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
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iations.
Copyright: © 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
Microorganisms 2021, 9, 611. https://doi.org/10.3390/microorganisms9030611
1 Institute of Microbiology, Faculty of Biology, University of Warsaw, Miecznikowa 1, 02-096 Warsaw, Poland;
zyang@biol.uw.edu.pl (Z.Y.); asklodowska@biol.uw.edu.pl (A.S.);
marcinmusialowski@student.uw.edu.pl (M.M.)
2 School of Minerals Processing and Bioengineering, Central South University, No. 932 Lushan South Road,
Changsha 410083, China; lzh2019@csu.edu.cn (Z.L.); yinhuaqun_cs@sina.com (H.Y.)
3 Faculty of Geology, Geophysics and Environmental Protection, AGH University of Science and Technology in
Krakow, A. Mickiewicza 30, 30-059 Krakow, Poland; bajda@agh.edu.pl
* Correspondence: ldrewniak@biol.uw.edu.pl; Tel./Fax: +48-22-55-41-219
Abstract: Management of excessive aqueous sulfide is one of the most significant challenges of
treating effluent after biological sulfate reduction for metal recovery from hydrometallurgical leachate.
The main objective of this study was to characterize and verify the effectiveness of a sulfide-oxidizing
bacterial (SOB) consortium isolated from post-mining wastes for sulfide removal from industrial
leachate through elemental sulfur production. The isolated SOB has a complete sulfur-oxidizing
metabolic system encoded by sox genes and is dominated by the Arcobacter genus. XRD analysis
confirmed the presence of elemental sulfur in the collected sediment during cultivation of the SOB in
synthetic medium under controlled physicochemical conditions. The growth yield after three days
of cultivation reached ~2.34 gprotein /molsulfid , while approximately 84% of sulfide was transformed
into elemental sulfur after 5 days of incubation. Verification of isolated SOB on the industrial
effluent confirmed that it can be used for effective sulfide concentration reduction (~100% reduced
from the initial 75.3 mg/L), but for complete leachate treatment (acceptable for discharged limits),
bioaugmentation with other bacteria is required to ensure adequate reduction of chemical oxygen
demand (COD).
Keywords: sulfide-oxidizing bacteria; sulfur production; sulfide depletion; sulfate reduction
1. Introduction
Hydrometallurgical effluents containing sulfate and heavy metals are produced widely
from streamlines of, for example, electronics, mining, and electroplating industries [1].
Metal sulfide precipitation followed by biogenic sulfide generation of sulfate-reducing bac-
teria was frequently applied to metal recovery from industrial effluents instead of chemical
approaches, such as ion exchange, adsorption, and electrolysis, etc. [2–4] because of its
low cost and high efficiency [5]. However, the high concentration of sulfide significantly
restricts the activity of sulfate-reducing bacteria [6]. In addition, sulfide is a highly toxic
substance concerned with odor and corrosion and can be released into the air through
volatilization as H2 S gas, posing a risk to human health. Methods like adding biochar
were proposed to decrease its toxicity to the microbial community in the sulfate-reducing
system [7]. However, free sulfide cannot be completely contained because a lack of sulfide
also notably limits metal removal by precipitation [8]. On the other hand, 1 mg/L of sulfide
is already considered corrosive [9], with an odor threshold at 0.18 mg/L [10]. Therefore,
it is necessary to treat the remaining sulfide after metal sulfide precipitation before such
water enters the environment.
Removing sulfide often refers to its oxidation either to elemental sulfur or sulfate [11,12].
Sulfate is produced when the complete oxidation of sulfide is realized. This is in contrast
https://www.mdpi.com/journal/microorganisms
Microorganisms 2021, 9, 611 2 of 17

to the production of biogenic sulfides, and it is less attractive to treat excessive sulfide after
sulfate reduction. While the aim of a circular economy focuses on sustainable production
from waste, transforming sulfide to elemental sulfur by partial oxidation is attracting
more attention. Elemental sulfur shows considerable application potential in soil amend-
ment [13]. Chemical oxidants such as KMnO4 and H2 O2 have been successfully used to
produce sulfur from sulfide [14]. However, the biological oxidation of sulfides to sulfur
with sulfide-oxidizing bacteria (SOB), which are also known as sulfur compound oxidizing
bacteria, is more commonly used to reduce the cost of this process [15–17].
Many studies reported the application of SOB to produce sulfur by oxidizing sulfide,
of which chemolithoautotrophs are of the greatest interest [17,18]. With the presence of
limited oxygen supply, these bacteria utilize inorganic reduced sulfur, especially sulfide,
as the electron donors and energy source for metabolic activities, and CO2 as the carbon
source. Studies with a special focus on removing sulfide in the gas phase were conducted.
A good example is the THIOPAQ® technology that oxidizes sulfide producing sulfur
in a synthetic medium after washing off H2 S gas adsorbed in a column [19]. The SOB
work effectively in a controlled system, where sulfur is formed as the terminal product
instead of sulfate. The optimal parameters, e.g., pH, oxygen level, and temperature,
for chemolithoautotrophic-producing sulfur were well-elucidated by Krayzelova et al.
(2015) [18]. However, actual industrial wastewater presents a more difficult and complex
environment for the survival of SOB, which has to compete with other bacteria. Not
only sulfide but other contents need to be depleted, such as high COD (chemical oxygen
demand). Changing the physicochemical conditions, in this case, for spontaneous removal
of all contaminants becomes relatively difficult.
This work aims to evaluate an SOB consortium isolated from post-mining wastes
and explore new strategies for biological sulfide depletion and sulfur production in terms
of aqueous industrial effluent. We focused on the metabolic functions and physiological
characteristics of the microbial consortium. Particular emphasis was placed on its use
in the treatment of water loaded with sulfides and high COD after biological reduction
of sulfates. Specifically, we sought to address the following questions: (i) What are the
sulfide oxidizers in the consortium; (ii) how does the SOB consortium produce sulfur under
optimal conditions in the synthetic medium, and (iii) how can the SOB consortium be used
for treating industrial effluent?

2. Materials and Methods

2.1. Microbial Consortium for Sulfide Oxidation
Samples were taken from post-mining wastes deposited in a dump located near a
uranium mine in Radoniow, Poland. The characteristics of these wastes were described
by Rewerski et al. (2014) [20]. Serial passaging of the SOB consortium was carried out on
a modified Beijerinck medium [21] (MB medium): Na2 S2 O3 (10 g/L), KH2 PO4 (4 g/L),
K2 HPO4 (4 g/L), NH4 Cl (1.6 g/L), MgSO4 ·7H2 O 0.8 g/L. In MB medium, thiosulfate was
used (instead of sulfide), which is often applied for the isolation of obligate chemolithoau-
totrophic SOB [22]. It is easily soluble and non-toxic, while the external sulfur in S2 O3 2−
ion has an oxidation state of −2 as sulfide ion. For the isolation process of SOB consortium,
the production of S[0] is not the targeting aspect. Further, standard potential (in volts) for
the oxidation of thiosulfate to sulfate is lower than sulfide to sulfate [23]. Thus, employing
thiosulfate as the substrate of electron donor for SOB enables complete oxidation to sulfate
S[6+], which is easier for process control: (i) thiosulfate causes no volatilization; (ii) no
controlled oxygen supply is needed. After a series of 10 passages on the MB medium, the
consortium was subjected to detailed metagenomic analysis dedicated to illustrating the
sulfur microbial structure and metabolic pathways.
For the first passage, 10 g of sample was added to a 500 mL Erlenmeyer flask filled
with 200 mL of MB medium. The flask was shaken at room temperature (120 rpm) for three
days. Afterwards, 100 mL of cell suspension was collected and centrifuged. The cell pellet
was then resuspended in 200 mL of fresh MB medium poured into a new Erlenmeyer flask
Microorganisms 2021, 9, 611 15 of 17

utilize these low molecular weight compounds. The sulfate-reducing bacteria might be
one of the functional microbes as the effluent provides them the perfect electron acceptor
(sulfate) and carbon source (VFA). However, the sulfate concentration was stable, while
the sulfide concentration decreased. This indicates that complete sulfide oxidation to
sulfate partially took place in the first stage, which approximately balanced the sulfate
consumption by microbial sulfate reduction. This is confirmed by the result of the control
variant, that is, the sulfate concentration slightly increased in the first stage. At the second
stage, VFA concentration decreased rapidly, ending at 98.3 mg/L. In the control variant,
the COD and VFA concentration changes as a function of time were similar to that in the
bio-oxidation. In the first 5 days, very limited COD and VFA removal was realized. At
day 6, the COD and VFA removal markedly raised, followed by the increase of oxygen
concentration from 1% to 100%. The final concentration of COD and VFA after 14 days
reached 2054 and 215 mg/L, respectively, indicating that oxygen strikingly improves the
activity of indigenous bacteria, decomposing the organic substrates in the effluent. These
results suggest that most of the organic substrates, especially VFA, in the effluent can be
simply removed by increasing the oxygen concentration. On this basis, the application of
the bacteria consortium considerably improves their removal efficiency.

4. Conclusions
In conclusion, a SOB consortium that can produce elemental sulfur by microbial sulfide
oxidation was isolated from post-mining wastes. Metagenome analysis of the SOB showed
that Acrobacter may be the main functioning genus responsible for sulfur production,
because it contains a complete set of genes encoding sulfide-oxidizing system and is the
most represented among the consortium. The activity of the SOB consortium was confirmed
in both synthetic medium and industrial effluents. However, to manage industrial effluents
after sulfate reduction, the depletion of both sulfide and COD is needed. By controlling the
oxygen supply, we developed a two-stage strategy using the SOB consortium and another
commercial bacterial consortium, whereas fast removal of sulfide and COD (including
VFA) were achieved. Our work presents a sustainable model for aqueous sulfide depletion
in industrial effluent containing significant organic matter. However, few questions are
still unclear with our concerns: (i) how to separate/collect the produced sulfur from the
industrial effluent; (ii) what changes in the microbial community take place after sulfur
production, and (iii) is it possible to effectively adopt SOB in a passive system with lower
costs and lower energy demand.

Author Contributions: Conceptualization, Z.Y. and L.D.; methodology, Z.Y., L.D., and A.S.; val-
idation, Z.Y., L.D., and A.S.; formal analysis, Z.Y., Z.L., M.M., T.B., and L.D.; investigation, Z.Y.,
Z.L., M.M., T.B., and L.D.; resources, A.S. and L.D.; data curation, Z.Y., H.Y., and L.D.; writing
(original draft preparation), Z.Y. and L.D.; writing, review and editing, Z.Y., A.S., M.M., H.Y., and
L.D.; visualization, Z.Y., Z.L., and M.M.; supervision, L.D.; project administration, L.D.; funding
acquisition, L.D. All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by Foundation for Polish Science, grant number POIR.04.04-
00-00-2053/16 (TEAM TECH 2016-2/9) as a part of Measure 4.4 of the 2014-2020 Smart Growth
Operational Programme, EU.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Sequencing data generated within this study are deposited in NCBI
Genbank under the BioProject ID PRJNA705003.
Acknowledgments: We gratefully thank Witold Uhrynowski, Bartosz Rewerski, and Robert Stasiuk
for their excellent laboratory assistance.
Conflicts of Interest: The authors declare no conflict of interest.
 TYPE Review
PUBLISHED 07 March 2023
DOI 10.3389/fmicb.2023.1089630

Reaching unreachables: Obstacles

OPEN ACCESS and successes of microbial
cultivation and their reasons
EDITED BY
Romy Chakraborty,
Berkeley Lab (DOE),
United States

REVIEWED BY Gabriela Kapinusova †, Marco A. Lopez Marin † and Ondrej Uhlik *

Slava Epstein,
Department of Biochemistry and Microbiology, Faculty of Food and Biochemical Technology, University
Northeastern University,
of Chemistry and Technology, Prague, Czechia
United States
Brendan Paul Burns,
University of New South Wales,
Australia In terms of the number and diversity of living units, the prokaryotic empire
*CORRESPONDENCE
is the most represented form of life on Earth, and yet it is still to a significant
Ondrej Uhlik degree shrouded in darkness. This microbial “dark matter” hides a great deal of
ondrej.uhlik@vscht.cz potential in terms of phylogenetically or metabolically diverse microorganisms,
These authors have contributed equally to this
†
and thus it is important to acquire them in pure culture. However, do we know
work what microorganisms really need for their growth, and what the obstacles are
SPECIALTY SECTION to the cultivation of previously unidentified taxa? Here we review common
This article was submitted to
and sometimes unexpected requirements of environmental microorganisms,
Terrestrial Microbiology,
a section of the journal especially soil-harbored bacteria, needed for their replication and cultivation.
Frontiers in Microbiology These requirements include resuscitation stimuli, physical and chemical factors
RECEIVED 04 November 2022 aiding cultivation, growth factors, and co-cultivation in a laboratory and natural
ACCEPTED 10 February 2023 microbial neighborhood.
PUBLISHED 07 March 2023

CITATION
KEYWORDS
Kapinusova G, Lopez Marin MA and
Uhlik O (2023) Reaching unreachables: environmental microbiome, microbial ecology, dormancy, VBNC, growth factors,
Obstacles and successes of microbial cultivation techniques, improved cultivation, difficult-to-culture microorganisms
cultivation and their reasons.
Front. Microbiol. 14:1089630.
doi: 10.3389/fmicb.2023.1089630

COPYRIGHT
© 2023 Kapinusova, Lopez Marin and Uhlik.
This is an open-access article distributed under
the terms of the Creative Commons Attribution
License (CC BY). The use, distribution or
reproduction in other forums is permitted,
provided the original author(s) and the
copyright owner(s) are credited and that the
original publication in this journal is cited, in
accordance with accepted academic practice.
No use, distribution or reproduction is
permitted which does not comply with these
terms.
Introduction
The planet we know today is largely the result of the microbial activity in the biosphere.
Earth’s smallest and simplest organisms created the conditions for the development of the vast
number of life forms we all know. The microscopic world is even vaster, and its diversity is
stunning, but it is very difficult to reach. Even though its existence has been acknowledged for
several centuries, it has been very challenging to study its roles. A crucial advance in the study
of “the unreachables” arose in the days of Robert Koch at the end of the 19th century.
He established a causative relationship between a microbe and its impact on a host (disease).
Koch’s postulates demanded the presence of a microorganism in pure culture, isolated from the
host, to confirm the link between the pathogen and the disease. From this point on, microbes
were no longer considered scientific curiosities, but rather modelers of our bodies and Earth’s
ecosystems (Turnbaugh et al., 2007; Graham et al., 2016; Gilbert et al., 2018). Much more efforts
have been taken over the following decades to study microorganisms: these progressed from the
description of and fight against the most critical human and plant pathogens, which dramatically
improved our quality of life, to the later investigations on the community composition of
different environments, the most advanced of which used marker gene or metagenome
sequencing (Lane et al., 1985; Lynch et al., 2012). In recent years, sequencing technologies have
addressed many environmental and human health-associated issues, such as the analysis of
microbial responses to contamination (Hemme et al., 2010), the discovery of novel taxa to
be used for bioremediation, the discovery of novel producers of antibiotics (Ling et al., 2015),

Frontiers in Microbiology 01 frontiersin.org

Kapinusova et al.
or revealing the co-occurrence of antibiotic resistance genes in
different environments (Li et al., 2015), to name a few.
Microorganisms live in virtually any environment, including
those considered extreme due to their high temperature, pH, salinity,
or concentration of pollutants (Mirzaie et al., 2015; Mehetre et al.,
2018; Panda et al., 2018; Power et al., 2018; Maza et al., 2019). The
physiological and biochemical potential of microbes living within
these extreme environments is enormous. Thriving at the limits of life,
extremophilic and extremotolerant microorganisms can provide
enzymes such as the widely used Taq polymerase isolated from
Thermus aquaticus (Brock, 1967; Brock and Freeze, 1969); or
uncommon metabolites, such as previously unknown lipids
(Schneider et al., 2019), unusual polyunsaturated fatty acids (Řezanka
et al., 2019), antioxidants, pigments (Asker et al., 2012), bioactive
natural compounds and other secondary metabolites with a wide
range of applications (Lewis et al., 2010; Manivasagan et al., 2014).
Microbes can also offer improved bioremediation possibilities
(Pascoal et al., 2020), can assimilate unusual substrates (Xu R. et al.,
2018) including toxic compounds, or resist and detoxify several
antibiotics (Rettedal et al., 2014; McLain et al., 2016).
In order to fully describe these microorganisms and reveal their
vast potential, it is necessary to obtain them in pure culture. Moreover,
cultivation provides context to the metagenomic data (Nichols, 2007)
and helps us verify metagenome-based conclusions on microbial
interactions (microbe-microbe, microbe-plant, microbe-
environment). However, bringing environmental microbes to pure
culture under standard laboratory conditions has proven to be a very
challenging task. Cultivation can be labor-intensive, tiring, time-
consuming, and may not ensure success; but it can be rewarding if all
the factors required for microbial growth are included (Figure 1). Here
we discuss some generalities that elucidate the phenomenon of
unculturability, with special attention paid to soil, being a habitat that
harbors the greatest diversity of microorganisms, to build a foundation
upon which to review some of the recent strategies to better reach
“the unreachables.”
Why do you not grow?
If it is alive, no microorganism is unreachable: we just do not
know how to recreate their natural environment in order to obtain a
pure culture (Watve et al., 2000; Stewart, 2012). With this in mind, the
key step toward successful cultivation would be to replicate essential
aspects of the microorganism’s natural existence as thoroughly as
possible (Figure 1). Some of the environmental variables are easily
discovered and can be readily incorporated into cultivation
methodologies, but many other factors that influence growth are
much more obscure, and including them in cultivation strategies is
not as straightforward.
The environment in which microorganisms exist is usually
different from the one we create for them in the laboratory.
Microorganisms live under what Koch (1971) called a “feast and
famine existence.” As a consequence, the growth dynamic observed
under nutrient-rich laboratory conditions does not necessarily exist
in nature, where environmental changes are common and poor
nutritional conditions need to be withstood for longer periods of time
(Koch, 2001; Pinto et al., 2015). Microorganisms can be categorized
by their resource intake characteristics either as oligotrophs or
Frontiers in Microbiology
10.3389/fmicb.2023.1089630
copiotrophs (Meyer, 1994; Fierer et al., 2010). The main distinguishing
parameters between these categories, as Ho et al. (2017) states, are
their growth kinetics, substrate affinity, and efficiency at substrate
utilization. Copiotrophs have higher Michaelis–Menten kinetics and
maximal growth rate. Conversely, oligotrophs are slow-growing but
have higher substrate utilization efficiency, and thus higher biomass
yields per substrate molecule utilized. Oligotrophs thrive in
environments with low nutrient flows, but not in substrate-rich/
diverse environments. Copiotrophs, on the other hand, can utilize
highly concentrated substrates rapidly and react promptly to substrate
changes; they nevertheless lack the necessary regulatory mechanism
of starvation, and are thus generally unable to grow in nutrient-poor
sites (Ho et al., 2017).
The proportion of copiotrophs to oligotrophs in the environment,
as well as under laboratory conditions, is governed by a dynamic
process called succession (Fierer et al., 2010). Microbial communities
change over time after they colonize a certain environment. For
heterotrophic bacteria, organic carbon can be constantly supplied, i.e.,
exogenous succession, or present all at once at the initial colonization
point, i.e., endogenous succession (Fierer et al., 2010). In the initial
stage of endogenous succession, when nutrients are plentiful,
copiotrophs are more abundant in the community; oligotrophs
become dominant when highly concentrated substrates are depleted
(Song et al., 2016). Both the changing environmental conditions in
nature and an inappropriate choice of growth conditions in the
laboratory hinder the ability of microorganisms to replicate and could
thus render them dormant and seemingly unculturable.
Do not wake up until it is beautiful
outside
The low number of microbes cultivated in the laboratory
compared with the total number of microorganisms observed under
the microscope hinted at the existence of other states in which
microorganisms may exist in nature, apart from being alive
(replicating) or “dead” (non-replicating). This discrepancy, known as
the “great plate count anomaly,” is a large difference, by several orders
of magnitude, between the viable plate counts and the total direct
microscopic counts (Staley and Konopka, 1985). This phenomenon
reveals our failure to isolate all cells from a particular environment in
pure cultures. Just as cells wait in a quiescent state for environmental
conditions to be favorable again and start replicating (Kaprelyants
et al., 1994), they can be waiting for these optimal conditions when
deposited in the laboratory environment. Grandly said, microbes can
be unreachable because they are “sleeping” (Xu et al., 1982).
The term “sleeping cells” encompasses several dormancy or
quiescence phenomena that can cause unculturability under
laboratory conditions. Dormancy is “any rest period or reversible
interruption of the phenotypic development of an organism”
(Sussman and Halvorson, 1966), or simply a state of metabolic
inactivity as defined by Kell et al. (1998): cells exhibit negligible
metabolic activity but can later transit to a growing state. This
inactivity can be caused by the advent of unfavorable conditions, for
example, the famine period in the dual feast-famine existence. Several
dormancy phenomena have been identified, which suggests the
existence of a “dormancy continuum,” where some states of dormancy
can be deeper than others (Ayrapetyan et al., 2015). The most
02 frontiersin.org
Kapinusova et al.
changes or pH changes (Du et al., 2007). The inoculation of cells from
their environment into artificial media can potentially trigger such a
state. For example, when cultivating oligotrophs, the usage of a
nutrient-rich medium can lead to cellular death; this may be a result
of a depletion of energy for balanced growth or by osmotic shock
caused by the sudden intake of non-metabolic complex substrates
(Ho et al., 2017). In the VBNC state, cells do not replicate but remain
viable after being exposed to stressful conditions (Xu et al., 1982).
VBNC cells are also different from metabolically active and dividing
cells, since they perform respiration and gene expression at low rates
(del Mar et al., 2000; Shleeva et al., 2004; Li et al., 2014). They are also
able to change their adhesion properties and virulence potential
(Rahman et al., 1994; Du et al., 2007). Furthermore, their lower
metabolic rate, strengthened cell wall and higher peptidoglycan
cross-linking confer them better physical and chemical resistance, as
opposed to normally dividing cells (Signoretto et al., 2000). When
activity rates are reduced, VBNC cells also reduce their size (Biosca
et al., 1996), increase their surface-to-volume ratio (Li et al., 2014),
and, as a consequence, their nutrient intake increases (Baker et al.,
1983). This size reduction was observed in Burkholderia pseudomallei
and Vibrio cholerae cells when changing from rods during exponential
growth to cocci in the VBNC state (Inglis and Sagripanti, 2006; Senoh
et al., 2010).
Dormancy can be one of the many reasons for unculturability,
which biases the picture of the community observed via culturing
methods. Fortunately, dormant cells are not totally unculturable but
can be more challenging to culture because not only must their
growth conditions be elucidated but also their resuscitation
mechanisms. Two different mechanisms are thought to resuscitate
microorganisms from dormant stages: either they depend on some
environmental queue to do so or they do not, the latter situation
being called the scout hypothesis (Epstein, 2009; Buerger et al., 2012).
This stochastic reactivation of growth is the consequence of
phenotypic variation within the dormant population (Sturm and
Dworkin, 2015), which resembles the idea of the “dormancy
continuum” previously mentioned. In both cases, knowing which
factors are present in the environments where microorganisms dwell
can teach us what is necessary for their effective culturing in the
laboratory (Figure 2). If they wake up stochastically, they still need
environment-resembling conditions where they can thrive after
awakening. If they need environmental stimuli, then these would
need to be included in in vitro cultivations for microorganisms to
resuscitate and grow (Figure 2).
The stimuli needed to resuscitate microorganisms from
dormancy include physical and chemical stimuli, which can
be provided by the environment or by organisms to which
yet-unculturable microbes are associated (Zhang et al., 2021). In this
sense, the conditions needed to support growth in the laboratory
medium can overlap with those to resuscitate microbes from
dormancy but both phenomena correspond to different physiological
processes, namely the exit from a reduced metabolic existence, after
which comes the ability to replicate. Zhang et al. (2021) reviewed the
factors that play a role in the resuscitation of VBNC organisms such
as the addition of metabolites to minimize oxidative stress, quorum
sensing autoinducers or temperature changes. In the present
manuscript, the focus will be on those factors aiding the growth of
microorganisms in the laboratory environment and what cultivation
implies for modern microbiology.
Frontiers in Microbiology
10.3389/fmicb.2023.1089630
A helping hand from the environment
– Physical and chemical factors
Temperature, pH, osmotic pressure, and oxygen and nutrient
concentrations are ever-changing factors in the environment (Puspita
et al., 2012). These changing conditions are stress factors that shape
the composition of microbial communities as well as the environments
in which they live. Soil pH has a major impact since it influences soil
chemistry, including the availability of organic matter, redox
conditions, and oxygen availability (Anderson et al., 2018). Energy-
yielding metabolisms such as microbial respiration (Jin and Kirk,
2018) and the hydroxylated lipid membrane composition (Wang et al.,
2016) also respond strongly to pH changes. The impact of pH on soil
chemistry even shapes the assembly of microbial communities on a
global scale (Feng et al., 2014; Tripathi et al., 2018). In this regard,
according to a cross-continental phylogenetic survey of over eighty
soils representing a wide range of ecosystems, soil pH was significantly
correlated with the overall bacterial community composition (Lauber
et al., 2009). The pH has been shown to significantly influence the
community structure of other environments such as lakes (Ren et al.,
2015), permafrost (Ren et al., 2018), and animal microbiomes (Sylvain
et al., 2016). Even small changes in this variable can thwart growth on
an artificial medium since some microorganisms have a very narrow
zone of pH tolerance (Rousk et al., 2010). Adamberg et al. (2003) used
a pH-auxostat to study the growth rate decrease of different lactic acid
bacterial strains. A pH decrease from 6 to 4.3 was enough to slow
down the bacterial growth rate, and ATP production was also lowered.
However, microbial growth is not only affected by drastic changes in
pH disabling microbial growth, but also by suboptimal pH, at which
cell growth is detectable but the growth rate is significantly decreased,
as was shown in the cultivation study of Bacillus termoamylovorans
when pH changes by ~1.5 from the optimal pH for its growth caused
a significant reduction in the growth rate and thus caused a reduction
in energy yield per glucose molecule consumed (Combet-Blanc et al.,
1995). However, there are cases when the microbes themselves,
intentionally and unintentionally, are able to adjust the pH of their
near environment, even by excreting basic metabolites or enzymes,
and thus shape the microbial community and subsequently determine
the interactions between individual species of the consortium (Ratzke
and Gore, 2018).
Oxygen concentration also shapes the composition of entire
microbial niches: whether it is oxygen-requiring algae, microaerophilic
or facultatively anaerobic purple non-sulfur photoheterotrophs,
anaerobic green-sulfur bacteria, or any chemotrophs, the development
of individual subpopulations is impacted based on their relationship
to oxygen. Not just the simple dichotomy of aerobic and anaerobic
conditions is important, but also small, specific changes in oxygen
concentration matter. For instance, Coxiella burnetii, the intracellular
pathogenic agent of Q-fever, infects mammalian cells at a microaerobic
concentration of O2 ~ 3%. Omsland et al. (2009) successfully cultivated
an axenic culture of Coxiella burnetii on an improved acidified citrate
cysteine medium under an oxygen tension of 2.5%–5%. Because of its
ability to grow at lower oxygen levels, the hitherto uncultured Coxiella
burnetii was able to utilize up to 17 different substrates and form
visible colonies in the absence of host cells. Recently, C. burnetii was
cultured in a modular hypoxic chamber that maintains the required
O2 concentration (2.5%) without constant airflow, which greatly
reduces the evaporation of the medium (Miller et al., 2020).
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FIGURE 2
The list of factors affecting microorganisms in their environment (inner circle), and strategic approaches reflecting these factors in the cultivation (outer
circle). Created with BioRender.com.
Oxygen concentration also induces oxidative stress caused by
reactive oxygen species. Generally, the ideal oxygen conditions depend
on oxidative stress sensitivity and the need for a reduced form of a
nutrient (Vallejo Esquerra et al., 2017). Since reactive oxygen species
often have a lethal effect on cells, it is desirable to reduce their
concentration to a minimum. Oxidative stress during cultivation can
be reduced by procedures such as autoclaving the agar and the
phosphates separately (Tanaka et al., 2014; Kato et al., 2018, 2020) or
by adding catalase or pyruvate to media (Bogosian et al., 2000; Tanaka
et al., 2014).
Another decisive factor that enhances cultivation success is the
choice of substrates and notably their concentration. Differing carbon
concentrations create niches that are occupied by different bacteria
(Eichorst et al., 2011; Wu et al., 2020). In environments prone to
drastic environmental changes such as soil or water, selective pressure
favors cells with a low metabolic cost existence (Mukamolova et al.,
2003). Diluted, low-carbon media favor slow-growers and increase the
overall diversity, thus increasing the chances of culturing unknown
taxa. Low-carbon media have successfully increased the culturing of
microorganisms coming from a wide range of environments, such as
sea sponges (Karimi et al., 2019; Gutleben et al., 2020), aquatic
environments (Imazaki and Kobori, 2010; Sun et al., 2019), or soils
(Janssen et al., 2002; Molina-Menor et al., 2021). Aquatic environments
offer the advantage of using the water directly from the source as part
of the cultivation media. Applying this strategy, Kapinusova et al.
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(2022) isolated over 100 bacterial species, including several novel
species of Alphaproteobacteria, Betaproteobacteria, Flavobacteriia, and
even a member of a novel genus of Thermoleophilia (Kapinusova et al.,
2022). A similar strategy combined with a prolonged incubation time
was used for the culturomics of the world-renowed thermal springs of
Karlovy Vary (Smrhova et al., 2022) and led to the acquisition of
several thermotolerant strains of the Bacillota phylum and isolation of
novel microorganisms of Bacilli, Gammaproteobacteria, and
Actinomycetia classes. The dilution-to-extinction technique, based on
the cultivation of soil oligotrophic microorganisms on media
containing 100-fold diluted nutrients, resulted in the isolation of a
wide spectrum of the most abundant soil representatives, and also of
members of two previously undescribed actinobacterial lineages
(Bartelme et al., 2020). The combination of the above-mentioned
factors into one modified cultivation procedure, namely an adjusted
N2/CO2 atmosphere (80:20), low substrate concentrations, the
temperature corresponding to the original environment, etc., led to
the successful isolation of members belonging to the OP5 phylum
(Mori et al., 2008), first described by the 16S rRNA gene analysis in a
hot spring in Yellowstone National Park (Hugenholtz et al., 1998).
Since many unreachables are slow-growers, prolonged incubation
times can lead to their successful cultivation. Prolonged cultivations,
usually coupled with culturing diluted cell suspensions, have proved
to be useful in many studies (Eilers et al., 2001; Connon and
Giovannoni, 2002; Rappé et al., 2002; Kakumanu and Williams, 2012;
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Adam et al., 2018; Bender et al., 2020). In a study by Davis et al.
(2005), autochthonous soil cells, as well as non-native cells from
constructed consortia, were counted on six different media at 7-day
intervals. Cell counts increased even after 12 weeks of incubation.
Another successful example of prolonged cultivation, and an
important microbiological milestone, was the isolation of the
previously uncultured archaeon Candidatus Prometheoarchaeum
syntrophicum MK-D1 (Imachi et al., 2020). This extremely slow-
growing Asgard archaeon, related to the Lokiarchaeota, was isolated
from a 2,533 m deep-water sediment in the Nankai trough, Japan.
Aiming to achieve deep-sea microbial cultivation, Imachi et al. (2020)
set up a methane-fed-continuous bioreactor in which the enrichment
cultivation ran for 2,000 days, resulting in the isolation of this archaeon
from a symbiotic culture. The growth of some organisms from cold
and oligotrophic environments, such as those isolated from Antarctica,
can only be seen in culture after prolonged incubation times (Pulschen
et al., 2017; Tahon and Willems, 2017). These organisms form very
small colonies which often have to be observed under a microscope
(Pulschen et al., 2017).
Longer incubations in a Petri dish or batch liquid medium can
be problematic because the composition of the medium tends to
change over time, either because of the action of the organism’s
metabolism or other processes, such as water evaporation. Even
though microbial species with apparently long cultivation times can
have these incubations shortened upon subculturing (Buerger et al.,
2012), their initial isolation from the natural environment could fail if
they are cultured together with a faster-growing species. Slow-growing
microorganisms can be disadvantaged mainly when microorganisms
from complex consortia are attempted to be cultured together.
Physically separating or sorting the microorganisms before their
culturing is a helping strategy to overcome this problem and it is
discussed further in the text.
Periodically varying conditions exist in nature, from the feast and
famine cycles (Koch, 1971) to alternating oxic and anoxic periods
(Dorofeev et al., 2019) and seasonality (Steiner et al., 2020), all of
which can affect microbial communities. Besides culturing in
continuous cultures (open systems) or batch cultures (closed systems),
cyclic cultivation can be useful for microorganisms with a cyclic type
of metabolism. This metabolism is divided into two phases: first,
energy and carbon sources are accumulated, which are then used in
the second phase to biosynthesize biomass (Dorofeev et al., 2014). Any
of the above-mentioned culture parameters (e.g., temperature, oxygen,
or substrate concentration) can be the cycling factor in the cultivation
strategy (Dorofeev et al., 2014).
Some growth-influencing factors can be more enigmatic. One
such factor is acoustic vibration, which is useful as a cultivation
enhancement in several biotechnological studies (Bochu et al., 2003;
Avhad and Rathod, 2015; Huang et al., 2017). By causing (i) cavitation
and repairable damage in microbial cells, (ii) loosening of microbial
aggregates in liquid cultures, and (iii) an increase in cell membrane
permeability, ultrasonic low-intensity waves (∼20 kHz) can increase
the substrate intake in microbial cells and subsequently enhance
microbial proliferation (Huang et al., 2017, 2021), and thus can help
in the cultivation of the unreachables.
Similarly, all the aforementioned culturing parameters can
be combined in a high-throughput fashion to describe as much of the
community composition as possible using cultivation, with each
condition used being “a different aspect of the community’s picture.”
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This approach is referred to as culturomics (Greub, 2012). Bacteria
obtained in culture are massively characterized using MALDI
TOF-MS, or 16S rRNA gene sequencing (Strejcek et al., 2018;
Nowrotek et al., 2019).
A helping hand from the surroundings
– Carrier particles
Many prokaryotes prefer to live attached to surfaces rather than
in a dispersed, single-celled planktonic state (Mills, 2003; Flemming
and Wingender, 2010; Hemkemeyer et al., 2018). In soils, different
particle size fractions (PSFs) have a different impact on the
concentration, chemical composition, and availability of organic
matter (Christensen, 1992; Hemkemeyer et al., 2018). Organic matter
is associated with fine-sized particles such as silt and clay; nevertheless,
the sand fraction contains most of the free particulate organic matter
(POM; Christensen, 2001), and therefore represents the fraction with
the highest availability of substrates. The reported reduction in
diversity among larger-sized fractions can be caused by low nutrient
availability, protozoan grazing, and competition with fungi (Sessitsch
et al., 2001). Hence, Hemkemeyer et al. (2018) observed the suitability
of different PSFs and their associated POM to harbor microbial
communities differing in their structure, functional potential, and
sensitivity to environmental conditions. Genetic fingerprinting
showed very strong preferences of the observed bacterial communities
(up to 56% OTUs) for specific PSFs, while the archaeal populations
did not exhibit significant preferences. Members of Bacteroidota and
Alphaproteobacteria preferred the sand-sized fraction with POM,
while Actinomycetota and Betaproteobacteria preferred fine silt,
Planctomycetales clay, and Gemmatimonadales coarse silt
(Hemkemeyer et al., 2018).
If cells prefer living in close contact with surfaces, it can result in
it being difficult for them to grow in liquid media. Surfaces composed
of different materials such as glass, steel, or synthetic polymeric
substances such as polyurethane foams can enhance the cultivation of
biofilm-forming bacteria from different natural environments
(Yasumoto-Hirose et al., 2006; Gich et al., 2012; Dellagnezze et al.,
2016). Liquid media provide many advantages compared to solid
media: they guarantee a homogenous distribution of nutrients and
oxygen, while also facilitating the manipulation of cultures. Aiming to
combine the benefits of liquid media while meeting the requirements
of microorganisms that live attached to surfaces, liquid media can
be improved by adding a small amount of gelling agents such as gellan
gum, xanthan gum, or carrageenan (Das et al., 2015), glass beads
(Nguyen et al., 2005; Droce et al., 2013), or sand (Suman et al., 2019).
Adding these supplementary solid agents can help the microorganisms
to attach to the surface but still live and divide in the liquid or
semiliquid medium.
A helping hand from your neighbors
– Growth factors
Trace elements from the environment, apart from the carbon
source, are necessary to guarantee growth in vitro. To give a simple
example, genera of the slow-growing Acidobacteriota living in
manganese-enriched environments benefit from the addition of this
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element into their growth medium (Costa et al., 2020). Complex
matrices, such as soil, harbor many phylogenetically diverse
microorganisms (Bahram et al., 2018) that not only participate in
important biogeochemical cycles (Louca et al., 2019), but also create
conditions that enable the growth of other microorganisms by
sharing metabolites and essential growth substances (Schink, 2002).
These molecules include those that play a role in quorum sensing,
biofilm community cooperation, or in the mutualism between plants
and plant-growth promoting organisms (Jacoby et al., 2017), such as
rhizobacteria and endophytes (Papik et al., 2020). If a metabolite is
available in the environment, microorganisms can lose the metabolic
capability of producing it and thus become metabolically dependent
on their neighborhood (Pande and Kost, 2017). The absence of
neighbors in pure culture, and consequently the absence of the
necessary metabolites, is then one of the reasons behind
unculturability (Pande and Kost, 2017). Bacteria living in certain
environments, such as endophytes, benefit from the use of highly
specialized growth medium containing the environment’s original
metabolites (Gerna et al., 2022).
With the above said, some bacteria can only grow in a pure
medium when in co-culture with another community member, also
called a helper strain, which can be a phylogenetically different
bacterium or even a different organism such as an amoeba (Boilattabi
et al., 2021). Co-culturing can be achieved either by direct culturing
of the helper strain together with the bacterium of interest or by using
spent supernatants as a proxy for the helper strain (Stewart, 2012).
Spent supernatants are the media where the helper strain grew, so the
supernatants contain the metabolites that are potentially essential for
other members of the community. Microbes can also be cultured
together with the host from their natural environments (Knobloch
et al., 2019; Lopez Marin et al., 2021). High-throughput co-culture is
also now possible with devices such as microscale microbial
incubators (Ge et al., 2016), micro-petri dishes (Ingham et al., 2007),
microfluidic devices (Frimat et al., 2011; Burmeister et al., 2019), or
agarose-based microwell chips (Zhang et al., 2019), where hundreds
of single cells can grow in parallel in individual compartments,
sharing metabolites and necessary substances for growth. The latter
approach has proved very helpful in culturing bacteria directly
related to human health, such as antibiotic-resistant pathogens from
the human gut (Versluis et al., 2019).
Metabolites from associated bacteria can provide nutrients or
trigger other stimuli necessary for growth. As was previously
mentioned, when water and nutrients are on the wane and the
surrounding conditions are unfavorable, some cells can enter
dormancy. Dormant cells can be resuscitated by different resuscitation
stimuli (Pinto et al., 2015). There can be many sources of such stimuli,
but they often include substances such as amino acids and peptides
(Nichols et al., 2008; Pinto et al., 2011), metabolites such as N-acyl
homoserine lactones (Batchelor et al., 1997), or resuscitation
promoting factors (Mukamolova et al., 2006; Pinto et al., 2013; Lopez
Marin et al., 2021). For example, in a study by Bruns et al. (2002), the
signaling molecules cAMP and N-(butyryl)-DL-homoserine lactone
(BHL) increased total bacterial counts in highly diluted inocula from
aquatic environments by several orders of magnitude. Thanks to this
effort, the previously uncultured bacterial clone G100, Citreicella
manganoxidans, belonging to the Rhodobacteraceae family, was
cultured (Bruns et al., 2002; Wirth and Whitman, 2018). Less
ambitious but still hopeful results were provided by the follow-up
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studies of Bruns, where the addition of cAMP led to a 10% increase
in MPN values (Bruns et al., 2003). Yet, in several studies where
signaling compounds were used for increasing cultivation yields, the
influence of cAMP on culturability has been disproven (Pernthaler
et al., 2003; Sangwan et al., 2005).
The resuscitation promoting factor (Rpf) produced by
Micrococcus luteus promotes bacterial resuscitation and growth in
the same producing organism (Mukamolova et al., 2006), but can
influence taxa distributed along several other phyla, such as
Pseudomonadota and Bacteroidota (Su et al., 2018; Lopez Marin
et al., 2021; Su et al., 2021). This small protein (16–17 kDa) with a
lysozyme-like structure (Cohen-Gonsaud et al., 2005) promotes
bacterial cell growth even at picomolar concentrations (Mukamolova
et al., 1998; Sexton et al., 2015). Rpf-like encoding genes are
distributed among other prokaryotic genomes, especially in G + C
rich gram-positive Actinomycetota (Nikitushkin et al., 2016), but
Rpf-like proteins extend to other bacterial phyla, such as Bacillota
(Shah and Dworkin, 2010) and Pseudomonadota (Li et al., 2020). The
addition of Rpf during cultivation has resulted in the isolation of
novel bacteria, such as organisms of the genera Rhodococcus and
Arthrobacter, or of the family Alcaligenaceae (Su et al., 2013, 2015,
2018, 2021). Lopez Marin (Lopez Marin et al., 2021) isolated 51
novel bacterial species belonging mainly to the phyla Actinomycetota,
Pseudomonadota, and Bacteroidota on reasoner’s 2A (R2A) agar and
an agar made from the soil’s water-soluble fraction after
supplementing Micrococcus luteus Rpf-containing supernatant to
soils. Some of these species were members of novel genera, such as
Pedomonas mirosovicensis of the family Sphingosinicellaceae, or
Solicola gregarius of the family Nocardioidaceae (Lopez Marin et al.,
2022, 2023). Spent supernatants containing growth factors have also
aided the cultivation of Chloroflexota strains (Xian et al., 2020) or
Leucobacter, the growth of which was supported through the action
of zincmethylphyrins and coproporphyrins produced by
Sphingopyxis sp. (Bhuiyan et al., 2016).
Do you want to stay in your
neighborhood?
The identification of specific substances promoting cell growth is
not an easy task. To bypass the search for crucial growth factors,
microorganisms can be co-cultured with growth-promoting
microorganisms or can be cultivated in situ in the environments they
come from Bollmann et al. (2007) and Remenár et al. (2015). In situ
cultivation allows for the isolation of microorganisms that are more
adapted to the original environment than those originating from the
same habitat but obtained on standard agar media (Jung et al., 2016).
Several innovative devices have been envisioned to deal with in situ
cultivation. In an early attempt, Kaeberlein et al. (2002) developed a
diffusion chamber that allowed the nutrients from the natural
environment to migrate to the site where bacteria were inoculated.
Seawater solidified with agar was sandwiched between two
polycarbonate membranes, which allowed the flow of nutrients from
the natural environment to the agar while at the same time isolating
the inoculum from the natural environment (Kaeberlein et al., 2002).
Diffusion chambers have since increased the diversity of culturable
bacteria (Bollmann et al., 2007), including those that are difficult to
culture, such as members of the phylum Verrucomicrobiota (Pascual
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