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Feb 20, 2013 (4 years and 10 months ago)

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AMITY UNIVERSITY

RAJASTHAN

AMITY INSTITUTE OF BIOTECHNOLOGY

MICROBIOLOGY ASSIGNMENT

ON

METHANOGENESIS, ACETOGENESIS &
NITROGEN FIXATIO
N



SUBMITTED BY:
-


Deepak Kumar
Tanwar


Btech. Bioinformatics


METHANOGENESIS


Methanogenesis is a microbial process, involving many complex, and differently interacting
species, but most notably, the methane
-
producing bacteria. Th
e biogas process is shown
below in figure 1, and consists of three stages; hydrolysis, acidification and methane
formation.


Figure 1: The process of methanogenesis
).

In the first stage of enzymatic hydrolysis, the extracellular enzymes of microbes, such

as
cellulase, protease, amylase and lipase externally enzymolize organic material. Bacteria
decompose the complex carbohydrates, lipids and proteins in cellulosic biomass into more
simple compounds. During the second stage, acid
-
producing bacteria convert

the simplified
compounds into acetic acid (CH
3
COOH), hydrogen (H
2
), and carbon dioxide (CO
2
). In the
process of acidification, the facultatively anaerobic bacteria utilise oxygen and carbon,
thereby creating the necessary anaerobic conditions necessary for methanogenesis. In the
final stage, the obligatory anaerobes that are involved in methane
formation decompose
compounds with a low molecular weight, (CH
3
COOH, H
2,
CO
2
), to form methane (CH
4
) and
CO
2
(Gate, 1999).

The resulting biogas, sometimes referred to as 'gobar' gas, consists of methane and carbon
dioxide, and perhaps some traces of other

gases, notably hydrogen sulphide (H
2
S). Its exact
composition will vary, according to the substrate used in the methanogenesis process, but as
an approximate guide, when cattle dung is a major constituent of fermentation, the resulting
gas will be between

55
-
66% CH
4
, 40
-
45% CO
2
, plus a negligible amount of H
2
S and H
2

(KVIC, 1993). Biogas has the advantage of a potential thermal efficiency, given proper
equipment and aeration, of 60%, compared to wood and dung that have a very low thermal
efficiency of 17%
and 11% respectively (KVIC, 1993).

Methanogenesis or more particularly, the bacteria involved in the fermentation process are
sensitive to a range of variables that ultimately determine gas production, and it is worth
briefly outlining these factors. Tempe
rature is perhaps the most critical consideration.
Gasification is found to be maximised at about 35
o
C, and below this temperature, the
digestion process is slowed, until little gas is produced at 15
o
C and under. Therefore in areas
of temperature changes,
such as mountainous regions, or winter conditions that may be more
accentuated inland, mitigating factors need to be taken into account, such as increased
insulation (Kalia, 1988), or the addition of solar heaters to maintain temperatures (Lichtman,
1983).

Loading rate and retention period of material are also important considerations. In the KVIC
model, retention ranges between 30
-
55 days, depending upon climatic conditions, and will
decrease if loaded with more than its rated capacity (which may result in

imperfectly digested
slurry). KVIC state that maximum gas production occurs during the first four weeks, before
tapering off, therefore a plant should be designed for a retention that exploits this feature.
Retention period is found to reduce if temperatu
res are raised, or more nutrients are added to
the digester. Human excreta, due to its high nutrient content, needs no more than 30 days
retention in biogas plants (KVIC, 1983).

Other factors likely to affect methanogenesis are pH; gas production is found
to decrease with
increasing acidity, and can result from over
-
loading the plant, which may stimulate the more
fecund acidophiles, at the expense of the more tardy methane
-
producing microbes. Improved
nutrient content, also, as mentioned above will increase

the digestion process, and can be
manipulated by the addition of animal (and male human) urine, while toxic substances, such
as heavy metals may inhibit gas production (KVIC, 1983).

Understanding the process of methanogenesis allows manipulation, which c
an serve to
maximise gas production in the field. Workers over the last twenty years have experimented
with the digestion process, and have made strides in increasing gas yields, using techniques
that can be similarly employed in a rural environment. Sharm
a et al (1988), have shown that
biogas generation is increased when the particle size of organic material is small, in this case,
less than 1mm. The workers recommend that a physical pre
-
treatment, such as grinding
would improve a system's performance, and

could also reduce the size of digester needed. A
manual machine for physical pre
-
treatment of material would be a viable piece of equipment
in a rural environment; indeed, there may be a similar piece of equipment already in use.

Other workers have found
that biogas production is accelerated by the presence of metal ions
in biomass (Geeta et al, 1990). The species principally researched was water hyacinth
(
Eichornia crassipes
Solms.), which flourishes in eutrophic water bodies. The plant
characteristically

grows at high densities, which often leads to clogging, and is therefore
considered an environmental pest. Water hyacinth, however, also concentrates nickel from
eutrophic environments, upto 0.27 kg h/day, which, when mixed with bovine excreta upto 25
par
ts per million (ppm) was found to enhance gas production by 40%. The use of
E. crassipes
in biogas systems can both increase gas production, and contribute to environmental
management, by way of controlling a pest.

Research in other areas has focused on th
e composition on the substrate, and its effect on gas
production. Habig (1985), fermented a range of organic materials from marine macroalgae to
vegetables and discerned that carbohydrate and protein are the principal components utilised
during methanogene
sis.

Such work is invaluable in enabling a sound management and manipulation of
methanogenesis, and can be of use to users in a rural environment.




NITROGEN FIXATION

Nitrogen fixation

is the natural process, either biological or abiotic, by which
nitrogen

(N
2
) in
the
atmosphere

is converted into
ammonia
. This process is e
ssential for life because fixed
nitrogen is required to
biosynthesize

the basic building blocks of life, e.g.
nucleotides

for
DNA and RNA

and
amino
acids

for
proteins
. Formally, nitrogen fixation also refers to other
abiological conversions of nitrogen, such as its conversion to
nitrogen dioxide
.

Nitrogen fixation is utilized by numerous
prokaryotes
, including
bacte
ria
,
actinobacteria
, and
certain types of
anaerobic

bacteria. Microorganisms that fix nitrogen are called
diazotrophs
.
Some higher plants, and some animals (
termites
), have formed associations (
symbioses
) with
diazotrophs. Nitrogen fixation also occurs as a result of non
-
biological processes. These
include
lightning
, industrially through th
e
Haber
-
Bosch Process
, and
combustion
. Biological
nitrogen fixation was discovered by the Dutc
h microbiologist
Martinus Beijerinck
.

Biological nitrogen fixation




Biological nitrogen fixation (
BNF
) occurs when atmospheric nitrogen is converted to
ammonia by an enzyme called
nitrogenase
. The formula for BNF is:


N
2

+ 6 H
+

+ 6 e


→ 2 NH
3

The process is coupled to the hydrolysis of 16 equivalents of
ATP

and is accompanied by the
co
-
formation of one molecule of H
2
. In free
-
living diazotrophs, the
nitrogenase
-
generated
ammonium is assimilated into
glutamate

through the glutamine synthetase/glutamate
synthase pathway.

Enzymes responsible for nitrogenase action are very susceptible
to destruction by oxygen. (In
fact, many bacteria cease production of the enzyme in the presence of oxygen) Many
nitrogen
-
fixing organisms exist only in anaerobic conditions, respiring to draw down oxygen
levels, or binding the oxygen with a protein such a
s
Leghemoglobin
.

Plants that contribute to nitrogen fixation include the
legume

family



Fabaceae



with taxa
such as
clover
, soybeans,
alfalfa
,
lupines
,
peanuts
, and
rooibos
. They contain
symbiotic

bacteria called
Rhizobia

within
nodu
les

in their
root systems
, producing nitrogen compounds
that help the plant to grow and compete with other plants. When the plant dies, the fixed
nitrogen is released, making it available to other

plants and this helps to fertilize the
soil
.
The
great majority of legumes have this association, but a few genera (e.g.,
Styphnolobium
) do
not. In many traditional and organic farming practices, fields are rotated through various
types of crops, which usually includes one consisting mainly or entirely of clover or
buckwheat (family
Polygonaceae
), which were often referred to as "
green manure
."

Non
-
leguminous nitrogen
-
fixing plants

Although by far the majority of nitrogen
-
fixing plants are in the legume family
Fabaceae
,
there are a few non
-
leguminous plants, such as alder, that can also fix nitrogen. These plants,
re
ferred to as "
actinorhizal plants
", consist of 24

genera of woody shrubs or trees distributed
among in 8 plant families. The ability to fix nitrogen is not universally
present in these
families. For instance, of 122 genera in the
Rosaceae
, only 4 genera are capable of fixing
nitrogen. All these families belong to the
orders

Cucurbitales
,
Fagales

and
Rosales
, which
together with the
Fabales

form a clade of
eurosids
. In this clade, Fabales
were the first lineage
to branch off; thus, the ability to fix nitrogen may be
plesiomorphic

and subsequently lost in
most descendants of the original nitrogen
-
fixing plant; alte
rnatively, it may be that the basic
genetic

and
physiological

requirements were present in an incipient state in

the
last common
ancestors

of all these plants, but only evolved to full function in some of them:

There are also several nitrogen
-
fixing symbiotic associations that involve
cyanobacteria

(such
as
Nostoc
). These include some lichens such as
Lobaria

and
Peltigera
:



Mosquito fern

(
Azolla

species)



Cycads



Gunnera




A sectioned alder tree root nodule.



A whole alder tree root nodule.

Microorganisms that fix nitrogen



Diazotrophs



Cyanobacteria



Azotobacteraceae



Rhizobia



Frankia

Nitrogen fixation by cyanobacteria

Cyanobacteria

inhabit nearly all illuminated environments on Earth and play key roles in the
carbon and
nitrogen cycle

of the
biosphere
. Generally,
cyanobacteria

are able to utilize a
variety of inorganic and

organic sources of combined nitrogen, like
nitrate
,
nitrite
,
ammonium
,
urea

or some
amino acids
. Several cyanobacterial strains are also capable of
diazotrophic

growth.
Genome sequencing

has provided a large amount of information on the
genetic bas
is of
nitrogen metabolism

and its control in different cyanobacteria.
Comp
arative
genomics
, together with functional studies, has led to a significant advance in this field over
the past years. 2
-
oxoglutarate has turned out to be the central signalling molecule reflecting
the carbon/nitrogen balance of cyanobacteria. Central pla
yers of nitrogen control are the
global transcriptional factor NtcA, which controls the expression of many genes involved in
nitrogen metabolism, as well as the P
II

signalling protein, which fine
-
tunes cellular activities
in response to changing C/N condit
ions. These two proteins are sensors of the cellular 2
-
oxoglutarate level and have been conserved in all cyanobacteria. In contrast, the adaptation to
nitrogen starvation involves heterogeneous responses in different strains.
[4]

Nitrogen fixation
by cyanobacteria in
coral reefs

can fix twice the amount of nitrogen than on land

around
1.8

kg of nitrog
en is fixed per hectare per day.

.
Symbiotic Nitrogen Fixation

Symbiotic nitrogen fixation occurs in plants that harbor nitrogen
-
fixing bacteria within their
tissues. The best
-
studied example is the association between
legumes

and
bacteria

in the
genus
Rhizobium
.

Each of these is able to survive independently (soil nitrates must then be available to the
legume), but life together is clearly beneficial to both. Only together can nitrogen fixation
take place.

A symbiotic relationship in which both partne
rs benefits is called
mutualism
.


Rhizobia

Rhizobia are
Gram
-
negative bacilli

that live freely in the soil (especially where legumes have
been

grown). However, they cannot fix atmospheric nitrogen until they have invaded the
roots of the appropriate legume
.

The Infection Thread

The interaction between a particular strain of rhizobia and the "appropriate" legume is
mediated by:



a "Nod factor" se
creted by the rhizobia and



transmembrane receptors

on the cells of the
root hairs

of the legume.



Different strains of rhizobia produce different Nod factors, and



different legumes produce receptors of different specificity.

If the combination

is correct, the bacteria enter an epithelial cell of the root; then migrate into
the cortex. Their path runs within an intracellular channel that grows through one cortex cell
after another. This
infection thread

is constructed by the root cells, not the
bacteria, and is
formed only in response to the infection.

When the infection thread reaches a cell deep in the cortex, it bursts and the bacteria are
engulfed by
en
docytosis

into endosomes. At this time the cell goes through several rounds of
mitosis


without
cytokinesis



so the cell becomes
polyploid
.

The electron micrograph on the right (courtesy of Dr. D. C. Jordan) shows a rhizobia
-
filled
infection thread growing into the cell (from the upper left to the lower right). Note

how the
wall of the infection thread is continuous with the wall of the cell. Once the thread ruptures,
rhizobia are engulfed into membrane
-
bound
symbiosomes

within the cytoplasm (dark ovals).

The cortex cells then begin to divide rapidly forming a
nodul
e
. This response is driven by the
translocation of
cytokinins

from epidermal cells to the cells of the cortex.

The photo on the left (courtesy of The Nitragin Co. Milwaukee, Wisconsin) shows nodules
on the roots of the birdsfoot trefoil, a legume.

The rhizobia also go through a period of rapid multiplication within the nodule cells. Then
they begin to change shape

and lose their motility. The
bacteroids
, as they are now called,
may almost fill the cell. Only now does nitrogen fixation begin.

The electron micrograph on the right (courtesy of R. R. Hebert) shows bacteroid
-
filled cells
from a soybean nodule. The hori
zontal line marks the walls between two adjacent nodule
cells.

Root nodules are not simply structureless masses of cells. Each becomes connected by the
xylem and phloem to the vascular system of the plant. The photo below on the left shows a
developing la
teral root on a pea root. On its right is a segment of a pea root showing a
developing nodule

12 days after the root was infected with rhizobia. Both structures are
connected to the nutrient transport system of the plant (dark area extending through the ce
nter
of the root). (Photomicrographs courtesy of the late John G. Torrey.)

Thus the development of nodules, while dependent of rhizobia, is a well
-
coordinated
developmental process of the plant.

Although some soil bacteria (e.g.,
Azotobacter
) can fix nit
rogen by themselves, rhizobia
cannot. Clearly rhizobia and legumes are mutually dependent.

The benefit to the legume host is clear. The rhizobia make it independent of soil nitrogen.

But why is the legume necessary? The legume is certainly helpful in tha
t it supplies nutrients
to the bacteroids with which they synthesize the large amounts of
ATP

needed to convert
nitrogen (N
2
) into ammonia (NH
3
)

In addition, the legume host supplies one critical component of
nitrogenase



the key
enzyme for fixing nitrogen.

The bacteroids need oxygen to make their ATP (by
cellular respiration
). However,
nitrogenase is strongly inhibited by oxygen. Thus the bacteroids must walk a fine line
between too much and too little oxygen. Their job is made easier by another contribution
from their host:
hemoglobin
.

Nodules are
filled with hemoglobin. So much of it, in fact, that a freshly
-
cut nodule is red.
The hemoglobin of the legume (called
leghemoglobin
), like the hem
oglobin of vertebrates,
probably supplies just the right concentration of oxygen to the bacteroids to satisfy their
conflicting requirements.

The metal
molybdenum

is a critical component of
nitrogenase

and so is absolutely essential
for nitrogen fixation.

But the amounts required are remarkably small. One ounce of
molybdenum broadcast over an acre of cropland in Australia was found to be sufficient to
restore fertility for over ten years.

The photo at the right shows that the legume clover grows normally
only where the supply of
molybdenum is adequate. The soil shown here (in eastern Australia) is naturally deficient in
molybdenum. Although the entire fenced
-
in plot was seeded to clover, the plant was able to
flourish and fix nitrogen only where molybdenum

fertilizer had been added (foreground).


Because of the specificity of the interaction between the Nod factor and the receptor on the
legume, some strains of rhizobia will infect only peas, some only clover, some only alfalfa,
etc. The treating of legume

seeds with the proper strain of rhizobia is a routine agricultural
practice. (The Nitragin Company, that supplied one of the photos above specializes in
producing rhizobial strains appropriate to each leguminous crop.)

How did two such organisms ever wor
k out such an intimate and complex living
relationship? Assuming that the ancestors of the rhizobia could carry out the entire process by
themselves


as many other soil bacteria still do


they must have gained some real
advantage from evolving to share t
he duties with the legume. Perhaps the environment
provided by their host, e.g., lots of food and just the right amount of oxygen, enabled the
rhizobia to do the job more efficiently than before.

Nitrification

Ammonia can be taken up directly by plants


usually through their roots. However, most of
the ammonia produced by decay is converted into
nitrates
. This is accomplished in two steps:



Bacteria of the genus
Nitrosomonas

oxidize NH
3

to
nitrites

(NO
2

).



Bac
teria of the genus
Nitrobacter

oxidize the nitrites to
nitrates

(NO
3

).

These two groups of autotrophic bacteria are called
nitrifying bacteri
a
. Through their
activities (which supply them with all their energy needs), nitrogen is made available to the
roots of plants.

Both soil and the ocean contain archaeal microbes, assigned to the
Crenarchaeota
, that
convert ammonia to nitrites. They are more abundant than the nitrifying bacteria and may
turn out to play an important role in the nitrogen cycle.

Many legumes, in addition to fixing atmospheric nitrogen, also perform nitrification


converting some of their organic nitrogen to nitrites and nitrates. These reach the soil when
they shed their leaves.

Denitrification

The three processes above remove
nitrogen from the atmosphere and pass it through
ecosystems.

Denitrification reduces nitrates to nitrogen gas, thus replenishing the atmosphere.

Once again, bacteria are the agents. They live deep in soil and in aquatic sediments where
conditions are
anaerobic
. They use nitrates as an alternative to oxygen for the final electron
acceptor in their
respiration
.

Thus they close the nitrogen cycle.








ACETOGENESIS

Although some acetate (20%) and H
2

(4%) are directly produced by acidogenic fermentation
of sugars, and amino acids, both products are primarily derived from the acetogenesis and
dehydrogenation

of higher volatile fatty acids.

Obligate H
2
-
producing acetogenic bacteria are capable of produc
ing acetate and H
2

from
higher fatty acids. Only
Syntrophobacter wolinii,

a propionate decomposer (3) and
Sytrophomonos wolfei,

a butyrate decomposer (4) have thus far been isolated due to technical
difficulties involved in the isolation of pure strains, s
ince H
2

produced, severely inhibits the
growth of these strains. The use of co
-
culture techniques incorporating H
2

consumers such as
methanogens and sulfate
-
reducing bacteria may therefore facilitate elucidation of the
biochemical breakdown of fatty acids.


Overall breakdown reactions for long
-
chain fatty acids are presented in Tables 4
-
1 and 4
-
2.
H
2

production by acetogens is generally energetically unfavorable due to high free energy
requirements (

”G
o,
> 0; Table 4
-
1 and 4
-
2). However, with a combination
of H
2
-
consuming
bacteria (Table 4
-
2, 4
-
3), co
-
culture systems provide favorable conditions for the
decomposition of fatty acids to acetate and CH
4

or H
2
S (

”G
o,
< 0). In addition to the
decomposition of long
-
chain fatty acids, ethanol and lactate are also
converted to acetate and
H
2

by an acetogen and
Clostridium formicoaceticum,

respectively.

The effect of the partial pressure of H
2

on the free energy associated with the conversion of
ethanol, propionate, acetate, and H
2
/CO
2

during methane fermentation is

shown in Fig. 4
-
2.
An extremely low partial pressure of H
2

(10
-
5

atm) appears to be a significant factor in
propionate degradation to CH
4
. Such a low partial pressure can be achieved in a co
-
culture
with H
2
-
consuming bacteria
.