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Process Biochemistry & microbiological

conditions required for Anaerobic process - I


Prof SK Maiti
Department of Environmental Sc. & Engg
ISM, Dhanbad
INTRODUCTION
Anaerobic wastewater treatment is an oldest concept that dates back to 1964
(McCarty, 1964), but it is only after the extensive works of researchers like
Lettinga (1979), Pette (1979), Jewell (1981) etc, anaerobic treatment
technology for treatment of domestic sewage and highly concentrated industrial
waste have been successfully used.
Anaerobic biological treatment processes offer several, advantages over
aerobic system like
1) A high degree of waste stabilization is possible at high loading rate;
2) Significantly lower production of microbial sludge due to lower microbial cell yield
(approximately 0.1-g VSS/g COD vs. 0.5 to 1.0 g VSS/g COD in aerobic treatment),
3) Excess sludge has good dewatering characteristics,
4) Low nutrient requirement,
5) No requirement of aeration;
6) Production of useful end products in terms of methane at a theoretical rate of 0.33 m
3
CH
4
/
kg COD stabilized;
7) Well developed sludge can be preserved for a period of one year or more without any
appreciable deterioration and
8) May be less sensitive to toxic compounds than aerobic process.
Major disadvantages
1. Slow growth rate of methane bacteria, which means that low
substrate utilization rate, hence require relatively very long detention
time in the reactor.
2. Low growth rate resulting long start-up time (this is not only a
problem at the beginning of the operation but also after mechanical
disturbance and inhibition, which requires recovery).
3. The anaerobes are considered more efficient at temperature of 35
o
C
and higher, requiring some of the energy from methane to be used
for heating the content of the reactor and
4. Effluent from anaerobic treatment needs some post-treatment
before discharge because of lower oxygen content.

ADVANTAGES Disadvantages
1. Less energy required Anaerobic processes may be net
energy producers instead of energy users.
2. Less biological sludge production Because the energetics of
Anaerobic processes results in lower biomass production by a
factor of about 6 to 8 times, sludge processing and disposal
costs are reduced greatly.
3. Fewer nutrients required- Many industrial wastewaters lack
sufficient nutrients to support aerobic growth. The cost of
nutrient addition is much less for anaerobic processes because
less biomass is produced.
4. Methane production, a potential energy source
5. Smaller reactor volume required - Anaerobic processes
generally have higher volumetric organic loads than aerobic
process; Organic loading rates (OLR) of 3.2 to 32 kg
COD/m
3
.day may be used for anaerobic process, compared to
0.5 to 3.2 kg COD/m
3
.day for aerobic processes.
6. Elimenation of gas air pollution;
7. Rapid response to substrate addition after long periods without
feeding.

1. Longer start-up time to develop necessary
biomass inventory (months for anaerobic
treatment versus days for aerobic processes),
2. May require alkalinity addition The most
significant negative factor that can affect the
economics of anaerobic versus aerobic
treatment is the possible need to add alkalinity.
Alkalinity concentration of 2000 to 3000 mg/L
as CaCO3 may be needed in anaerobic
processes to maintain an acceptable pH.
3. May require further treatment with an aerobic
process to meet discharge requirements;
4. Biological nitrogen & phosphorus removal is
not possible;
5. Much more sensitive to the adverse effect of
lower temperatures on reaction rates;
6. May be susceptible to upsets due to toxic
substances;
7. Potential for production of odours and
corrosive gases.
Biodegradation pathway
Hydrolysis process
Protease
Peptidase
Lipase
FATS
FATS Fatty Acids +
Glycerols
Cellulase
Carbohydrate
(starch, cellulose)
Polysaccharides
Monosaccharides
(sugar)
Protein
Polypeptides
Amino acids
ACIDOGENESIS (non-methanogenic)
The organisms are known as non-methanogenic, consists of facultative
and obligate anaerobic bacteria also known as Acitogens or Acid formers.
Among the non-methanogenic bacteria that have been isolated from
anaerobic digester are Clostridium spp., Peptococcous anaerobus,
Bifidobacterium spp., Desulphovibrio spp., Cornybacterium spp.,
Lactobacillus, Actinomycetes and Escherichia coli.
Other physiological group present includes those producing proteolytic,
lipolytic, ureolytic or cellulytic enzyme.
The sugar and amino acids are farther metabolized to acetic acid, H
2
or
higher volatile fatty acid (VFA) like- butyric acid, propionic acid, and
alcohol like methanol and ethanol.
The major routes of these metabolisms are via Pyruvic acid fermentation.
Among these, only CO
2
, H
2
and acetic acid can be converted to CH
4
by
methane bacteria.
The other two important products, VFA and alcohols must be converted to
acetic acid before conversion to CH
4
.
ACETOGENSIS Process
The VFAs and alcohols are converted by acetogens to the H
2
,CO
2
,
formate and acetate as follows:
(i) Propionate Acetate
CH
3
CH
2
COOH + 2H
2
O CH
3
COOH + CO
2
+ H
2
. [G
o
= +76.1 kJ]

(ii) Butyrate Acetate
CH
3
CH
2
CH
2
.COOH +2H
2
O 2CH
3
COOH + 2H
2
[G
o
= +48.1 kJ]

(iii) Ethanol Acetate
CH
3
CH
2
OH + 2H
2
O CH
3
COOH + H
2
[G
o
= +9.6 kJ]

(iv) Lactate Acetate
CH
3
CHOH.COOH + 2H
2
O CH
3
COOH+ CO
2
+ 2H
2
. [G
o
= - 4.2 kJ]
METHANOGENESIS
These organisms are strict anaerobes, called as "methanogens" or
"methane former".
The principle genera of microorganisms are rod shaped bacteria
(Methanobacterium. Methanobacillus), spherical shaped
(Methanosarcina, Methanoccocus).
These organisms convert Acetic acid, H
2
, and CO
2
into Methane.
The Acetoclastic methanogens convert acetic acid to methane as follows:
CH
3
COOH + H
2
O CH
4
+ CO
2
[G
o
= -104.6 kJ]

The Hydrogenclastic methanogens converted CO
2
and H
2
to methane as follows:
CO
2
+ 4H
2
CH
4
+ 3H
2
O [G
o
= -135.6 kJ]
It is important to note that methane bacteria can only use a
limited number of substrates for the formation of CH
4
.

Currently, it is known that, methanogens use following
substrate: CO
2
+ H
2
, Formate, Acetate, Methanol, Methalamine
and CO for methane processors.

(i) Formic acid Methane
4HCOOH CH
4
+ 3CO
2
+ 2H
2
O

(ii) Methanol Methane
4CH
3
OH 3CH
4
+CO
2
+ 2H
2
O.

(iii) Methalamine Methane
4(CH
3
)
3
N + H
2
O 9CH
4
+ 3CO
2
+ 6H
2
O + 4NH
3

METHANOGENESIS
In the anaerobic environment, 70% of methane is formed
from acetic acid and rest from CO
2
and H
2
as shown below.
Complex
organics
Higher
organic
acids
H
2

Acetic
acid
CH
4

28%
72%
24%
52%
76%
20%
4%
Carbon and hydrogen flow during CH4 production
Hydrolysis
Acidogenesis
Complex
substrate
Simple soluble
substrate
Methanogenesis
Ace
tate Acetogenesis
H
2
, CO
2
,
Formate
Acetate
CO
2
, CH
4

VFA
Alcohol
product
s
H2,
CO2,
Formate
Single Phase digestion
Hydrolysis
Simple
soluble
substrate
Acidogenesis
H
2
, CO
2

Methanogenesis
Acetogenesis
VFA
H
2
, CO
2
,
Formate
Acetate
Pha
se 1
Phase 2
CO
2
, CH
4

Two Phase digestion
Acetate,
Formate
Figure 1: Principle sequence of anaerobic biodegradation
Complex
substrate
OPTIMUM ENVIRONMENTAL ONDITIONS
FOR ANAEROBIC TREATMENT
Biologically, it may stated that the optimum conditions for anaerobic treatment are
prevalent when the microorganisms are living in an environment allowing the highest
possible growth rate Biological optimum conditions for anaerobic treatment are
given below:
Parameters Optimum conditions
1. Wastewater
Balanced in carbon source, macro and
micronutrients.
2. Composition
Absence of oxidized compounds prone to
reduction (O
2
, NO
3
, H
2
O
2
, SO
4
); Absence of toxic
material
3. Temperature 55 to 60
0
C
4. pH 6.8-8.0
Q4. Enumerate the optimal conditions required for CH
4
production.
OPTIMUM REACTORS CONDITIONS FOR GROWTH OF (AF) AND (MF)
Parameters Acid Former (AF) Methane Former (MF)
Temperature
Acid formers (AF) favors the
growth in 30
0
C .
For mesophilic methane former (MF),
the optimum temperature is 35-37
0
C
pH
AF, a more acidic pH range is
desirable. At pH 7.0 acid
formations is inhibited.
MF are very pH sensitive and reaction
should held in the pH range of 6.8-7.2.
The optimum pH MF reported is 6.6-7.6
(McCarty, 1964). At pH below 6.2, the
efficiency drops significantly.
Different Hydrogen
pressure:
AF developed under low partial
pressure of hydrogen. If such
condition does not maintained,
they can not survive.
In case of MF, bacteria require
hydrogen as an electron donor and also
relatively insensitive towards higher
hydrogen concentration.
Stirring velocity:
Through mixing of the substrate -
bacterial mixture is a condition
of the optimal progress of the
hydrolysis and acid-forming
reactor.
Very slow internal mixing is require.
Different growth
condition:
AF are fast grower than MF MF are slow grower
Therefore, phase separation of AF and MF is envisaged for anaerobic degradation of waste.
Q5. How temperature, pH, stirring velocity and different hydrogen pressure effects the growth of acid formers
and methane former during anaerobic degradation of sewage.
PHASE SEPARATION
1. In the two-stage process, the hydrolysis and fermentation/acidogenesis are contained in the first phase and the
acetogenesis/methanogenesis reactions in the second phase.
2. The two -phase processes isolate both potentially rate-limiting steps and thereby enhances each.
3. The first phase can be enhanced by more contact between extra-cellular enzyme and complex substrate.
4. A smaller reactor volume containing primarily acidifying organisms can create higher concentration of enzymes.
5. In the second-phase, acetogenic and methanogenic reactions are promoted as well as biological reactions that
require symbiotic relationship.
6. The major advantages and disadvantages of phase separation is shown in table 1 (Fox and Pohland, 1994).
ADVANTAGES
Isolates and optimize potential rate-limiting steps;
o Hydrolysis is encouraged during first phase, while methanogenesis is
encouraged during second phase.
Improves reaction kinetics and stability
o pH control in each phase;
o improve reactor stability to shock load
o select for faster -growing microbe
DISADVAN-
TAGES
Disruption of symbiotic relationship
More difficult to implement, engineer and operate_
Lack of process experience and applicability to variety of waste
Uncertainty of linkage between substrate type and reactor configuration
Table 1: Major advantages and disadvantages of phase seperation

FACTORS AFFECTS THE ANAEROBIC PROCESS

These are the following factors affect the performance of
anaerobic process:
1. Organic loading and the type of waste,
2. SRT and HRT,
3. Temperature,
4. pH and alkalinity,
5. Stirring velocity,
6. Nutrient content and
7. Inhibitors.
V
So Q.
load Organic
Xe
Xr
Xe Q
Xr V
SRT
c
.
.
.
) (


6.1 ORGANIC LOADING
Organic loading is usually measured as COD or BOD, which is depends on the type of
organics. The Organic load (COD) is applied per volume of the reactor and expressed as Kg
COD/m
3
/day.

; Where. Q= Influent flow rate (m
3
/day); So = influent waste concentration (g/m
3
) and V =
reactor volume, m
3
.
If So is constant, load is proportional with the flow. Hence, by reducing the flow, the load
can be reduced in the reactor. The organics i.e., sugar waste are degraded rapidly and
therefore, high organic loading can be used.

6.2 SRT and HRT
The potential parameter used for designing of anaerobic treatment process is the SRT. It is
the average time that the microorganisms remain in the reactor during the treatment. It is
de-fined as:


Xe
Xr
Xe Q
Xr V
SRT
c
.
.
.
) (


[3]. TEMPERATURE
Bacteria may be divided into the following groups based on their temperature growth
ranges;
Cryophilic able to grow rapidly below 20
0
C;
Thermophilic species grow best at temperature above 55
0
C;
Mesophilic are grow best at intermediate temperature range between cryophilic
and thermophilic (22 55
0
C).

Bacterial formation of methane has been demonstrate between 0
0
C to 97
0
C .It is well
known from the literature that, microorganisms participating in anaerobic treatment,
divide faster at higher temperature.
Optimal temperature for biomethananation is at least 55 60
0
C.
Since SRT, is a measure of the amount of microorganism per amount of substrate, an
increase in SRT can be compensated for a lower temperature.
During treatment, constant temperature is desirable, and it should not fluctuate more
than 2
0
C/day (Mosely, 1974).
The need for a constant temperature is explained by the different kinetic behavior of
acidogenic and methanogenic organisms.
Owing to their faster growth rates, the acidogens acclimatize more rapidly to changed
conditions, causing an accumulation of metabolic products, which result is an overall
imbalance.
[4]. pH and ALKALINITY
Simply, the anaerobic process may be regarded as an acid producing step (hydrolysis
+acidification) and followed by acid consuming step (methanogenesis).
The methanogenic bacteria have stricter requirement for pH than acidogenic bacteria.
Optimal pH for methanogens is 6.8-8.0 , and if pH drops below 6.2 methane production rate
drops. In an anaerobic digestion system, pH is controlled by carbon dioxide- bicarbonate
equilibrium (MaCarty et.al, 1964):
3
3 2
H
HCO
CO H K
t

Where, K
t
= ionization constant for carbonic acid.
H
2
CO
3
= carbonic acid concentration, related to percentage of CO
2
in the reactor.
HCO
3
= bicarbonate ion concentration, forming of the total alkalinity.
The bicarbonate alkalinity is approximately equivalent to the total alkalinity for most waste,
when VFA concentration is low. When VFA begins to increase in concentration, the pH is
buffered by the bicarbonate alkalinity.
It is found that, CO2 gas and bicarbonate alkalinity are closely related. Hence, low alkalinity
values in an anaerobic reactor do not allow much safety factor for increase in volatile acid.
It is therefore, desirable to have bicarbonate alkalinity value in the range of 2000 5000 mg/L,
in order to provide a buffer capacity to increase with a minimum decrease of pH.
It is recommended that VFA concentration should be less than 250 mg/L. The alkalinity in the
reactor can be controlled by- (i) by reducing the feed rate, which will allow to utilized the VFA
in the reactor and (ii) addition of alkaline materiel such as lime or sodium hydroxide.
[5]. NUTRIENTS
i. For any biological system to operate, inorganic nutrients
required for bacteria growth must be supplied.
ii. The minimum requirement of C:N:P ratio is 100:6:1. However
several investigations reported in the range of 200:5:1 to
800:5:1 (Matta, 1985).
iii. When the loading rate is high, the ratio is usually 100:5:1 to
500:6.7:1.
iv. For complex waste, especially higher carbohydrate contents,
the ratio of 300:6.7:1 should be preferred (Frostell, 1985).
v. Besides nitrogen and phosphorus, a large number of other
elements have been shown to be necessary for optimum
anaerobic growth like, Ni, Co, No, Mg, Fe, K etc.
[6.] OXIDANTS
It has been long recognized that absence of free oxygen is an obligatory prerequisite for
stable anaerobic treatment. The chemically bound oxygen interferes negatively, if available to
anaerobes. The interferes of compounds like, NO
3
and SO
4
/SO
3
in gaining wider importance
in anaerobic treatment.
(i) Nitrate: If wastewater contains high concentration of NO3, by induction of pre-
denitrification can improve the performance.
(ii). Sulfate: Sulfides requires special attention in case of all anaerobic processes. Sulfide may
come from industrial process or may formed in the anaerobic unit as a result of reduction of
sulfate present in wastewater. Sulfur reducing bacteria utilized sulfate as an electron acceptor
with H
2
S being a end product. Elemental sulfur or organic sulfur compound may also appear
in product.
The role of sulfite and sulfate is to divert the electron from methanogenesis thus resulting in a lower
methane production.
Another influences is the production of H2S gas, which inhibits anaerobic treatment when present at
higher concentration. The inhibition of acetoclastic methanogens by sulfide was studied & found that
more than 40% inhibition of methanogenesis at free H
2
S level of 50 mg/L, and increases to 100%
inhibition at 250 mg/L.
It has been showed that, when TOC level is less than 170 g/L (equivalent to 500 mg/L COD), sulfate
concentration up to 160 mg/L do not effect the TOC reduction. Therefore, when TOC/sulfate ratio = 1,
there will be no problems, which is common in majority of sewage.
[7]. Inhibitors/ toxic materials
A large number of toxicants are known to affects the anaerobic biological process; like-
1. Heavy metals;
2. Detergent and disinfectant used in food industry clean up
3. Solvent from degreasing operation,
4. Inhibitors formed as secondary products (e.g, cyanide in coal coking operation).
5. Chemical inhibitors for food preservation.

The available strategies to decrease the impact of toxic material have been listed as follows:
1. Remove toxic material from waste
2. Dilute the waste,
3. Allow a long adaptation period,
4. Use detoxification stage
5. Design for a longer SRT,
6. Form an insoluble complex or precipitate,
7. Antagonize toxicity with another materials.

Removal of toxic materials or dilutions of the waste are rather obvious solutions, but
the possibility of NH
3
, SO
2
, H
2
S stripping is mentioned by Takesshita et.al (1982).
MONITORING AND CONTROL OF ANAEROBIC TREATMENT
PARAMETER FREQUENCY
(i) Influent (rate. concentration
composition, toxicity)
+++
(ii) SRT +++
(iii) Temperature +++
(iv) pH +++
(v) Gas production and composition ++
(vi) Treatment efficiency ++
(vii) Volatile fatty acid and alkalinity +
+++= Should continuously monitored and controlled
++ = daily monitored; + =Intermittent monitoring.
Q8. List the important parameters to be monitored for the improvement of anaerobic
process efficiency and their frequency of monitoring.
8.0 Points to be remembered
1. Anaerobic treatment definitely has the potential to treat low strength
wastewater.
2. Major advantages are low energy requirement, low nutrient
requirement, low sludge production, however slow growth of
anaerobes has disadvantages that causes higher solid retention time
in the reactor.
3. Separate reactor conditions are needed for anaerobic process,
however partial phase separation could be achieved in UASB,
anaerobic filter, and expanded bed reactor.
4. Organic loading, pH and alkalinity, SRT and HRT, nutrients, oxidant
and toxic substances affect the performance of the anaerobic process.
5. Frequency of monitoring is important for process improvement. A drop
in pH, increase in VFA/alkalinity ratio and increase of VFA in effluent
alarms the process failure.