The Historical Development of Cultivation Techniques for Methanogens and Other Strict Anaerobes and Their Application in Modern Microbiology

Abstract

The cultivation and investigation of strictly anaerobic microorganisms belong to the fields of anaerobic microbial physiology, microbiology, and biotechnology. Anaerobic cultivation methods differ from classic microbiological techniques in several aspects. The requirement for special instruments, which are designed to prevent the contact of the specimen with air/molecular oxygen by different means of manipulation, makes this field more challenging for general research compared to working with aerobic microorganisms. Anaerobic microbiological methods are required for many purposes, such as for the isolation and characterization of new species and their physiological examination, as well as for anaerobic biotechnological applications or medical indications. This review presents the historical development of methods for the cultivation of strictly anaerobic microorganisms focusing on methanogenic archaea, anaerobic cultivation methods that are still widely used today, novel methods for anaerobic cultivation, and almost forgotten, but still relevant, techniques.

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Citation: Hanišáková, N.; Vítˇezová,
M.; Rittmann, S.K.-M.R. The
Historical Development of
Cultivation Techniques for
Methanogens and Other Strict
Anaerobes and Their Application in
Modern Microbiology.
Microorganisms 2022,10, 412.
https://doi.org/10.3390/
microorganisms10020412
Academic Editor: Marcell Nikolausz
Received: 20 January 2022
Accepted: 9 February 2022
Published: 10 February 2022
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microorganisms
Review
The Historical Development of Cultivation Techniques for
Methanogens and Other Strict Anaerobes and Their
Application in Modern Microbiology
Nikola Hanišáková1, Monika Vítˇezová1, * and Simon K. -M. R. Rittmann 2, *
1Laboratory of Anaerobic Microorganisms, Section of Microbiology, Department of Experimental Biology,
Faculty of Science, Masaryk University, 62500 Brno, Czech Republic; 446645@mail.muni.cz
2Archaea Physiology & Biotechnology Group, Department of Functional and Evolutionary Ecology,
Universität Wien, 1030 Wien, Austria
*Correspondence: vitezova@sci.muni.cz (M.V.); simon.rittmann@univie.ac.at (S.K.-M.R.R.)
Abstract:
The cultivation and investigation of strictly anaerobic microorganisms belong to the fields
of anaerobic microbial physiology, microbiology, and biotechnology. Anaerobic cultivation meth-
ods differ from classic microbiological techniques in several aspects. The requirement for special
instruments, which are designed to prevent the contact of the specimen with air/molecular oxygen
by different means of manipulation, makes this field more challenging for general research compared
to working with aerobic microorganisms. Anaerobic microbiological methods are required for many
purposes, such as for the isolation and characterization of new species and their physiological exam-
ination, as well as for anaerobic biotechnological applications or medical indications. This review
presents the historical development of methods for the cultivation of strictly anaerobic microorgan-
isms focusing on methanogenic archaea, anaerobic cultivation methods that are still widely used
today, novel methods for anaerobic cultivation, and almost forgotten, but still relevant, techniques.
Keywords: methane; anaerobes; methanogens; biogas; cultivation methods
1. Introduction
In the field of microbiology, anaerobic cultivation methods differ from the classic meth-
ods for aerobic cultivation. The main reason for the difference is that while manipulating
strictly anaerobic microorganisms, it is necessary to prevent molecular oxygen (O
2
) expo-
sure to the organisms. This is due to the fact that O
2
is toxic to anaerobic microorganisms,
to varying degrees and depending on the microbe [
1
], and on their oxidation-reduction
potential (ORP), the optimal value of which differs among anaerobic species. As awareness
of microorganisms that are not capable of growing while exposed to air was increasing
towards the end of the 19th century, the need for special methods for their cultivation
and manipulation was realized. The first efforts began more than a century ago and con-
tinued until the present variety of anaerobic cultivation methods and procedures was
established [
2
–
5
]. For the most common anaerobic and aerotolerant microorganisms, culti-
vation techniques are often manageable under ordinary laboratory conditions, but more
sophisticated cultivation methods are required for the study of strictly anaerobic microor-
ganisms, such as methanogens, whose importance and biotechnological applications are
now of utmost interest [6–9].
The relatively demanding procedures for handling strictly anaerobic microorganisms
and technical requirements often prevent the cultivation of these organisms in common
microbiological laboratories. Apart from this, there are laboratory limitations towards
many different anaerobic cultivation setups with regard to experimental conditions. That
means using high-throughput methods or studying requirements towards a particular
gas composition and applied pressure. In addition, the toxicity and flammability of some
Microorganisms 2022,10, 412. https://doi.org/10.3390/microorganisms10020412 https://www.mdpi.com/journal/microorganisms

Microorganisms 2022,10, 412 2 of 28
microbial substrates and product gasses alone is an important factor, which might render
cultivation difficult or impossible in standard microbiological laboratories.
It is of importance to know methods of anaerobic cultivation to perform such experi-
ments, study microorganisms responsible for anaerobic substrate conversion, and measure
their metabolic activity and degradation potential to enable us to fully understand their
physiological processes. Mastering anaerobic cultivation techniques is necessary to de-
scribe new species, for anaerobic research and development, and to advance anaerobic
microbiology and biotechnology.
The aim of this work is to present methods of anaerobic cultivation and their historical
development. Although the primary focus of these techniques is to capture broad methods
developed for cultivation of methanogenic archaea (methanogens), they are applicable to
other strictly anaerobic microorganisms, for example clostridia, sulphate-reducing bacteria
(SRB), or phototrophic bacteria. In addition, this review will introduce the methods of
anaerobic microbiology that have been almost forgotten or are no longer in use, and
show their potential applicability in modern microbiology and biotechnology. This insight
into the principles of anaerobic cultivation might help scientists to choose among these
methods to successfully isolate, cultivate and perform biotechnological experiments with
anaerobic organisms.
2. Milestones in the Historical Development of Anaerobic Microbiology
2.1. The Origin of Anaerobic Cultivation
Methods for cultivation of microorganisms have been developed and adapted since
their discovery. Initially, the studies were aimed at discovering the connection of microbes
to infection, illnesses and to biotechnological applications [
10
,
11
]. Understanding the cause
of the infections and diseases was crucial as it would lead to an understanding of their
transmission, prevention and treatment.
Since the first discovery of ‘animalcules’ in 1680 in Leeuwenhoek’s specimen, the
awareness of organisms smaller than the human eye can see brought a new perspective on
how the world and its processes might be dependent on microbes. Even then, the anaerobic
microorganisms noticed for the first time as some of the ‘animacules’ were growing in
sealed glass tubes and producing gas [
12
]. The existence of organisms that could not be
seen by eye was, of course, something that was never heard of before. However, the biggest
development in microbial cultivation techniques occurred in the 20th century, when the
importance of the natural processes caused by microorganisms increased [13–16].
It is understandable that the first cultivation techniques were aerobic and led to
the discovery and description of bacteria. The greatest advance in the field of microbial
cultivation was made by Robert Koch, who used prepared media for cultivation. The media
were made from broth based on boiled meat or bovine serum. He also used media solidified
by gelatine. However, working with gelatine was also unfruitful, as some bacterial species
are capable of degrading gelatine, and at temperatures higher than 30
◦
C it is impossible
to achieve gelatine solidification. Walter Hesse, with his wife’s suggestion, replaced the
gelatine with agar. Gelatin-like agar is harvested from the red algae Gelidium sp. and
Gracilaria sp. (division Rhodophyta). Agar is more stable, a dilute solution solidifies at 37
◦
C,
does not affect the growth, and is not degraded by most of the microorganisms [
17
]. It was
a breakthrough for the cultivation of plateable organisms.
Anaerobic microorganisms came to Pasteur’s attention during his research on the
acidification of wine. The process was caused by contamination of bacteria that led to
the production of acids. He described the fermentation process and suggested that the
wine-making process was brought about by anaerobic microorganisms. Pasteur also
described the effect that the substrate uptake rate increases under anaerobic conditions
compared to aerobic conditions to produce the same amount of biomass, the so-called
Pasteur effect. He cultivated Clostridium butyricum using meat extract broth as the medium,
which was introduced to a vacuum. Under close examination of the microorganisms under
the microscope, he found that the exposure of the cells to O
2
resulted in a decrease in

Microorganisms 2022,10, 412 3 of 28
motility in some cases. He used the terms ‘aerobic’ and ‘anaerobic’ for the first time and
also developed the first techniques of anoxic media preparation. These involved boiling or
application of a vacuum [18,19].
The recognised significance of anaerobic microorganisms grew over the years, as they were
discovered to be the cause of intoxications and infections leading to gas
gangrenes [4,11,20,21]
.
Studies of the infections led to an increase in the number of anaerobes isolated and described.
In order to cultivate these anaerobes, modifications of the basic microbiological methods needed
to be carried out, and the number of reviews on that topic at that time also increased. In 1929,
I. C. Hall
extended his description of anaerobic bacteria isolation [
2
] and summarized the known
methods of anaerobic cultivation in one of his reviews [
13
]. These methods later led to the
isolation and cultivation of Clostridioides difficile. Thereafter, an interest in the study of C. difficile
emerged, as the organism had been described as an anaerobic, pathogenic microorganism [
22
].
A typical method for anaerobic cultivation used deep tube techniques, with the medium in a
semisolid or solid state (Table A1). For removing O
2
, boiling of the medium was recommended.
The depth of the media column ensured that O
2
reabsorption occurred only in a small surface
volume and anaerobiosis persisted at the bottom of the tube. To reduce the contact with air,
constricted tubes came to use, improved with a mechanical marble seal (Figure 1) [
23
]. Another
option is to apply a layer of mineral oil that seals the surface of the liquid medium and eliminates
contact with air. Since there are many types of oil, Hall suggests that paraffin oil is the most
sufficient to serve as seal [13].
Figure 1. Hall’s constricted tube with marble seal (Hanišáková).
Another anaerobic cultivation method is Buchner’s method, in which pyrogallic acid is
added onto the cotton plug inside the tube and the tube is then sealed with a rubber plug [
5
].
The method was later modified [
24
–
26
]. In addition, gas exhaustion and the use of inert
gas were also described one hundred years ago as a form of anaerobic cultivation [
4
,
21
,
27
].
During experiments with fermentation and anaerobic cultivation of mixed samples, gas
was produced, which aroused curiosity concerning the composition of this gas. The
main components produced from selected anaerobic species were described as carbon
dioxide (CO
2
), molecular hydrogen (H
2
) [
28
], and traces of hydrogen sulphide (H
2
S) [
29
].
Denitrification by bacteria was described as well as its inhibition by O2[30,31].
In the mid-20th century, interest in anaerobic cultivation increased, as it was discovered
that a large number of microorganisms possess strict anaerobic cultivation requirements
in contrast to the majority of species that had been anaerobically cultivated until that
time. Moreover, some of these anaerobic microorganisms were found to be responsible
for cellulose degradation in the digestive tract of ruminants, or they were isolated from
anaerobic infections in humans [10].

Microorganisms 2022,10, 412 4 of 28
Efforts to cultivate these strictly O
2
-sensitive organisms resulted in the further mod-
ification of anaerobic culturing techniques and prepared the space for the cultivation of
methanogens. The focus was on the cultivation of anaerobic bacteria from the rumen
environment, such as cellulose and xylene-degrading bacteria [
32
,
33
]. A pioneer in the
further development of anaerobic cultivation techniques was Hungate. His studies on
cellulolytic bacteria led to the isolation of species such as Fibrobacter succinogenes (formerly
Ruminobacter succinogenes) [33].
2.2. First Attempts of Methanogens Isolation and the Discovery of Archaea
In 1776, Alessandro Volta collected the gas formed bubbling in swamps [
3
,
34
]. He
discovered that the gas was flammable. Almost a hundred years later, the gas was named
methane by the German chemist August Wilhelm von Hofmann [
35
]. Further observations
and gas analyses revealed that the gas formed in swamps consists of methane, CO
2
and
N
2
. Thereafter, the microbiological role in methane formation started to be studied in
more detail.
Initially, methane formation and cellulose degradation were often associated with
each other, and cellulose decomposition and gas formation were deeply studied [
36
,
37
].
The presence of methane in the formed gas led to studies based on two topics. The first was
aimed at the isolation of methanogens [
3
,
38
], and second was the discovery of substrates
used by these methanogens [39].
At the turn of the 20th century, Omelianski studied the fermentation of cellulose
and noticed that some microorganisms produced H
2
and some methane [
36
]. He also
distinguished them based on temperature surveillance [
3
,
36
]. The methane bacilli were
killed by pasteurisation while the spores of the H
2
-producing bacilli survived, and using
the heating procedure, he obtained only the H
2
-producing consortium. In 1915, Mazé
found spherical microorganisms in his enrichment cultures that produced methane. They
fermented acetate to methane and formed aggregates [
28
,
38
]. In 1910, Söhngen discovered
two types of methanogens. He described methanogenic rods and also observed and
isolated acetate-utilizing methanogens that formed cell bundles and named them Sarcina
(sarcina (lat.) meaning bundle) [
3
]. Söhngen’s experiments also proved that added H
2
is
rapidly consumed by the microorganisms and leads to methane production.
Based on his observations and calculations, he confirmed Equation (1) proposed
by Omelianski:
CO2+ 4H2→CH4+ 2H2O (1)
His observations were published in his work with photographs of these microorgan-
isms, coining the genera Methanobacterium and Methanosarcina [3].
The isolation of pure cultures of methanogens is complicated due to their strictly
anaerobic nature. Agar plates could not be used, as the microorganisms did not grow on
them because they were not incubated in a strictly anaerobic atmosphere. Agar shake tubes,
however, proved efficient for isolating colonies.
The first methanogenic pure culture isolated was Methanobacterium omelianski by
Barker in 1936 [
38
]. In the same work, he described the isolation and description of
Söhngen’s methane-producing bacterium and named it Methanobacterium söhngenii (now
Methanothrix söhngenii). In that work, he also described the isolation of Söhngen’s Sarcina.
The isolated strain was able to form methane only from calcium acetate and was named
Methanosarcina methanica. He also succeeded in isolating Mazé’s cocci and named them
Methanococcus mazei (now Methanosarcina mazei). He emphasised that the latter three species
were not strictly pure, but were only purified by repeated transfers and applications of
techniques as a combination of agar shake tubes and special Hall’s tubes with marble seals.
Only later, the supposedly pure Methanobacterium omelianski, capable of oxidation
of ethanol and further described in Barker’s next work [
40
], was discovered to consist
of two different species. These were Methanobacterium bryantii and the “S” organism,
which oxidized ethanol to acetic acid and produced H
2
that served as a substrate for the

Microorganisms 2022,10, 412 5 of 28
syntrophically growing methanogen [
41
,
42
]. Other species of methanogens that were
isolated were Methanococcus vannielii [43] and Methanobrevibacter ruminantium [44].
The breakthrough in anaerobic cultivation was achieved by Hungate. He innovated his
techniques for cultivation of cellulolytic microorganisms, which led to new discoveries in
the field of microbiology. In 1969, Hungate published “A Roll Tube Method for Cultivation
of Strict Anaerobes”, which has become the most important source of techniques and
procedures for anaerobic cultivation. Many of the principles described by Hungate or
derivatives of these methods are widely used by many scientists today [
14
]. The application
of these modifications led to the further description of numerous other species, expanding
the entire group of methanogens [45–47].
In 1977, Woese and Fox published the seminal paper describing the Archaebacteria,
now referred to as Archaea, which raised quite a commotion, as the third domain of life was
established [
48
]. The study of the 16S rRNA structure was the key element for the distinction
between Archaea and Bacteria. At first, the difference was found in methanogens, and
later confirmed in halophilic archaea and extremely thermophilic archaea. More features
of archaea were noticed to have different characteristics, e.g., cell wall, cytoplasmatic
membrane, resistance to antibiotics and toxicity of the diphtheria toxin [
49
–
53
]. The
existence of the third domain of life was supported by this evidence and led to further
discoveries related to studies on the metabolism of these microorganisms, protein structure
and function and their evolution. It also raised an interest in the cultivation and description
of new species that might belong to the domain Archaea [54–58].
3. Anaerobic Cultivation Techniques
3.1. Laboratory Equipment for Cultivation of Methanogens
Anaerobic cultivation differs from aerobic cultivation in many aspects; one of these
concerns the instruments with which the laboratory should be equipped. These instruments
were established as novel methods of anaerobic cultivation to be introduced. The purpose
of maintaining an anaerobic atmosphere and avoiding exposure to O
2
led to a replacement
of some classical instruments and cultivation vessels with the new ones.
The most commonly used cultivation vessels are Hungate or Balch tubes and serum
bottles (Figure 2a–c). A Hungate tube is closed with a butyl stopper and a screw cap. Balch
tubes and serum bottles are closed with butyl stoppers and sealed with aluminium seals
by crimping. This allows the cultivation vessel to be pressurised with substrate gases [
59
].
However, it also allows the cultivation of gas-producing microbes. In the case of cultivation
of, e.g., hydrogenotrophic, autotrophic methanogens, repeated gassing is still necessary
because the gaseous substrate in the headspace is consumed according to the specific
gas uptake rate and the biomass concentration of gas-utilizing microbes. In the case of
thermophilic and hyperthermophilic methanogens, the headspace replenishment must be
performed at higher frequency compared to mesophilic or psychrophilic methanogens.
Cultivation in larger volumes can be performed using a modified bottle with a neck that
can be sealed with a stopper and aluminium cap (Figure 2d,f). The bottle can consist of
more than one opening for various purposes [
16
,
60
–
62
]. It is also possible to use a pressure-
resistant Schott bottle for anaerobic cultivation, which is closed with a GL45 screw cap with
an opening and the appropriate butyl rubber stopper (Figure 2e).
The use of anoxic gas or a gas mixture is necessary in anaerobic cultivation. To ensure
that there is no O
2
present in the gas, a copper column was used as another innovation to
remove O
2
from the used gas [
14
]. The tube filled with copper turnings or copper pellets is
heated while the gas flows through the column. When the apparatus is constructed using
copper turnings, the electric heating needs to exceed 350
◦
C. The copper pellets proved to
be more effective, as a lower temperature of around 150
◦
C was sufficient to remove trace
amounts of O
2
[
63
]. Before introducing H
2
, the column must be absolutely free of O
2
, so
it is gassed with N
2
or argon beforehand. Today, when gasses of purity 5.0 (99.999%) are
used in the laboratory, the removal of O
2
by heated copper columns is no longer necessary.

Microorganisms 2022,10, 412 6 of 28
Figure 2.
The most used cultivation vessels: (
a
) Hungate tube; (
b
) Balch tube; (
c
) serum bottle;
and culturing vessels for greater volume: (
d
) modified bottle with neck (according to Balch, 1979);
(
e
) pressure bottle with butyl stopper and GL45 opening; (
f
) modification of bottle with more openings
(Hanišáková, according to Miller and Wolin, 1974).
For the purpose of quantitative gassing, the gassing manifold was
invented [60,61,64]
.
The gassing manifold is necessary when a large number of media or cultures need to
be gassed. This is because the gassing of cultures or anaerobization of media is a very
time-consuming step. The gassing manifold divides the gas stream into several parallel
streams, so that parallel gassing is possible [
64
]. The gassing manifold can be assembled
from individual parts, or it is possible to buy prefabricated gassing manifold (Ochs Glas-
gerätebau, Bovenden, Germany). These manifolds are often operated manually. However,
an electric gassing manifold is also available, e.g., Deoxidized Gas Pressure Injector; IP-8;
Sanshin Industrial, Japan. The tubing exiting the gassing manifold can be terminated with

Microorganisms 2022,10, 412 7 of 28
a glass syringe containing sterile cotton or a Luer-Lock connector. In the latter case, a sterile
filter is inserted between the tubing and the needles. Due to these settings, it is possible to
connect the needle through the lock and manipulate closed vessels without exposing them
to O
2
. Since all operations with cultures are also performed using syringes and needles, it
is also recommended to flush the syringe with the N
2
or CO
2
gas before taking a sample of
the culture or solutions [14,15].
Some assignments require a strictly anaerobic environment for manipulation, and it is
complicated or even impossible to perform them without the use of the anaerobic glove
box (Figure 3). It should be noted that the anaerobic glove box is an anoxic environment,
but not a sterile one. Glove boxes with built-in HEPA filters are available (Labconco
corporation, Kansas City, MO, USA), or a HEPA filter system could be added additionally
(Coy Laboratory Products, Grass Lake, MI, USA).
Figure 3. Anaerobic box (Coy Laboratory Products, USA) (Photo: Laboratory).
3.2. Preparation of Anoxic Media for Methanogens
3.2.1. Composition of Medium for Methanogens
The preparation of media for methanogens can be a very time-consuming process,
as many steps are required to make the media suitable for their growth. In addition, the
media should reflect the growth conditions of each species in the natural environment,
adapting it to the specific requirements of the species for laboratory experiments or for
later biotechnological application.
The basis of the media is a mineral buffer solution. The salts in the media can be
chlorides or sulphates, although sulphates increase the possibility of SRB growing in the
media and competing with methanogens in enrichment cultures [
65
]. Another important
component is the trace element solution, which contains important cofactors of metabolic
enzymes. The most used solutions were described by Pfennig and often used by Widdel in
his works with phototrophs and sulphate reducers, e.g., SL6 or SL10 [
66
–
69
] (Table A1), or
Wolfe’s mineral solutions, made especially for methanogens [
60
]. Recently, 80 methanogens
were grown in 22 chemically defined and/or complex media to assess their methane pro-
duction and growth characteristics for the identification of high-performance methanogens.
The analysis also included multivariate statistical analysis of the medium’s constituents [
8
].

Microorganisms 2022,10, 412 8 of 28
Table 1.
Composition of the most common traceelement solution used in methanogenic media preparation.
Wolfe’s Solution 1
(g/L)
SL10 2
(g/L)
SL6
(g/L)
Nitrilotriacetic acid (NTA) 1.5 - -
MgSO4.7H2O 3 - -
MnSO4.H2O 0.5 - -
MnCl2.4H2O - 0.1 0.003
NaCl 1 - -
NiCl2.6H2O - 0.024 0.002
FeSO4.7H2O 0.1 - -
FeCl2.4H2O - 1.5 -
CoCl2.6H2O 0.1 0.19 0.02
CaCl20.1 - -
ZnSO4.7H2O 0.1 - 0.01
ZnCl2- 0.07 -
CuSO4.5H2O 0.01 - -
CuCl2.2H2O - 0.002 0.001
AlK(SO)4.12H2O 0.01 - -
H3BO30.01 0.006 0.03
Na2MoO4.2H2O 0.01 0.036 0.003
Reference [60] [68] [67]
1
dissolve NTA in 500 ml water and adjust pH to 6.5 with KOH, then add the rest of the compounds.
2
dissolve
FeCl
2
.4H
2
O in 10 ml 25% HCl, add deionized water and dissolve the rest of the salts. Fill to volume of 1000 mL.
Vitamin requirements differ among species. Many methanogens can be grown without
the addition of vitamins [
8
,
56
,
70
,
71
], but previous studies have reported that the presence
of vitamins supports their growth [
72
,
73
]. Other species might be fastidious and require
a whole range of different vitamins, e.g., Methanimicrococcus blatticola [
72
,
74
,
75
]. Since
vitamins are often thermolabile, their filter-sterilized stock solutions are added to media
after the sterilization of a basic solution. Similarly to trace elements, complete solutions
of vitamins are often prepared in advance and used in various media, e.g., Wolfe’s [
60
] or
Widdel’s [69] vitamin solutions (Table 2).
Table 2. The most common vitamin solution used in methanogenic media preparation.
Wolfe’s Solution
(mg/L)
Widdel’s 5 Vitamin Solution
(mg/L)
Pyridoxine-HCl 10 15
Thiamine-HCl 5 -
Riboflavin 5 -
Nicotinic acid 5 10
Calcium pantothenate 5 5
p-Aminobenzoic acid 5 4
α-Lipoic acid 5 -
Biotin 2 1
Folic acid 2 -
Cyanocobalamin 0.1 -
Reference [60] [69]
The use of reducing agents in the media to keep the ORP within a specific range is
required for the successful cultivation of anaerobic organisms, not only methanogens [
76
].
For methanogens, the ORP should not exceed -330 mV. Sodium sulphide (Na
2
S), sodium
thioglycolate, dithiothreitol, L-cysteine or sodium dithionite could be used as a reducing
agent, which should be added to the media prior to inoculation [
76
]. The most used re-
ducing agent is Na
2
S. On the other hand, L-cysteine is also used as a source of sulphur for
microorganisms instead of a pure reducing agent because an ORP of -340 mV cannot be
achieved. The ORP is then set by the final addition of the reducing agent to the medium.

Microorganisms 2022,10, 412 9 of 28
If a redox indicator is present in the medium, the coloration of the medium will indicate
whether the ORP is within the intended boundary. There are a number of different indica-
tors with resazurin being the most commonly used [
76
]. However, when methanogens are
frequently cultivated in the laboratory, redox indicators are not applied any more [8,64].
Since the organisms are adapted to live in a complex environment in the presence of
various microorganisms, this may lead to the necessity of a component that is specific to
particular environment and cannot be substituted
in vitro
. For the successful cultivation
of these auxotrophic microorganisms, which initially appear unculturable, additional
enhancement of the medium is required. The additive could be yeast extract, ruminal fluid,
or sludge fluid [
77
–
79
]. Media containing carbonate buffer ensure that the pH remains
stable. This is quite inconvenient when a wide pH range has to be measured and the
optimal pH of the species determined. Different buffer systems may have to be tested.
This is the basic characterization of media for methanogens. Nowadays, there are
different media depending on the species or environment of the microorganism of inter-
est, which are freely available on the websites of microorganism collections such as the
Deutsche Sammlung für Mikroorganismen und Zellkulturen (DSMZ) or the American
Type Culture Collection (ATCC). Unfortunately, the recipes are updated without historical
documentation of their former versions, which makes their use somewhat unreproducible.
Moreover, the medium recipes are sometimes written in an unnecessarily complicated
manner, especially when it comes to the recipe for the cultivation of methanogens. There-
fore, it is recommended to assess up-to-date literature in order to select the appropriate
medium. In addition, an assessment of growth characteristics of methanogens on different
media was recently performed, which could help to select a medium for the cultivation
of methanogens [8].
3.2.2. Process of Anaerobization of the Medium
Hungate’s technique (1969) for the preparation of O
2
-free media depends on the heat
stability of the solution. Heat-stable media undergo a boiling process in which the dissolved
O
2
is expelled from the media. Heat-labile solutions should be purged with gas bubbles for
about 30 min to one hour to remove residual O2.
Dispersal of the media can be performed prior to sterilization, which is recom-
mended [
14
,
64
], or after sterilization, which carries the risk of contamination [
80
]. Pre-
viously, Hungate’s method of pipetting the media into the vessels while simultaneously
gassing the media and the final vessel was quite difficult to manage, as one person must
hold the pipette while orally pipetting the media and hold the Hungate tube at the same
time (Figure 4a). The vessel is then closed with butyl stopper. A detailed description of
this method of media preparation with slight modifications is also provided by Bryant [
81
].
Not only is the procedure complicated and difficult to perform, but oral pipetting is also
prohibited nowadays. Later, apparatus to simplify the process of dispersing was invented,
which can be used in the preparation of non-pressurised media (Figure 4b) [63].
Balch proposed to prepare the media in an anaerobic box, which simplified the whole
pipetting process. The boiled medium is purged with a gas mixture of N
2
/CO
2
(4:1 (v/v)),
then the reducing agent is added, the medium is transferred into the anaerobic box and
distributed into the cultivation vessels without being exposed to air. In the final step, the
closed media are removed from the anaerobic box, the gas atmosphere is replaced, and the
media are autoclaved [
60
]. In this way, the anoxic conditions are maintained and a better
adjustment of pH is achieved, as it is carried out after the addition of Na2S.
Another modification as well as the first use of serum bottles in cultivation was devised
by Miller and Wolin in 1974. The medium is prepared, then boiled and purged with O
2
-free
gas while another stock solution is added. Afterwards, the medium is dispersed into serum
bottles that are constantly purged with O
2
-free gas and closed with a butyl robber stopper
while still being gassed. In this way, the O
2
cannot enter the vessel. The media are sterilized
by autoclaving.

Microorganisms 2022,10, 412 10 of 28
Figure 4.
(
a
) Media pipetting described by Hungate and Bryant. (
b
) Dispersing media illustrated
according to Sowers (Hanišáková).
When cultivating strictly anaerobic SRB and phototrophs, Widdel had also developed
methodological variations and procedures. Since these microorganisms do not require a
gas atmosphere, the apparatus was designed to fill the entire culturing vessel. The purpose
of his designs is to autoclave the medium in the vessel simultaneously with the dispersing
apparatus. After autoclaving, the medium is purged with gas while being stirred and
dispersed into the culture vessels. The first model was a bottle with a butyl cap for gas
purging and with an opening for dispersing the medium. Another model consisted of a
conical vessel with a butyl cap, holes in the stopper for thermolabile solution addition, a
gassing tube and a dispersing tube. During the process of dispersing, the vessel is turned
upside down [69].
The final design is the Widdel flask (Figure 5). The reverse conical shape with a
flat base ensures efficient stirring and the possibility of dispersing media from the whole
volume of the flask. The individual openings for inserting and taking samples reduce the
risk of contamination. At first, the vessel is filled with mineral solution and autoclaved.
Then, it is continuously purged with the necessary gas or gas mixture while cooling, and the
thermolabile components and reducing agents are added to the media. Due to the pressure
created inside through gassing, the medium is poured through the dispensing apparatus
into sterile vessels and closed. This method is suitable for media containing thermolabile
components or for the media where the precipitation of salts during autoclaving occurs. The
disadvantage of this method could be considered the contact with air during dispensing
of the media or possible microbial contamination. If this method is used for cultivation of
microorganisms that require a headspace, it is difficult to pour the exact volume of media
and ensure anoxicity due to the filling mechanism, but it is not a problem to purge the
culturing vessels with gas before and immediately after filling the media. Manipulation

Microorganisms 2022,10, 412 11 of 28
with this vessel is also described in detail elsewhere [
82
]. For the purpose of the cultivation
of methanogens, this method of media preparation was used in the work on the isolation
of Methanobacterium aarhusense [83].
Figure 5.
Widdel’s flask.
1

Openings for input/output of media samples.
2

Opening for gas entry.
3

Central opening for media filling.
4

Clamp for media filling.
5

Filling funnel for media.
6

Magnetic stirrer (Hanišáková).
In 2010, Wolfe published a method for the preparation of anoxic solutions and media
in small volumes. The main advantage of this method is the speed that leads to anoxic
solutions. Using the classic purging-with-gas method would require more time. The combi-
nation of vacuum–gas cycling and vortexing the entire system to increase the surface area
of the liquid effectively expels O
2
from the culturing vessel [
84
]. However, even without
boiling or vortexing, vacuum–gas cycling prior to autoclaving has been proven efficient for
media preparation and cultivation and is used as a proper method nowadays [
85
,
86
]. The
number of cycles before autoclaving can range from three [
85
,
86
] to five [
64
]. The flushing
of the aerobically dispersed media before autoclaving is also efficient [75].
To briefly summarize, the anaerobization of the media could be accomplished via
different approaches depending on the objective of the media. The easiest and the most
progressive, as well as the most suitable for manipulation and the least time-consuming,
is the vacuum–gas cycling method combined with gassing manifold. This method does
not require an anaerobic glove box, although a vacuum pump is required. Without a
vacuum pump, flushing the media is also an efficient possibility, though slightly more
time-consuming. For complicated media consisting of more solutions, the Widdel flask
is still a present option that would save material such as needles, syringes and butyl
stoppers, which could be destroyed by repeated piercing. These variants can substitute for
an anaerobic glove box, which is not present in every laboratory; the manipulation is more
difficult or which could be reserved for different tasks.

Microorganisms 2022,10, 412 12 of 28
3.3. Cultivation and Pure Cultures Isolation Techniques
Using solid media for strictly anaerobic microorganisms and isolation of pure cultures
may be seen as the most difficult part of anaerobic microbiology and biotechnology. The
main obstacles are the slow growth rate of some methanogens and maintaining an anaerobic
atmosphere. During cultivation, the agar could dry out quickly before the colonies become
visible. Not every species is cultivable on solid media, and the higher temperature cannot
be achieved with agar, so alternatives, e.g., Gellan Gum, have to be used [
63
]. Moreover,
Petri dishes are not airtight. These are the reasons why anaerobes are mostly cultivated in
liquid media.
3.3.1. Petri Dishes Cultivation in Anaerobic Jar
While cultivating anaerobic microorganisms on agar in a Petri dish, it is possible to
incubate the plates in the anaerobic glove box. The atmosphere here is provided by the gas
from the glove box, but it does not contain a sufficient amount of H
2
if methanogens are to
be cultivated, as the headspace gas contains up to 4.5 Vol.-% H
2
in N
2
(forming gas). The
low partial pressure of H
2
could slow down the generation time of methanogens. Anaerobic
jar/pressure cylinders can be used to incubate inoculated Petri dishes inside (Figure 6).
Some jars are equipped with gassing inlets to fill the headspace with substrate gas. The
other option is to use a palladium catalyst inside the jar that uses H
2
to reduce O
2
. It should
be noted that palladium is deactivated by H
2
S, so the catalyst must be replaced before
every experiment [
87
]. There are several commercially available anaerobic pack systems
(e.g., AnaeroPouch-Anaero; Mitsubishi Gas Chemical Company, Japan) that have been used
and described as support systems for anaerobic cultivation [
88
]. Some hydrogenotrophic,
autotrophic methanogens could be cultivated on agar plates without a H
2
atmosphere by
using, for example, formate as a substrate [85].
Figure 6. Illustration of anaerobic jar for anaerobic Petri dish cultivation (Hanišáková).
When cultivating methanogens on agar, in addition to all the above-mentioned aspects,
it is also essential to pay attention to the used agar, its type and brand, its concentration and
the amount of Na
2
S [
89
]. The agar should be washed several times before use to remove all

Microorganisms 2022,10, 412 13 of 28
impurities. All factors could affect the morphology of the colonies and the specific growth
rate. It is preferable to perform inoculation into deep agar or layered agar [90–92].
Special attention should also be paid to the material from which the anaerobic jar is
constructed. For long-term cultivations, plastic materials do not have to be O
2
-tight. For
this reason, even anaerobic jars with a set atmosphere should also be better kept in an
anaerobic box if they are not made of metal [
63
]. The use of a modified canning jar instead
of an anaerobic jar has also been described [
90
]. The agar plates have been used to isolate
Methanocaldococcus jannaschii [93] or Methanothermococcus thermolithotrophicus [94].
3.3.2. Hungate’s Roll Tube Technique
The most used technique for obtaining pure cultures of methanogens is Hungate’s
roll tube technique [
14
,
95
–
97
]. This technique is based on the formation of a thin layer of
inoculated agar, adhering to the walls of the Hungate tube. The gas phase is anaerobic.
During growth, colonies become visible on the agar and as subsurface colonies. These are
picked and further dispersed in the new dilution series or cultivated. Bryant preferred
to inoculate the picked colonies into a slant medium rather than a liquid medium. The
concentrated inoculum was found to be better adapted to the conditions in the new envi-
ronment, as inoculation into liquid media is sometimes unsuccessful [32,81,98]. Applying
this method, the microorganisms grow through the agar and form surface and subsurface
colonies. Due to this effect, the morphology of the colonies could differ and that could be
wrongly considered as a mixed culture [
14
]. It is also possible to apply thin layer of agar on
the walls and inoculate the roll-tube after the solidification, as it was carried out during the
isolation of Methanogenium marinum [
99
]. The purpose of the later inoculation was to avoid
exposing the microorganisms to temperatures higher than 15 ◦C.
A variation of this technique was performed in 1973 by Miller and Wolin with the
use of serum bottles instead of Hungate tubes. This method proved to be efficient for the
isolation of anaerobic microorganisms such as Selenomonas ruminantium,Ruminococcus albus
or Methanobrevibacter ruminantium [16].
3.3.3. Agar Shake Dilution Tube Method
Before the Hungate roll-tube method was developed, the agar shake dilution tube
method was used for the cultivation of anaerobic microorganisms [
4
,
27
]. The latter method
differs from Hungate’s method in the use of the column of semisolid agar in the tube,
closed by a butyl stopper. During the growth of methanogens, visible colonies form inside
the column. When the methanogens consume a liquid substrate (acetate or methylated
compounds), or the technique is used for gas-producing anaerobes, such as Clostridia, they
produce an excess volume of gas and the agar in the column is ruptured by the gas bubbles
(Figure 7b) [
100
]. If there are facultatively anaerobic microorganisms present, they grow at
the interface with the gas and consume the traces of O
2
, so the tube is completely anaerobic.
During the preparation of these agar shake tubes as well as the preparation of Hungate
roll tubes, the temperature of agar must be optimal, as it should still be liquid but not too hot
for microorganisms. For this method, modifications also have been introduced, changing
the shape of the test tube slightly [
101
]. This method has been repeatedly used to isolate
SRB [
80
,
102
] and has also been proven as a method for testing the antibiotic susceptibility of
anaerobic microorganisms [
103
]. Even Hungate initially used the method [
33
] and it has also
been repeatedly used for pure colony isolation in rather more recent publications [
83
,
100
],
or in serum bottle modification [75].
3.3.4. Hermann’s Flat Flask Method
As mentioned above, cultivation on a Petri dish is not convenient due to evaporation
of the water and drying of the agar. In 1986, Hermann published a method for anaerobic
cultivation on poured agar in a flat flask used originally for phototrophic cultivation
(Figure 8c) [
108
]. Later, in 1992, the method was modified by Olson. An additional opening
was added that can be closed and sealed with aluminium. The second opening is used to

Microorganisms 2022,10, 412 14 of 28
flush the bottle with anoxic gas during manipulation and inoculation (Figure 8d). Both
openings are then sealed and the flask is cultivated [
109
]. The advantage of this method is
that the presence of an anaerobic box is not necessary, the manipulation could be carried out
on a laboratory bench and the individual bottles can be checked separately, in comparison
to the cultivation of Petri dishes in an anaerobic jar. This method was used, for example,
for cultivation of Methanobrevibacter millerae and Methanobrevibacter olleyae [79].
Figure 7.
Test tube for agar shake. (
a
) Test tube with butyl stopper. (
b
) Photo of gas bubbles inside
the agar (Hanišáková, photo: laboratory).
3.3.5. Lee Tube Method
Another interesting method for the enumeration and cultivation of anaerobic microor-
ganisms is the use of the Lee tube (Figure 8a) [
104
]. Colonies grow in the reduced agar
present in the layer between the outer and inner tubes. The original Lee tube is made of a
single piece of glass and sealed with a screw cap, which is more appropriate; a modified
Lee tube consisting of two pieces and sealed with a cotton plug, which is not suitable for
methanogens using a gaseous substrate, has also been described (Figure 8b) [
105
]. The Lee
tube method has been used repeatedly to isolate and characterise anaerobic bacteria [
106
]
and to maintain the cultures [107], but has not yet been used for methanogens.
Figure 8.
Cultivation vessels for pure colony isolation. (
a
) Lee tube; (
b
) modified Lee tube; (
c
) flat
flask — original used by Hermann; (
d
) flat flask modification with additional opening (Hanišáková).

Microorganisms 2022,10, 412 15 of 28
3.3.6. Single Cell Isolation Methods
For the isolation of strict anaerobes, micromanipulation and single-cell cultivation is
also a possible solution used in anaerobic cultivation. The purpose of this method is to take
a single cell from the sample and inject it into the media. The isolation of the cell could
be performed manually using the microinjectors BactoTip [
110
] or by laser microscope
where the laser beam isolates the cell [
111
]. For some anaerobic microorganisms, this could
be quite time consuming, as they are known for a longer generation time [
112
,
113
] and
the cultivation of the extracted cells sometimes proves unsuccessful. The recovery rate
has been studied and optimization of the technique for different common bacteria was
recently accomplished [
114
]. This method proved successful not only for the isolation of
the methanogen Methanobrevibacter sp. from termite gut [
110
], but also for extremophilic ar-
chaea such as Metallosphaera sedulla and Saccharolobus solfataricus [
115
] or hyperthermophilic
archaea [116].
3.3.7. Dilution to Extinction Method
In case the microorganisms are fastidious and cannot be isolated by agar techniques,
one option is dilution to extinction. The technique is based on serial dilutions in liquid
media, ensuring the loss of most cells until the desired cells are the only present. This
method is widely used, although it requires number of cultivation vessels to perform
the method [
71
,
117
,
118
]. To remove contaminating microorganisms from an enrichment
culture containing methanogens, the addition of appropriate antibiotics, such as ampicillin,
vancomycin, clindamycin or kanamycin, could be used to simplify the process of purifica-
tion [
54
,
66
,
119
]. To exclude fungal contamination, amphotericin B could also be added for
the isolation of methanogens from the intestinal sphere [120,121].
3.4. Novel Insights in Cultivation Techniques
The anaerobic cultivation methods described by Hungate and their variants are still in
use today and are widely applied in anaerobic microbiology and biotechnology. However,
there are still many obstacles, and the cultivation of anaerobes is still considered to be very
difficult and not always successful, even by following procedures step by step. In order
to improve cultivation methods, reduce the difficulty level, increase experimental results
and expand the application possibilities, the methods need to be further adapted to today’s
requirements and novel devices need to be used for automation, although the improvement
of the techniques has not been an issue in the last two decades.
3.4.1. The Six-Well Method
One of the novel methods for cultivation of strictly anaerobic microorganisms was
published in 2011 by Nakamura et al. The six-well method using the AnaeroPack system
was found to be efficient for the cultivation of a number of methanogens, SRB and syn-
trophic bacteria [
88
]. The AnaeroPack system is based on the use of special catalyst sachets
without the presence of water or H
2
production. In the work of Nakamura, inoculated
plates in aerobic and anaerobic conditions were placed into a bag with the AnaeroPack
system and the gas was recharged with H
2
/CO
2
(4:1 (v/v)) mixture. Both variants proved
to be successful, although anaerobic conditions for inoculation showed better results.
3.4.2. Growth in Syntrophic Communities
A completely different approach to anaerobic cultivation was published in 2009 by
Sakai. There are a number of microorganisms that cannot be cultured or require specific
conditions. Most methanogens are cultivated under an atmosphere of H
2
/CO
2
(4:1 (v/v))
and an overpressure of 1 to 2 bar. The natural environment can provide even higher
pressure and H
2
concentrations, but there are also environments that have a low H
2
concentration in which methanogens are also encountered. They often require a lower H
2
concentration as they are accustomed to grow dependently on the H
2
-producing bacterial
species, forming a syntrophic relationship between the two species. It is difficult to simulate

Microorganisms 2022,10, 412 16 of 28
the complex relationship in a laboratory. Co-cultivation is the key to successful cultivation
and obtaining new species. Instead of adding direct substrate, pre-substrate such as ethanol,
butyrate or propionate is consumed by bacteria and H
2
is gradually released, which is
then utilized by hydrogenotrophic, autotrophic methanogens. This method results in the
capture of a wider range of methanogens, while the high H2concentration tends to target
fast-growing species [
122
]. The obstacle in the method is connected with the follow-up
purification of the culture. Sometimes it is enough to apply the techniques mentioned
above and cultivate the microorganism under conditions of higher H
2
concentration using
the liquid dilution method [
123
] or deep agar dilution tubes [
124
,
125
], but the purification
is not always accomplished and the whole process is very time-consuming.
3.4.3. Microplate Reader Technique
One of the obstacles to anaerobic cultivation is the testing of chemical substances. In
aerobic cultivation, it is possible to obtain results using microtiter plates for which different
conditions can be set in one run. Several devices are designed for the continuous mea-
surement of optical density during growth, e.g., Bioscreen C (Oy Growth Curves Ab Ltd.,
Helsinki, Finland) or Sunrise (Tecan, Männedorf, Switzerland) [
126
]. This method could
be adapted to anaerobic conditions with the use of an oil layer to ensure a microaerobic
environment [
127
], or some of the instruments may provide a Gas Control Module for
changing the atmosphere composition. However, strictly anaerobic microorganisms are not
able to grow using these methods because the oil layer is not absolutely gas-impermeable,
or the gas composition does not favour the growth of methanogens. The third option is to
place the entire device inside an anaerobic box to anaerobically measure the optical density
continuously or just for a one-time measurement [128,129].
For quantitative experiments, a great amount of serum vials or Hungate tubes must be
prepared, which is time-consuming and material-heavy. For this reason, various scientific
groups have taken an interest in the microplate reading technique to enable large-scale
experiments with methanogens. In 2012, Bang et al. tested antimicrobial peptides on
methanogens. The microplate was prepared anaerobically in an anaerobic box, cultivated
in an anaerobic jar under an atmosphere of H
2
/CO
2
(4:1 (v/v)) and then read with a device
placed in the anaerobic box to maintain anaerobic conditions [
130
]. This procedure resulted
in the cultivation of hydrogenotrophs and the measurement of their susceptibility to various
antimicrobial peptides. A similar experiment was performed to test chemical substances
using a microtiter plate, but with the differences that the microtiter plate was cultivated
directly in an anaerobic box and the microorganisms were growing on a substrate other
than H2[131].
3.4.4. Microfluidic Techniques
In recent years, microfluidics has achieved more recognition as an efficient and high-
throughput method for cultivation and testing different conditions. It is obvious that its
applications are broad, as it offers a variety of conditions in a small space. Applications
of microfluidics in anaerobic conditions are focused primary on human gut microbiota
and capturing species that are not yet culturable [
132
–
134
]. This technology was used to
determine the optimal conditions for the growth of Methanosaeta concilii, using an anaer-
obic microbioreactor under a N
2
/CO
2
atmosphere [
135
]. However, the aforementioned
experiments were conducted in an anaerobic chamber, and hydrogenotrophic, autotrophic
methanogenesis has not yet been investigated.
At the borderline between microfluidics and the classic microtiter plate method is
a device designed for anaerobic cultivation in a 48-well plate (e.g., BioLector, m2p-labs
GmbH, Baesweiler, Germany). It allows measurements of optical density, pH, biomass and
fluorescence, as well as continuous feeding of the reactors. The instrument has been tested
for fermentation of e.g., Clostridium sp. and Lactobacillus sp. [
136
], although the potential
use for strict anaerobes such as methanogens has not yet been tested.

Microorganisms 2022,10, 412 17 of 28
4. Quantification Techniques
During the cultivation of microorganisms, it is necessary to measure their growth, use
these data to calculate growth parameters to define these microorganisms, and compare
different growth conditions. Knowledge of the growth kinetics is also important for
biotechnological applications, to relate the substrate conversion or product formation
to biomass, or to directly calculate gas production using pure methanogenic cultures.
Methods for quantification of growth of anaerobic microorganisms were recently reviewed
by Mauerhofer et al. [76]. These quantification methods are non-growth-dependent.
4.1. Optical Density Measurement Technique
Optical density measurement is still a widely used and classic method to measure
biomass and calculate specific growth rate (Equation (2)) [137]:
µ=lnOD2−lnOD1
t2−t1
(2)
where
µ
is the specific growth rate (h
−1
), OD
1
and OD
2
are the optical density values
determined at the respective times t1and t2(h).
Optical density of methanogens is often measured at wavelength 578 nm [
138
–
140
]
but it could be also measured at wavelength 600 or 660 [
74
,
141
]. The measured optical
density has to be multiplied by a conversion factor, which is specific to every organism
and must be experimentally determined, to obtain data for biomass or number of cells per
volume (Equation (3)): x
V=a·OD (3)
where x represents the number of cells in the sample, V is the volume of the sample (mL or
L), OD is optical density and ais the conversion factor.
In some cases, this method cannot be used due to cell aggregation, the presence of
precipitates or possible interference of resazurin in the medium. For the latter complication,
it is recommended to add a reduction agent before measurement, which results in decol-
orization of the medium, or even better to omit resazurin addition into the media at all if
OD measurement is considered.
4.2. ATP Determination Method
When optical density measurement is not an option for biomass quantification, there
are other possible methods, such as the calculation based on direct cell counting [
66
,
71
]
or adenosine triphosphate (ATP) determination. ATP determination could be also used
to determine the growth of the culture [
142
,
143
]. Nowadays, there are many commercial
products for ATP assay available, based on luminescence (luciferin/luciferase system) or
colorimetric methods.
4.3. Methods Requiring the Cultivation of Anaerobic Microorganism
4.3.1. Methane Production Measurement Techniques
Methanogens have the advantage of not only measuring biomass, but also releasing
methane as a metabolic end product. Methane, as the main component of biogas, carries
the conserved energy and it is highly important to obtain the biggest energy yield from the
different used substrates. The growth rate can be calculated from methane production with
the use of Equation (4) [117,144,145]:
µCH4=ln(M2/M1)
t2−t1
(4)
where
µCH4
is the specific growth rate (h
−1
) calculated based on methane production. M
1
and M
2
represent the produced methane (mmol) measured at the respective times t
1
and
t2(h).

Microorganisms 2022,10, 412 18 of 28
When the culture’s growth slows down and enters the stationary phase, the optical
density does not change rapidly. On the other hand, methane production still occurs at
high rate. To capture the methane produced over time, methane evolution rate (MER)
(Equation (5)) is used [64,146]:
MER =∆nCH4
(t2−t1)·V(5)
where MER is methane evolution rate (mmol L
−1
h
−1
),
∆
n
CH4
is the difference between the
produced methane (mmol) measured at the respective culturing times t1and t2(h), and V
is the volume of the media (L).
During hydrogenotrophic, autotrophic methanogenesis, the methane is produced
according to the molar equation as mentioned above in Equation (1).
Based on the equation, four moles of H
2
and one mole of CO
2
is consumed as one
mole of methane is produced. Since the substrates are both in the form of gas, it is possible
to use the ideal gas law (IGL) (Equation (6)) for calculation the methane concentration in
the headspace of the culture vessel:
n=p·V
R·T(6)
where n is amount of the substance of gas (mol), p is pressure (Pa), V is the volume of
the headspace (m
3
), R is the universal gas constant (J K
−1
mol
−1
), and T represents the
temperature (K).
For the application of this law, two ways of measurement exist. The first is based
on the change in pressure in the headspace, which is measured with a manometer. The
pressure drop represents the four moles of consumed H2(Equation (7)):
nCH4=(p0−p)·V
R·T·4(7)
where n
CH4
is the amount of produced methane in the headspace (mmol), p
0
represents
the initial pressure in the vessel (Pa), p represents the actual pressure in the vessel (Pa), and
V is the volume of headspace (L).
The other option is to combine the actual pressure of the vessel to calculate absolute
moles of gas in the headspace and the percentual concentration of methane measured by
gas chromatography (GC) (Equation (8)):
nCH4=p·V
R·T.% GC
100 (8)
where n
CH4
is the amount of produced methane in the headspace (mmol), p represents the
actual pressure in the vessel (Pa), V is the volume of headspace (L), and % GC represents
percentual representation of methane in the headspace.
The first way is much faster but depends heavily on the pressurization of the vessels. It
is also recommended to flush the headspace with the gas after every measurement to set the
same conditions and pressure. This is because gas leaks may occur when measuring with a
manometer and the results may be greatly affected during the second measurement. The
use of GC is dependent on the actual pressure and the repeated flushing is not required if
there is still a significant amount of substrates, respectively H
2
, present in the headspace [
64
].
The use of GC is also the only option to directly measure methane produced from formate
or by methylotrophic or acetoclastic methanogens.
4.3.2. Manometric OxiTop Measurement
Apart from typical reactors, a simple device for measuring pressure in the vessel
is the anaerobic OxiTop (OxiTop
®
-IDS, WTW GmbH, Germany). With two openings
for manipulation through needles, the substrate can be added or the samples removed.
Although it has not been yet directly used on pure cultures, potential application on cultures
is possible. Continuous pressure measurement gives insight into gas formation kinetics.

Microorganisms 2022,10, 412 19 of 28
4.3.3. Indirect Quantification of Produced Methane via Weight Gain
Another indirect method to determine produced methane uses the weight increase due
to the formation of water during hydrogenotrophic methanogenesis [
64
]. This method is
based on repeated measures of weight in regular intervals (Figure 9). The weight increment
represents the two moles of water formed on the one mole of methane and is described as
the water evolution rate (WER) (Equation (9)).
WER =∆mH2O
(t2−t1).MrH2O.V (9)
where WER is the water evolution rate (mol L
−1
h
−1
),
∆
m
H2O
is the mass difference (g)
measured in the respective culturing times t
1
and t
2
(h), V is the volume of the media (L),
and MrH2O is the molar mass of water (g mol−1).
Figure 9.
The process of quantification of methane via produced water during hydrogenotrophic
methanogenesis.
1

The weight of inoculated serum bottle is measured.
2

Incubation of the serum
bottle.
3

The weight of the serum bottle after incubation is measured.
4

The pressure in the
headspace is determined.
5

The serum bottle is flushed with gas. (1) The weight of the serum bottle
is again measured, presenting the weight difference and gained mass of water (Taubner & Rittmann,
2016, redrawn by Hanišáková).
In combination with the pressure measurement, this approach presents the metabolic
activity of hydrogenotrophic, autotrophic methanogens. It is applicable to fast-growing
methanogens where the weight increase is easily measurable and noticeable. For slowly
growing methanogens, it is recommended to set a longer interval of measurement. In
addition, drops of the suspension may leak when handling the serum bottles, leading to
inaccuracies in the measurement and a slow decrease in the weight in the control serum
bottle [147].
5. Up-Scaling Process during Anaerobes Cultivation
Classic use of serum bottles is required for cultivation of species, capturing new species
of microorganisms, and studying of material degradation in the primary steps of research.
The setup is called closed-batch cultivation. However, the repeated gassing results in
unbalanced growth and the maximal pressure of the headspace should not exceed 3 bar.
Therefore, cultivation in an environment where the culture could achieve balanced growth
according to a growth curve with enough substrate is necessary. Furthermore, in order to

Microorganisms 2022,10, 412 20 of 28
simulate processes in industrial reactors, up-scaling is needed to achieve more accurate
results. It is also the way to obtain microbial cell mass for further analyses.
Batch, Fed-Batch and Continuous Cultivation
Bioreactors can be operated in batch, fed-batch, and continuous modes. This division
is based on the experimental design and on the level of intervention in the reactor. Batch
mode requires the least intervention. The medium, substrate and culture are prepared at
the beginning and harvested at the end of the experiment. The fed-batch operation mode
differs from the batch system as the carbon and energy source are added during the run of
the reactor at a defined rate. This setup is limited by the volume of the reactor as there is
no outflow of the excess liquid (in the case of a liquid feed). Continuous cultivation, as the
name suggest, depends on the continuous inflow of substrate and nutrients and outflow of
suspension and products.
Up-scaling the cultivation of methanogens encounters several different obstacles.
Batch cultivation of pure cultures depends on the amount of substrate, which does not have
to be sufficient to obtain the requested yield of biomass. The greatest advantage of this
setup is the possibility of pressurisation of the reactor and setting conditions for observing
the growth of the population. The more the reactor is pressurised, the more substrate
is available to the culture and substrate conversion could be observed through pressure
measurement [
146
,
148
]. In special occasions, the gas could be removed and the reactor
repeatedly pressurised.
A fed-batch system ensures continuous substrate addition; in the case of hydrogenotrophic,
autotrophic methanogens, this means a flow of H
2
/CO
2
(4:1 (v/v)) mixture (Figure 10).
Continuous cultivation also removes suspension and ensures a stable medium volume [
76
].
Reactors with a continuous supply of H
2
/CO
2
gas mixture are hard to pressurise to increase
the solubility of the gases. One way to solve the problem is to stir the suspension to increase
the liquid-phase interface. Not every species of methanogen is able to withstand agitation
and atmospheric pressure. There were some cases where the continuous reactor was
pressurised to an absolute pressure of 1.22 bar [
149
] or 1.75 bar [
150
], but this does not reach
the pressure achievable in closed batch cultivation, where a pressure of tens of bars could be
easily set. There is one exception described of a constructed, pressurised fed-batch reactor,
with a pressure 10 to 20 bars, which had been operated with M. marburgensis [
151
]. Due to
water formation during hydrogenotrophic, autotrophic methanogenesis, it is also suggested
to take a notice of increasing volume of medium during the planning of the experiment. If
the expected production is greater, removal of the excess volume and addition of medium
should be considered.
In 1968, Bryant et al. successfully cultivated Methanobacterium strain M.o.H. in a fed-batch setup
with a reactor volume of 12 L and under continuous gassing [
152
]. To date, the experiments of
fed-batch or continuous operation setups of different volumes have been successfully carried out
with mainly thermophilic microorganisms, such as
Methanothermobacter species
[
138
,
149
,
153
–
156
], or
microorganisms from the order
Methanococcales [138,157]
. The exceptions are mesophilic Methanococcus
maripaludis [158] and the mentioned Methanobacterium strain M.o.H [152].
Acetoclastic and methylotrophic species are not dependent on pressure, and it is pos-
sible to cultivate them under a H
2
-free atmosphere. In the case of closed batch cultivation,
the pressure would increase instead [145].

Microorganisms 2022,10, 412 21 of 28
Figure 10.
Model of fed-batch bioreactor.
1

Gas mixture inflow.
2

Gas outflow.
3

Acid-base and
sulphide solution input.
4

Stirring mechanism.
5

Opening for inoculum input.
6

Sample output.
7
Temperature sensor and pH electrode (Hanišáková).
6. Conclusions
Cultivation techniques for anaerobic microorganisms, especially strictly anaerobic
ones such as methanogens, vary greatly, from classic methods of aerobic cultivation to
modern anaerobic high-throughput methods. Because of the difficulty of dealing with the
fastidious anaerobic microorganisms and the required equipment for the cultivation, not
many laboratories focus on anaerobic methods and anaerobic microorganisms. With this
review, the historical development of culturing methods and the potential applications are
presented in order to introduce anaerobic cultivation to the scientific community and to
assist in reflecting on these methods to develop a new generation of anaerobic cultivation
methods. This review also emphasises the importance of persevering in the knowledge and
pitfalls of anaerobic cultivation techniques in anaerobic microbiology and biotechnology.
Author Contributions:
Writing—original draft preparation, N.H.; figures design, N.H.; writing—
review and editing, M.V. and S.K.-M.R.R. All authors have read and agreed to the published version
of the manuscript.
Funding: Open Access Funding by the University of Vienna.
Acknowledgments: Open Access Funding by the University of Vienna.
Conflicts of Interest: The authors declare no conflict of interest.
Appendix A
Table A1. Milestones in historical development of cultivation of anaerobic microorganisms.
Year Technique Description Reference
1898 Agar shake tubes Agar in test tube mixed with culture and cultivated after
solidification of the agar [4]
1900 Pyrogallic acid (Buchner’s method) The use of pyrogallic acid on the cotton plug together with
alkaline solution to absorb oxygen in the tube [5]
1921 Hall’s marble seal Special constricted tube with concave marble seal that is
overpoured with sterile medium [23]

Microorganisms 2022,10, 412 22 of 28
Table A1. Cont.
Year Technique Description Reference
1929 Paraffin oil seal Layer of paraffin oil on top of culture medium in test tube [13]
1969 Roll tube method
Layer of agar-culture mixture on walls of Hungate tube made
by rolling the tube with liquid mixture until
solidification occurs
[14]
1969 Copper column
Application of copper column for expelling O
2
from used gas
[14]
1969 The use of reducing solutions Reducing the redox potential in the medium for creating a
more suitable environment [14]
1972 Syringes and needles
Preservation of anaerobic environment by application of
needles and syringes without the oxygen exposure
during manipulation
[15]
1974 Serum bottle modification of
Hungate technique
Application of serum bottles for cultivation of anaerobic
microorganisms in liquid and solidified medium [16]
1976 Pressurization of culture vessels
Increasing partial pressure in the cultivation vessel leads to
lessening of the gassing frequency and an increase in the
methanogenesis rate
[59]
1979 Gassing manifold Special manifold for parallel gassing of culture vessels [60]
1979 Lee’s tube Layer of agar mixed with culture between two glass walls of
special tube [104]
1981 Widdel flask Special conical flask for preparation media containing
thermolabile solutions [69,80]
1986 Modified Lee’s tube Layer of agar mixed with culture between two walls of two
tubes inserted in each other [105]
1986 Hermann´s flat flask method The usage of closed flat flask for cultivation of anaerobic
microorganisms on agar [108]
1992 Modified Hermann´s flat flask
Addition of an opening for gassing the flask while picking up
the colonies from medium [109]
1999 Single cell isolation technique
First application of a single-cell isolation technique which
enables the picking up of single cells from sample
on methanogens
[110]
2007 Microfluidics application
The first usage of microfluidics in anaerobic microbiology
cultivating pure culture of methanogenic Methanosaeta concilii
under N2/CO2(4:1 (v/v)) conditions
[135]
2009 Vacuum-gas method The dispersed medium is put through cycles of gassing and
gas exhaustion to set anaerobic conditions in the vessel [86]
2010 Vacuum-vortex method
Vortexing the dispersed medium in vessel while applying
cycle gassing and gas exhaustion to set anaerobic conditions
in the vessel
[84]
2011 Six-well method
Anaerobic cultivation and isolation using a six-well plate and
supporting anaerobiosis generating system [88]
2012
Application of microplate technique
on methanogens cultivation
The first usage of microplate reader technique to cultivate
methanogens under H2/CO2(4:1 (v/v)) atmosphere and to
measure their optical density
[130]
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