TECHNICAL DIRECTION

Technical Document

Writer : Thierry ARNAUD

Department Energy & Sludge Valorization

Industrial methanization
Technical Guidelines
for Physico-Chemical Parameters
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> UPDATES

Follow-up
Version Date By Content
A 13/06/2013 Thierry ARNAUD First draft

Checked by
Version By Signature
A Biothane

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> CONTENT

Updates ......................................................................................................................................................... 2
Content .......................................................................................................................................................... 3
Figures ........................................................................................................................................................... 4
Tables ............................................................................................................................................................ 5
Preamble ....................................................................................................................................................... 6
I. Reminder concerning methanization ........................................................................ 7
II. General presentation of industrial anaerobic technologies in Veolia ..................... 10
III. PROCESS parameters............................................................................................... 13
A. COD & BOD5............................................................................................................. 13
B. OLR (organic loading rate) & SLR (sludge loading rate) .......................................... 14
C. TSS (total suspended solids) and VSS (volatile suspended solids) .......................... 15
D. HRT (hydraulic retention time)................................................................................ 16
E. Température............................................................................................................ 16
F. pH ............................................................................................................................ 17
G. Volatile fatty acids (VFA) and alkalinity ................................................................... 18
H. Nutrients requirements ........................................................................................... 20
IV. Bicarbonate equilibrium .......................................................................................... 22
A. Buffering capacity .................................................................................................... 22
B. Reduction of sodium hydroxide consumption by recirculation .............................. 24
C. Calcium carbonate precipitation ............................................................................. 24
V. Sulphate reduction .................................................................................................. 26
A. Sulphur compounds ................................................................................................ 26
B. Sulphide production ................................................................................................ 28
C. Calculation of H2S concentration in biogas ............................................................. 30
D. Hydrogen sulphide removal with ferric chloride..................................................... 34
VI. Toxicity & inhibition ................................................................................................ 36
A. Term explanation .................................................................................................... 36
B. Organic acids ........................................................................................................... 37
C. Nitrogen compounds ............................................................................................... 38
D. Heavy metals and other inhibitors .......................................................................... 40
To know more ............................................................................................................................................. 46

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> FIGURES

Figure 1: F420 observation - methanogenic archeas : Methanosaeta (A) Methanosarcina (B) ................... 7
Figure 2: principle of methanization ............................................................................................................. 8
Figure 3: field of applications for all anaerobic technologies ....................................................................... 9
Figure 4: Biothane Advanced® UASB: upflow anaerobic sludge blanket .................................................... 10
Figure 5: Biobed Advanced® EGSB : expanded granular sludge bed .......................................................... 10
Figure 6: Biobulk® : continuously stirred tank reactor ................................................................................ 10
Figure 7: Memthane®: anaerobic membrane bioreactor ........................................................................... 11
Figure 8: anaerobic technologies depending on COD & TSS ....................................................................... 12
Figure 9: example of membrane flux depending on the TSS concentration............................................... 15
Figure 10: Coefficient of average activity of methanogenic bacteria according to temperature. .............. 17
Figure 11: Repartition of the carbonate, bicarbonate and carbon dioxide depending on the pH value (T =
20°C). ........................................................................................................................................................... 23
Figure 12: Risk of calcium carbonate precipitation at 35°C. ....................................................................... 25
Figure 13: redox couples and anaerobic activity......................................................................................... 26
Figure 14: competition between AM & BSR................................................................................................ 27
Figure 15: Poet project - General process flow ........................................................................................... 28
Figure 16: S equilibrium in function of pH .................................................................................................. 29
Figure 17: Proportion of ammonia forms as a function of pH. ................................................................... 38

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> TABLES

Table 1: main differences between anaerobic technologies ...................................................................... 12

Table 2: OLR values for different anaerobic technologies (indication) ....................................................... 13
Table 3: OLR and COD values for different anaerobic technologies (indication) ....................................... 14
Table 4: TSS values for different anaerobic technologies (indication) ........................................................ 15
Table 5: Coefficient of average activity of methanogenic bacteria according to temperature. ................. 16
Table 6: Definitions of VFA concentrations ................................................................................................. 19
Table 7: Definition of VFA/TA ratios ............................................................................................................ 19
Table 8: Trace elements concentrations necessary for bacteria growth (Biothane’s recommendation). . 21
Table 9: Toxicity overview of heavy metals and other compounds ............................................................ 41
Table 10: IC50 concentrations for anaerobic and aerobic microorganisms ............................................... 45

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> PREAMBLE
The aim of this manual is to help operators and project engineers to better understand the
physico-chemical parameters which control the high rate anaerobic reactors used to treat
industrial wastewaters.

This manual covers all anaerobic technologies including:

 Granular sludge technologies (UASB, EGSB, IC, etc.).
 Biofilm technologies (biofilters, fluidized beds, RBCs, AMBBR)
 Technologies with flocculated sludge (CSTR, AnMBR)

If parameters are specific to certain technologies, it will be precised in the manual, such as a
degranulation due to a lack of calcium that concerns mainly UASB, EGSB, etc.

Readers are strongly encouraged to provide their comments to the authors if they have a
feedback that can be beneficial to enhance this manual.

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I. REMINDER CONCERNING METHANIZATION

Methanization or “anaerobic degradation” is a biochemical process carried out by several
types of micro-organisms in an oxygen free environment. Suitable substrates for this process
are mainly organic soluble substrates (liquid form).

It is also possible to use the path of anaerobic digestion for degrading solid organic substrates
(biological sludge from wastewater treatment plants, solid waste from food processing
industries or agriculture, etc.). But this path is not in the scope of this process note (see process
notes about digestion and co-digestion).

Concerning methanization of wastewater, there are four main process steps in the following
order:

1-Hydrolysis: exoenzymes hydrolyse biopolymers (polysaccharides, proteins, grease, etc.) into

monomers (sugars, amino acids, fatty acids, etc.). This step is very important in presence of high
concentration of organic solids but quite inexistent if the wastewater has only soluble COD.

2-Acidogenesis: facultative or strict anaerobic bacteria convert monomers in Volatile Fatty

Acids, (“VFA”: mainly acetic acid, and a few quantity of propionic and butyric acid), H 2 and CO2.

3-Acetogenesis: by-products (volatile fatty acids and alcohols) are converted in acetic acid. The
acetogenic bacteria interact closely with the methanogens.

4-Methanogenesis: This step is the final conversion of acetic acid, H2 and CO2 into methane
and carbon dioxide by methanogenic archeas. These very special microorganisms can be
distinguished from the others by fluorimetric microscopy at 420 nm. The photo n°1 with 420 nm
filter below shows some typical methanogenic archeas called Methanosaeta (A)
Methanosarcina (B). These archeas produce methane from H2+CO2 (A) or from acetic acid (B).

Figure 1: F420 observation - methanogenic archeas : Methanosaeta (A) Methanosarcina (B)

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General parameters for methanization:

- Temperature: for industrial wastewaters, mesophilic range is used in more than 80 % of

the cases (optimal: 37°C), possible applications in thermophilic (55°C) or psychrophilic
(20 to 30°C) conditions.
- pH: methanization is optimal near neutrality (6,5 to 7,5). Acidification can be applied at
pH 5-6 in case of separated reactor.
- Global anaerobic sludge production : in average 0.03 to 0.05 kg VSS/kg COD removed
- Biogas production: theoretic ratio 0.35 Nm3 CH4/kg COD removed
- Methanization don’t like oxygen (lethal for methanogens if more than 0.05 mg/L oxygen
or chemical forms like NO3->50 mg/L)
- Methanization is quite sensitive to inhibitor and toxic compounds (contact Technical
Department for more information)

The figure n°2 below shows the principle of methanization and the conversion rates based on a
theoretical 100% COD degradation.

Figure 2: principle of methanization

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Methanization can be applied wherever the wastewater is biodegradable, but mainly for high
concentrations of COD (more than 3 g/L).

The figure n°3 below shows the field of applications for all anaerobic technologies in the 2012
reference list of Biothane:

Figure 3: field of applications for all anaerobic technologies

The main limit of methanization is the absence of nitrogen and phosphorus removal (except a
few quantities for anaerobic bacteria growth). This means that a complementary treatment is
always necessary if nitrogen and/or phosphorus removal is requested.

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II.GENERAL PRESENTATION OF INDUSTRIAL ANAEROBIC TECHNOLOGIES IN VEOLIA

Concerning the anaerobic treatment of industrial wastewaters, four main technologies are
supplied by Biothane (www.Biothane.com & http://Biothane.vwsintranet.net/ – Biothane is a
subsidiary of VWST, one of the world leaders in anaerobic treatments and referenced as the
Technology Center for Anaerobic Treatment of Industrial Wastewater for Veolia):

Figure 4: Biothane Advanced® UASB: upflow anaerobic sludge blanket

Conditioning UASB reactor
tank
Granular sludge bed technology
Loading rate: 5 -15 kg COD/m3.d Biogas
TSS inlet < 1000 mg/L
Anaerobic
Operated in mesophilic range (30- effluent
40°C) recirculation
Mixing

COD removal ~ 80%

Chemicals Sludge bed
Excess
Concrete rectangular or round steel Influent granular sludge
http://www.veoliawaterst.com/Bioth
Treated effluent
ane-uasb/en/?bu=Biothane.en

Figure 5: Biobed Advanced® EGSB : expanded granular sludge bed

Conditioning Biobed® EGSB
tank reactor
Granular sludge bed technology
Loading rate: 15 - 30 kg COD/m3.d Biogas

COD removal ~ 80%

TSS inlet < 500 mg/L Anaerobic
effluent
Operated in mesophilic range (30-40°C) recirculation
Mixing
Typically round steel construction
Higher upflow velocity than UASB Chemicals
Excess
Influent granular sludge
http://www.veoliawaterst.com/biobed-
Treated effluent
egsb/en/?bu=Biothane.en

Figure 6: Biobulk® : continuously stirred tank reactor

Contact reactor
Settler
Completely mixed (anaerobic flocs instead of Biogas
granules)
Loading rate: 3 - 5 kg COD/m3.d
COD removal ~ 50-80% Effluent
TSS inlet from 1000 to 30000 mg/L
Influent
Operated in mesophilic or thermophilic range
(30-40°C or 50-70°C)
Typically round steel construction Recirculation
Sludge
http://www.veoliawaterst.com/biobulk/en/?bu=
Biothane.en

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Figure 7: Memthane®: anaerobic membrane bioreactor

Memthane® technology can be simply defined as a Biobulk® associated to a UF membrane skid.

Several patents concerning this process are pending from Biothane USA and Biothane
Netherlands.
Memthane® is applied for industrial wastewaters with the main following characteristics:
- No TSS
- Good technology for influent with soluble COD highly concentrated (> 15 000 mg/L)
- Good biodegradability by anaerobic path (no inhibitors or toxic compounds)
- Anaerobic reactor characteristics :
o Completely mixed (anaerobic flocs instead of granules)
o Loading rate: 5-8 kg COD/m3.d
o COD & TSS removal > 99% after membranes
o Operated in mesophilic range (30-40°C) (development in progress in 2012 for
thermophilic applications)
o Typically round steel construction
- Membranes characteristics :
o Norit X-Flow – cross flow ultrafiltration membranes – PVDF
o 0.03 µm
o 10-20 l/m2/h
http://www.veoliawaterst.com/memthane/en/?bu=Biothane.en

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Table n°1 shows the main differences between each anaerobic technology and table 2 the fields of
application in function of the COD and TSS ranges in wastewaters.
Memthane® Biothane Biobed Advanced®
Biobulk® CSTR
AnMBR Advanced® UASB EGSB

Biomass Flocculant Flocculant Granular Granular

Loading rate (kg

3-5 5-8 5 – 15 15 - 30
COD/m3.d)

Reactor Height (m) 12 - 20 12 - 20 6–9 12 - 20

Surface Required + ++ ++ +++

Solids Tolerance +++ ++ + +

Operation Costs ++ - or ++++* +++ ++

T-COD Rem. Efficiency + ++++ ++ +++

Market Differentiation - +++ +/- ++

Experience ++ + +++ +++

* If OPEX includes Aerobic post-treatment. OPEX anMBR is ++++

Legend: - : bad +/-: tolerance +: low ++: sufficient +++: good ++++: excellent
Table 1: main differences between anaerobic technologies

Figure 8: anaerobic technologies depending on COD & TSS

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III.PROCESS PARAMETERS
A. COD & BOD5
Chemical and biochemical oxygen demands are the first parameters taken in account for the design of
an anaerobic reactor.

COD is mainly used in industry in its total or soluble form, depending of the technology.

For granular sludge reactors, biofilters and other high rate systems, the design parameter is
soluble COD.

For CSTR and AnMBR, total COD is more adapted, notably due to the longer SRT that permit to
treat a portion of the no soluble COD after hydrolysis.

BOD is not frequently used, more often for checking the discharge parameters, regarding the
limits requested by the laws.

The ration COD/BOD is still a good indicator concerning the biodegradability of the wastewater.
Designers consider that a COD/BOD > 3 reveals a good biodegradability (whatever is the
biological way – aero or anaerobic). When COD/BOD is above 4, it is considered that sign of
inhibition or toxicity risk and designers have to be careful and investigate to have further
information about the wastewater characteristics.

Table 3 give indications about COD values used to select an anaerobic technology. These values
have to be used complementary to other parameter for the final choice.

Table 2: OLR values for different anaerobic technologies (indication)

Parameters Biobulk UASB EGSB Memthane

Min COD (mg/l) > 3 000 > 1 500 > 1 500 > 20 000

Max COD (mg/l) 125 0 15000 - 25 000 15 000 - 25000 >150 000

COD = 1,5 g/L is considered as the lowest admissible concentration in an industrial reactor.
Under this value economic feasibility is not guaranteed and biomass loss risk is important
(degranulation or wash-out). For Memthane®, the minimal COD value is given around 20 g/L
mainly for economic reasons, due to the low OLR and the high CAPEX/OPEX with membrane
technologies.

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B. OLR (organic loading rate) & SLR (sludge loading rate)

OLR is the flow of pollution that can be injected in a reactor. It is expressed in kg COD/m3.d

SLR is the flow of pollution that can be applied to a given amount of biomass. It is expressed in
kg COD/kgVS.d.

SLR currently applied is around 0,3 to 0,6 kg COD/kgVS.d, depending also on the
biodegradability of the wastewater and the COD removal efficiency expected for the project.

For granular sludge reactors, biofilters and other high rate systems, the design parameter is
soluble COD.

For CSTR and AnMBR, total COD is more adapted, due to the longer SRT that permit to treat a
portion of the no soluble COD after hydrolysis.

Table 3: OLR and COD values for different anaerobic technologies (indication)
Parameters Biobulk UASB EGSB Memthane
OLR
3 2-5 8-15 15-30 6-8
(kg COD/m .d)
Min COD (mg/l) > 5 000 > 1 200 > 1 200 > 20 000
(1) 15 000 - >150 000
Max COD (mg/l) 125 000 15 000 - 25 000 (1)
25 000

Concerning Memthane®, recent internal studies (Poet project - November 2012 – USA) show
that a minimal OLR value of 6 kg COD/m3.d is required to allow a competitive offer to face the
concurrence in other anaerobic technologies. This value has to be checked through pilot tests
(even at lab scale) if the wastewater is unknown or supposed difficult to degrade. For easily
degradable and well known wastewater (cheese whey, winery wastewater), it is possible to
design with an OLR at 8 kg COD/m3.d. More than 8 kg COD/m3.d requires preliminary lab scale
test to check the design of the membrane skid because the membrane clogging risk increases
and some inhibitions can appear with the increase of some components concentrations inside
the reactor.

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C. TSS (total suspended solids) and VSS (volatile suspended solids)

Firstly, it is important to make a difference between TSS admitted in the influent and TSS content inside
an anaerobic reactor.

For example, a granular sludge bed is sensitive to TSS concentrations above 0,5 g/L in the influent but
can admit until 100 g TSS/L inside the reactor because these TSS are only biomass (granules) and not
“external” solids in suspension. Granular sludge is retained by the triphasic configuration but external
TSS can clog the reactor and wash the granules out.

Table 3 give values of TSS admitted in the influent.

Table 4: TSS values for different anaerobic technologies (indication)

Parameters Biobulk UASB EGSB Memthane
Influent TSS
1000 – 30000 max. 1000 max. 500 < 30000
(mg/l)
Inside TSS
(Biomass) 20000 - 60000 30000 - 100000 30000 - 100000 10000 – 30000
(mg/L)

Concerning Memthane, it is possible to increase the biomass concentration inside the reactor
until values around 15-30 g TSS/L. VS proportion is frequently from 70 to 90 % depending of the
sludge age and the precipitation of mineral compounds. Above 30 g TSS/L, with the selected
membrane technology (Norit-X-flow) the filtration capacity (membrane flux) is considered too
low and not competitive. The graph below shows an example of membrane flux depending on
the TSS concentration.

Figure 9: example of membrane flux depending on the TSS concentration.

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D. HRT (hydraulic retention time)

HRT depends mainly on the applied OLR and SLR and is not considered as a prior design
parameter. It is more or less a consequence of the applied OLR and SLR. For a given wastewater,
HRT decreases when OLR and/or SLR increase.

However, design rules suggest having always a minimum 4 hours HRT in a high rate reactor like
UASB or EGSB.

Also a design of CSTR reactor with less than 10 days HRT shows frequent problems of removal
efficiency.

E. Température
The anaerobic process is relatively sensible for temperature (variations) and the temperature in
the reactor must be maintained fairly constant to ensure optimum conditions for the bacteria.

The optimum temperature range for the mesophilic process is 35-38°C. A too high temperature
will result in a lethal effect against the biomass. A lower temperature is not as a dangerous as a
higher one and generate only a decrease of metabolic activity (table 4).

For example, at a temperature of 28°C the plant will operate normally. This is also dependent
on the amount of biomass present in the reactor (SLR). Below 25°C the activity is severely
reduced. Reduced biogas production and pH drop are typical indications of disturbances.
However wastewater can still be treated with acceptable efficiency under 25˚C as far as the
temperature is constant and the system is acclimatized to such environment.

The activity of methanogenic bacteria can be measured in kg soluble COD degraded per kg VSS
and per day. Table 5 gives the coefficient of average activity of mesophilic methanogenic
bacteria according to the temperature. These values also depend on the characteristics of the
wastewater that has to be treated and optimal environmental parameters.

Temperature Coefficient of activity Activity / max. activity (@35°C)

°C kg COD/kgVSS.d %
35 1,2 100
30 0,90 75
25 0,60 50
(20) (0,42) (35) not recommended
Table 5: Coefficient of average activity of methanogenic bacteria according to temperature.

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Figure 10: Coefficient of average activity of methanogenic bacteria according to temperature.

Granular sludge bed reactors (UASB, EGSB) are operated only in mesophilic conditions
(exceptionally in psychrophilic, but never under 20°C).

Currently, Memthane® is not designed to run in thermophilic conditions (50-70°C) but lab scale
pilot tests are in progress.

CSTR and Biofilters can be operated in thermophilic conditions.

Thermophilic conditions allows higher OLR (compared to mesophilic for the same wastewater)
but are considered as more sensitive to inhibitory or toxic parameters.

F. pH
The optimum pH for methanogenic bacteria is in the range 6.5-7.5, although methane
formation proceeds between pH 6.0 and 8.5. For stable maximal performance it is important to
keep the pH in the optimal range. During a process study, sometimes it is not possible to
maintain the pH within this range. It is then very important to keep the pH in the reactor always
above 6.0.
The conversion of complex organic matter into volatile fatty acids is far less sensitive to low pH.
For example the activity of acidifying bacteria stops only when pH is under 4.
In case the pH in the reactor accidentally drops below 6.0, the acidifying bacteria continue to
produce VFA, while methanogenic bacteria cannot tolerate low pH and stop methane
production resulting in VFA accumulation. The harmful effect of a low pH in such case is caused
by an increased concentration of undissociated VFA and hydrogen sulphide (H 2S) in the liquid
phase. These compounds can penetrate through the non-charged cell membrane and dissociate
inside the cell where they cause a destructive pH drop. Note that dissociated fatty acids as well
as HS- are not toxic for the bacteria.
In case the influent has low pH because of VFA production, it is very important that:
 the sludge/water mixture in the reactor has enough buffering capacity to
neutralize the VFA in the influent;
 the reactor is sufficiently mixed to avoid local areas with high VFA
concentration and low pH.
In a well operating anaerobic system, the biochemical reactions tend to balance the pH by:

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- VFA degradation resulting in H2CO3/CO2 production.

- Bicarbonate equilibrium (pKa = 6.36 at 25°C) and solubility of CO2 in the liquid phase.
- Release of ammonia contributes to the alkalinity.
As pH measurement is not precise enough to give a good indication of VFA content, it is
necessary to follow the VFA concentration. If necessary, pH is maintained at a pre-determined
level by caustic dosing: usually between 0.05 and 0.11 kg NaOH / kg COD removed.

In wastewater containing easily degradable substrate (i.e. food & beverage, bioethanol, etc.),
the fermentation process can start already in the equalization tank leading to pH drop to as low
as pH 4 (which is the lower level where fermentation is inhibited). If the low pH is due to the
fermentation process having already started before the wastewater enters the reactor, only
limited pH correction is required, as the VFA formed in fermentation are now degraded and the
bicarbonate produced can balance the alkalinity.

If the wastewater contains high concentrations of sulphate or ammonia, operating pH should be

selected to limit the toxicity of free ammonia and free hydrogen sulphide. pKa (@ 25°C) for
H2S/HS- is 6.99 and pKa (@ 25°C) for NH3/NH4+ is 9.24.

Concerning Memthane®, most of the projects don’t need pH regulation because the OLR is low
enough (and HRT high enough) to allow a natural increase of the alkalinity (and pH) inside the
anaerobic reactor.

G. Volatile fatty acids (VFA) and alkalinity

VFA are the most important substrate for the methanogens. Under regular operating conditions
in granular sludge bed reactors (UASB, EGSB), VFA concentration is less than 3-5 meq/L in the
anaerobic effluent at the outlet of the reactor. A higher concentration indicates that the
process is overloaded.

With the Memthane® process, due to specific parameters (low OLR, high HRT, high SRT), it is
possible to obtain much more VFA inside the reactor without affecting the COD removal
efficiency. For example, with bioethanol wastewater, 80 % COD removal has been reached at 5
kg COD/m3.d with around 40 meq VFA/L at the outlet.

The alkalinity quantifies the buffering capacity of the wastewater. Higher is the alkalinity, higher
is the resistance against pH fluctuations.
The buffering capacity is estimated with the concentrations of:
 weak acids (CO2, HCO3-, VFA, NH4+, H2S, HS-) ;
 weak bases (NH3, CO32-, S2-) ;
 salts of the acids and bases present.
Under good operating conditions, alkalinity varies between 1 500 and 3 000 mg/L as CaCO 3, or
30 - 60 meq/L (1 meq/L = 50 mg/L CaCO3 = 5°F = 2.8°D = 2.9 °US). Below 1 000 mg/L CaCO3 or 20
meq/L, biological activity can be reduced. During start-up, the alkalinity should be maintained
within this range.

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The effluent alkalinity must be maintained at least in the range of 10-15 meq/L, to prevent the
reactor pH from decreasing in case of sudden accumulation of acetic acid. To maintain this
alkalinity, the recirculation can be increased or caustic can be added to the influent. The above-
mentioned value is the lower limit. For concentrated wastewaters, the alkalinity can be much
higher.

From our feedback during start-up period, we consider the following levels:
VFA Impact on the reactor
(meq/L)
<3 The reactor running correctly
3 to 7 The reactor can work but the risk of instability is increasing.
>7 High instability of the reactor, with acidification risk and decrease of COD
removal efficiency.
Table 6: Definitions of VFA concentrations

However, the feedback has shown us that the only measure of VFA in the reactor outlet does
not reflect fully its start-up potential. In other words, some biogas plants may well continue to
rise over with 8 meq.L-1 of VFA and performance superior to 80% on COD removal efficiency
and in the same situation, others biogas plants fall in acidification from 5 meq.L-1. This highlights
the need to correlate the analysis with other parameters such as the TA (total alkalinity).

The TA shows the "buffering capacity" of an effluent, ie its ability to absorb the protons. When
effluent has a high buffering capacity (high TA), it is important to monitor the doses of injected
reagent (especially caustic soda) to obtain the desired pH.

We have seen cases of over-injection of caustic soda (NaOH - 30% diluted) to try to regulate the
pH to 6.5 in an acidification tank to treat effluent wine. We found that the dose of sodium
hydroxide solution was injected in the range of 0, 2 liters per kg COD to change the pH from 4.5
to 6.5 but rose to nearly 0.4 liters per kg COD to spend 6.5 to 7. This over-consumption of soda
even resulted in a loss on methane production in the second stage of digestion.

It is possible to take advantage of the benefits of a high TA in the monitoring of a digester,

particularly during its period of start-up. The TA typically play the role of "buffer" in front of the
risk of acidification and adverse effects of a too acidic pH that can inhibit the activity of
methanogenic archaea.

The ratio VFA/TA can be used to assess the stability of a digester as shown in the table below:
VFA/TA Impact on the reactor
< 0,4 The reactor can support low variations of pH.
0,4 à 0,8 The reactor can work but the risk of instability is increasing.
> 0,8 High instability of the reactor, with acidification risk and decrease of COD
removal efficiency.
Table 7: Definition of VFA/TA ratios

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H. Nutrients requirements
Cell material consists of proteins, carbohydrates and phosphorus containing macromolecules
like DNA and RNA. Elements like nitrogen, phosphorus and sulphur are therefore essential for
the synthesis of new cell material and need to be present in the wastewater to be treated.
Other macronutrients needed in relatively high concentrations for the proper functioning of the
bacterial cell are calcium, magnesium, iron and potassium. Nutrients required in very low
concentrations are referred to as trace elements or micronutrients. They include several heavy
metals like manganese, molybdenum, zinc, copper, cobalt, nickel, etc.

1) Nitrogen, phosphorus and sulphur

In anaerobic treatment, the required amount of nutrient will be in the range stated in the
examples below. The degree of acidification is the determining factor.

- In the case of anaerobic conversion for a low acidified wastewater (20-30% acidification
rate), specific biomass production will be relatively high. The acidifying bacteria will be
the main determinant of biomass production (0,15 gVSS/gCOD removed). Therefore, the
required ratio between COD and macronutrients will be: COD/N/P (/S) = 350/5/1 (/0.3)

- If the wastewater is highly acidified (> 50% acidification rate), biomass production will be
determined by the methanogenic population and be relatively low (0,05 gVSS/gCOD
removed). In that case, the ratio will be: COD/N/P (/S) = 800/5/1 (/0.3)

However, for a first approach, it is recommended to design new projects with the COD/N//P
ratio of 350/5/1.

In practice, the dosing of macronutrients is controlled to ensure that there is enough ammonia-
nitrogen and orthophosphate in the effluent. At least 5 mg/L of ammonia and 1 mg/L of
orthophosphate should be present in the effluent at the outlet of an anaerobic system. At COD
concentrations as high as 10 000 mg/L, these values can even be doubled since slight
fluctuations in the COD concentration may otherwise result in nutrient deficiencies.

The sulphide content of methanogens is unusually high compared to aerobic microorganism.

Optimal sulphide concentrations estimated for methanogenic growth vary from 1 to 25 mg/L.
Most process waters contain some sulphate. As a result, sulphur is seldom added. However,
spent demineralised water with an important lack of sulphate can be sent to the wastewater
treatment. In this case, sulphate dosing can be required.

2) Calcium, magnesium and potassium

Both calcium and magnesium have a stabilizing function in the cell wall. Negatively-charged
groups in cell walls macromolecules are neutralized by these divalent cations.

Calcium concentration in the wastewater should be in the range of 50-150 mg/L. Below these
values, lime milk (Ca(OH)2) should be added. Beyond these values, there is a risk that calcium

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carbonate precipitate in the granular sludge of the reactor system (and also on the internal
surfaces of tanks and pipes).

Magnesium, as well as potassium, plays a role in the stability of the ribosomes, the “protein
factories” of the bacterial cell. Several mg/L of magnesium are generally sufficient. In sodium
concentrated wastewater (Na+ > 5 000 mg/L), these concentrations should be increased in the
range of 10-20 mg/L.

Depending on the salt concentration, the potassium requirements are approximately:

 1.6% of biomass produced for wastewater with low salt concentration (Na + < 5 000
mg/L);
 8% of biomass produced for wastewater with high salt concentration (Na + > 5 000
mg/L).

Note: for specific wastewaters like those coming from PTA production units (Purified
Terephtalic Acid), yeast extract is also added to the influent to enhance the development of
biomass. It can be dosed as a slurry of Ca(OH)2, Mg(OH)2, KOH and Dry yeast. The dosing rate is
roughly 50 mg of dry yeast per liter of influent.

3) Trace elements
Micronutrients like iron, manganese, molybdenum, zinc, copper, cobalt, nickel and selenium are
necessary for growth as they are constituents of enzymes responsible for specific conversions.
These components are required in very small concentrations (see table below). They become
toxic at higher concentrations.

Concentrations COD range

in mg/L 0 – 10 000 mg/L 10 000 – 20 000 mg/L > 20 000 mg/L
Fe 2–5 5 – 10 10 – 20
Ca 0.3 0.7 1
S 10 15 20
Mg 5 10 10 – 15
K 10 10 – 15 10 – 20
Cu 0.05 0.10 0.15
Zn 0.1 0.2 0.3
Mn 0.05 0.10 0.15
Ni 0.035 0.07 0.10
Co 0.01 0.02 0.03
Mo 0.001 0.002 0.003
Se 0.0035 0.007 0.01
Al 0.05 0.10 0.15
Table 8: Trace elements concentrations necessary for bacteria growth (Biothane’s recommendation).

Biothane has developed its own micronutrients solution, called “Vithane Complete”, to fill those
needs. The common dosage is 3 ppm (3 mL/m3 of influent), maximum 10 ppm during start-up or
reseeding. Hydrex also provide a similar product with the reference “Hydrex 6993”.

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IV.BICARBONATE EQUILIBRIUM
A. Buffering capacity
In an anaerobic reactor, the conversion of carbonaceous molecules such as glucose gives
theoretically 50 % vol of CO2 and 50 % vol of CH4: C6H12O6 ↔ 3CH4 + 3CO2

However, biogas rarely has the molar composition corresponding to the chemical reaction.

There are two main reasons:

 A fraction of CO2 is released to the atmosphere during pre-acidification taking place in

the equalization tank and/or in the pre-acidification tank.

 CO2 can be dissolved in the liquid phase through bicarbonate (HCO3-) formation. Biogas
produced in anaerobic reactors contains around 30% carbon dioxide. This effectively
means that most of the carbon dioxide produced leaves the reactor system as
bicarbonate dissolved in the effluent.

In anaerobic systems, bicarbonate is the dominant buffer. The corresponding equations are:

CO2 + H2O  H2CO3

H2CO3  H+ + HCO3- pK1 = 6.31 at 35°C

HCO3-  H+ + CO32- pK2 = 10.25 at 35°C

At pH 6,31, 50% of the acid is undissociated. The methanogenic bacteria have their optimum
activity in between pH 6,5 to 7,5 i.e. above pK1.

If the buffering capacity of the influent is not sufficient to maintain the system within the
optimum operating range, caustic (NaOH) is normally added. Hydroxide ions OH- increase the
pH, leading to more carbonic acid dissolved. Sodium ion Na + added helps to increase the buffer
capacity of the system as it offers electron neutrality for dissolved carbonic acid/bicarbonate.

The equilibrium relations depend on the pH (buffer) as shown on Figure below.

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Figure 11: Repartition of the carbonate, bicarbonate and carbon dioxide depending on the pH value (T = 20°C).

When acid (H+ ions) are added to the system, the equilibrium shifts towards dissolved CO2,
which increase the CO2 content of the biogas and reduce the pH decrease. When hydroxide is
added, CO2 from the gas phase dissolve in the water, until a new equilibrium is reached,
thereby reducing the pH increase.

In anaerobic reactors alkalinity is increased by four phenomena, including the stripping of CO 2

which is by far the most important.

1. Conversion of nitrogen in proteins into NH4+ (act as a buffer).

2. Conversion of fatty acids into methane and bicarbonate.
3. Sulphate reduction into H2S and HS- (act as a buffer).
4. Stripping of CO2 from effluent to biogas.

The reactor effluent contains CO2 according to the following equilibrium:

2H  CO32-  H  HCO3  H2CO3  H2O  CO2 

In case the system needs recirculation, alkalinity can be increased with a partial stripping of CO2
from the effluent. This can be achieved in a slightly aerated conditioning tank with a closely
monitored redox potential. An excessive increase of the potential of the effluent could affect
methane production (should be kept under – 300 mV). By stripping CO2, the equilibrium shifts
to the right, H+ concentration drops and less caustic is needed to correct the pH.

Note that in systems with high calcium content, external stripping of CO 2 in recirculation stream
must be avoided as otherwise severe calcium carbonate precipitation can occur.

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Ca2  2HCO3-  CaCO3   CO2   H2O

B. Reduction of sodium hydroxide consumption by recirculation

As mentioned in the previous part, the pH of the reactor is often controlled by caustic injection.
The amount of sodium hydroxide required for neutralisation is directly related to the alkalinity
of the influent and its acidification rate. Alkalinity represents HCO3- available for buffering
whereas the acidification rate represents the potential HCO3- to be released after organic acids
conversion by methanogenic bacteria. For two influents with the same alkalinity, the one with
the highest acidification rate, hence the highest bicarbonate potential release, requires less
caustic dosing. For two influents with the same acidification rate, the one with the highest
alkalinity, hence the highest bicarbonate content, requires less caustic dosing. In those
conditions, recirculation is very effective.

C. Calcium carbonate precipitation

If the wastewater contains large amount of calcium, there is a risk that calcium carbonate
precipitate in the overall system (pipes, tanks, biomass, membranes).

The precipitation of calcium carbonate proceeds via several steps. At first amorphous
precipitate is formed in the bulk fluid, which may wash out of the reactor as colloidal material.
However, depending on the circumstances, the amorphous precipitate may also be converted
into crystalline forms like calcite and precipitate on inorganic surfaces. These crystalline forms
may lead to very high mineral content in the biomass and finally accumulation of inactive
material in the reactor.

Because accumulation of inactive material means efficiency loss, calcium carbonate

precipitation must be avoided.

Assuming the alkalinity in the anaerobic system and knowing the calcium concentration at the
inlet, the risk of precipitation can be theoretically calculated as below:

The following equations for dissociation and solubility should be considered:

H2CO3  H+ + HCO3- pK1 = 6.31 at 35°C

HCO3-  H+ + CO32- pK2 = 10.25 at 35°C
Ca2+ + CO32-  CaCO3 ↓ pKs = 8.54 at 35°C

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pH min for precipitation risk at 35 °C

8,2

7,8

7,6
Risk of precipitation
pH

7,4

7,2

6,8
10 20 30 40 50 60 70 80 90 100
TAC (meq/l)

Figure 12: Risk of calcium carbonate precipitation at 35°C.

The factors which determine the place of precipitation either bulk fluid or granules are:

 The hydraulic retention time (HRT).

 The size of the granules.
 The phosphate and sodium (or salt) concentrations in the wastewater.
 And the production of biomass in the system.

Obviously, the shorter is the HRT, the lower is the likelihood of equilibrium and scaling.

The bigger are the granules, the more scaling will take place. In fluidised-bed reactors, the
granules are much smaller due to high shearforces and fewer scaling problems are
encountered.

Low concentrations of phosphate (0.5-5 mgP/l) reduce the crystallisation rate of calcium
carbonate as a result of interference by co-precipitation. The ratio of sodium (or salt) to calcium
also interferes with the precipitation rate of calcium carbonate: the higher the sodium
concentration, the less scaling occurs.

A low sludge growth yield favours scaling in the granules and the amount of precipitation in
relation to biomass growth will be higher.

In practice, high calcium content wastewaters should preferably be treated in fluidised-bed

systems (EGSB, fixed biomass fluidized bed reactor).

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Note: When the anaerobic effluent containing calcium is treated in an aerobic post-treatment,
calcium carbonate precipitation increases in the aerobic stage. Carbon dioxide, which is
dissolved in the anaerobic reactor, is then stripped in the aerobic system by means of aeration.
As a result, pH goes up considerably – sometimes by more than half a pH unit – leading to
intense scaling in the aerobic system.

V.SULPHATE REDUCTION
A. Sulphur compounds
Industrial wastewater often contains important concentration of sulphur components. In this
case, the applicability of anaerobic treatment should become less profitable, due to more
potential sulphide toxicity. The toxic effect of sulphide is bacteriostatic. The inhibition is due to
the competition between methane producing bacteria and sulphate reducing bacteria, which
use the same substrate.

The biological reduction of sulphate is thermodynamically more advantageous than carbonate

reduction (methane production). This means that, in anaerobic reactors, sulphate will always be
reduced to sulphide prior to methane production (figure below).

Figure 13: redox couples and anaerobic activity

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In addition, the hydrogen sulphide (H2S) produced can inhibit methanogenic bacteria. But
bactericidal effect should not occur since sulphate reduction is inhibited with high hydrogen
sulphide concentration.

Inhibition can start from 30-50 mg/L of undissociated H2S in the liquid phase in the reactor.
Above 150 mg/L undissociated H2S, a significant reduction in activity will occur and it is
recommended to operate below this concentration. At pH 7 (pK = 6.99), this means that 300
mg/L is the maxi total sulphide concentration (H2S + HS-). Gradual adaptation to higher sulphide
concentrations has, however, been reported.

At COD/SO42- < 3 to 5 (based on the influent concentrations), the negative effect of sulphate-
reduction usually appears in anaerobic process. In case of lower COD/SO 42- ratio, it is
recommended to make an anaerobic biodegradability test to make sure of the expected
performance.

Figure 14: competition between AM & BSR

The best operating parameter for monitoring the H2S toxicity is the H2S content of the biogas.
When H2S content goes beyond 3%, immediate intervention becomes necessary:

 by increasing the pH of the reactor (adding Ca(OH)2, Na2CO3, NaOH) ;

 by adding ferric chloride (FeCl3) precipitating iron sulphide ;
 by decreasing the organic load ;
 by diluting with sulphide-free or sulphate-poor water or wastewater;
 by adding a micro-aeration system to the reactor.

Another solution to prevent inhibition due to H2S, is to recirculate “clean” biogas (free of H2S) to
increase the stripping-effect.

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A stripping solution has been proposed to improve the Memthane® performances for the
“Poet” project by Biothane LLC in USA (Nov. 2012). The figure below shows a Memthane® file
with a stripping tower installed in parallel loop with the anaerobic reactor. Lab scale pilot test
have proven that such stripping can guaranty more than 80% of COD removal despite a VFA
concentration around 40 meq/L, a highly concentrated wastewater (COD = 60 g/L, SO4 = 9 g/L)
and an OLR around 5-6 kg COD/m3.d.

Figure 15: Poet project - General process flow

B. Sulphide production

COD removal rates are slightly lower for a wastewater with high sulphate concentrations as
sulphides contribute to COD (analytical procedure) in the effluent. The COD created by organic
pollutants is replaced by sulphide COD (equivalence: 1 g H2S equal 2 g COD).

Biological sulphate reduction produces carbon dioxide in addition to the production by

methanogenesis, affecting the production and the composition of the biogas.

SO42- + CH3COO-  HS- + 2 HCO3-

HCO3- in equilibrium with CO2
(Theoretically 0.67 ppm COD is required to reduce 1 ppm of SO4)

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Another source of sulphide is the anaerobic conversion of proteins into amino acids: cysteine
and methionine are sulphur-containing amino acids. However, this contribution to total
sulphide production is generally negligible.

H2S  HS- + H+ pK = 6.99 (at 30°C)

Considering the pK for H2S/HS-, about 50% of the sulphide will be present as H2S at pH 7. As a
consequence, the production of H2S must be taken into account when designing anaerobic
plants (gas treatment and security).

1
0,9
0,8
0,7
0,6
Fraction

HS-
0,5
H2S
0,4
0,3
0,2
0,1
0
4 5 6 7 8 9 10
pH

Figure 16: S equilibrium in function of pH

H2S generates odour problems, corrosion and is also a toxin for methanogenic bacteria.

In order to avoid odour problems due to H2S emissions, the headspaces of tanks and reactors
can be ventilated. This vent air has to be treated to oxidise the odorous components.

The consequences of corrosive effect can be limited by selecting H2S resistant materials
(stainless steel, PVC, PE) for tanks, pipes and other equipment in contact with the gas or the
liquid phase. Biogas is often used as an energy source. This means that H2S has to be removed
from the gas to levels below 0,1–0,2 % (1000 – 2000 ppm) to prevent corrosion problems in
boilers ( Process Note N° 3NP90 Treatment and utilisation of biogas).

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C. Calculation of H2S concentration in biogas

It is important to be able to quantify the amount of H2S in the biogas to design the gas
treatment units.

The example of calculation below starts with the determination of the amount of sulphide
produced in the reactor. Next, the pH determines the concentration of undissociated H 2S in the
liquid phase. Then, this concentration is used to compute the amount of H2S in the gas phase
with Henry’s constant and assumed biogas absolute mean pressure (knowing the reactor
height). Those calculations don’t consider the presence of organo-sulphides.

Process parameter: - Temperature 30°C

- pH sludge bed 6,8
- Flow 194 m3/h
- COD 3 000 mg/L (582 kg/h)
- SO42- 480 mg/L
- conversion efficiency 80%
- specific biogas production 0,45 Nm3/kg COD converted
- wet reactor height 10 m
- mean absolute biogas pressure 1.5 bar (half water height)

Chemical constants: - Henry’s constant 600.74 bar/(mol/mol) (@30°C)

- pK H2S/HS- 6.99 (@30°C)
- S molar weight 32 g/mol
- SO4 molar weight 96 g/mol
- molar concentration H2O 5.56 x 104 mol/m3

Quantity of sulphide produced in the reactor:

It is assumed that the sulphates will have been converted completely (this assumption is close to
reality). The example of calculation below does not take into account the addition of ferric
chloride addition, which would lead to a reduction of the H2S concentration in both liquid and
gas phases.

[Stotal] = 480 x 32 / 96 = 160 mg S/l of total sulphur = 160/32 = 5 molS/m3

Quantity of undissociated H2S in the liquid phase:

[H2S] = 10-pH/(10-pK+10-pH)x[Stotal] = 0.61x [Stotal] = 3.05 mol/m3 = 97.6 mg/L H2S-S

[HS-] = 10-pK/(10-pK+10-pH)x[Stotal] = 0.39 x [Stotal] = 1.,95 mol/m3 = 62.4 mg/L HS-S

Quantity of H2S in the gas phase:

Henry’s law: pH2S = yH2S x H - pH2S partial pressure of H2S

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- yH2S mole fraction of H2S in water

- H Henry’s constant

Partial pressure: pH2S = xH2S x p - pH2S partial pressure of H2S

- xH2S mole fraction of H2S in gas
- p gas pressure

xH2S = yH2S x H / p = (3.05 / 5.56 104) x 600.74 / 1.5 = 0.022 mol/mol = 2.2% H2S

Accordance with the mass balance:

Biogas production: 582 x 0.8 x 0,45 =209.5 Nm3/h

There is 22.4 10-3 Nm3/mol in gas phase, so : 209.5 / (22.4 x 10-3) = 9 353 mol/h

The molar gas flow of H2S is:9353 x 0.022 = 206 mol/h

The molar liquid flow of sulphur components in the influent/effluent is: 5 x 194 = 970 mol/h
To account the difference between Sulphur that enter (970 mol/h) and Sulphur that leave
(206+970=1176 mol/h), due to the fact that we don’t take the progressive diminution of
sulphide in the water phase into account, we must take a corrective factor: 970/1176 = 0.82.

The final concentrations will be:

H2S in biogas 0.82 x 2.2% = 1.8% (0.9 mol/m3)

H2S in treated effluent: 0.82 x 3.05 = 2.5 mol/m3
HS- in treated effluent: 0.82 x 1.95 = 1.6 mol/m3

As the H2S constitutes only a small fraction of the total gas produced, mass balances may be
corrected in this manner. If H2S formation makes a considerable contribution to the total
amount of biogas, iterative calculations are appropriate.

These calculations make it clear that two process parameters are fundamental to determine H2S
content in the biogas:

 pH of the sludge bed.

 Height of the reactor ( absolute mean pressure).

The following examples quantify the amount of H2S in the biogas for the same previous
conditions, except for one parameter:

Ex1. higher pH value.

Ex2. higher reactor height.

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First calculation (pH 7,2 and reactor height 10 meters)

Quantity of sulphide produced in the reactor:

[Stotal] = 480 x 32 / 96 = 160 mg S/l of total sulphur components = 160/32 mol/m3 = 5 mol/m3

Quantity of undissociated H2S in the liquid phase:

[H2S] = 10-pH/(10-pK+10-pH)x[Stotal] = 0.38 x [Stotal] = 1.9 mol/m3 = 60.8 mg/L H2S-S

[HS-] = 10-pK/(10-pK+10-pH)x[Stotal] = 0.62 x [Stotal] = 3.1 mol/m3 = 99.2 mg/L HS-S

Quantity of H2S in the gas phase:

xH2S = yH2S x H / p = (1.9 / 5.56 104) x 600.74 / 1.5 = 0.014 mol/mol = 1.4 % H2S

Accordance with the mass balance:

Biogas production: 582 x 0.8 x 0.45 = 209.5 Nm3/h

209.5 x 1000 / 22.4 = 9353 mol/h

The molar gas flow of H2S is: 9353 x 0.014 = 131 mol/h
The molar liquid flow of sulphur components in the influent/effluent is: 5 x 194 = 970 mol/h
Sulphur at the inlet = 970 mol/h ≠ Sulphur at the outlet = 131+970 = 1101 mol/h

The final concentrations will be (with a corrective factor = 970 / 1101 = 0.88):

H2S in biogas 970 / 1101 x 1.4% = 1.2%

H2S in treated effluent: 970 / 1101 x 1.9 = 1.7 mol/m3
-
HS in treated effluent: 970 / 1101 x 3.1 = 2.7 mol/m3

 Higher pH significantly reduce H2S contents.

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Second calculation (pH 6,8 and reactor height 15 meters = mean pressure 1,75 bar)

Quantity of sulphide produced in the reactor:

[Stotal] = 480 x 32 / 96 = 160 mg S/l of total sulphur components = 160/32 mol/m3 = 5 mol/m3

Quantity of undissociated H2S in the liquid phase:

[H2S] = 10-pH/(10-pK+10-pH)x[Stotal] = 0.61x [Stotal] = 97.6 mg/L H2S-S = 3.05 mol/m3

[HS-] = 10-pK/(10-pK+10-pH)x[Stotal] = 0.39 x [Stotal] = 62.4 mg/L HS-S = 1.95 mol/m3

Quantity of H2S in the gas phase:

xH2S = yH2S x H / p = (3.05 / 5.56 104) x 600.74 / 1.75 = 0.019 mol/mol = 1.9 % H2S

Accordance with the mass balance:

Biogas production: 582 x 0.8 x 0.45 = 209.5 Nm3/h

209.5 x 1000 / 22.4 = 9 353 mol/h

The molar gas flow of H2S is: 9 353 x 0.019 = 178 mol/h
The molar liquid flow of sulphur components in the influent/effluent is: 5 x 194 = 970 mol/h
Sulphur that enter is 970 mol/h ≠ Sulphur that leave is 178+970=1148 mol/h

The final concentrations in the gas and fluid will therefore be:

H2S in biogas 970 / 1148 x 1.9% = 1.6%

H2S in treated effluent: 970 / 1148 x 3.05 = 2.6 mol/m3
HS- in treated effluent: 970 / 1148 x 1.95 = 1.6 mol/m3

 Bigger reactor heights reduce H2S contents.

 the lower the reactor height, the lower the partial pressure, the less H2S remains in solution
and the more H2S is stripped to the biogas.

A dimensioning tabulator making it possible to calculate the amount of H2S in the gas phase is
available (ref: Sulphur Balance BIOTHANE). Calculations are detailed below.

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D. Hydrogen sulphide removal with ferric chloride

Dosing of metal salts can cause a reduction of the hydrogen sulphide contents in the reactor
(liquid and gas phase). The most common method is the addition of ferric chloride (FeCl 3) to the
effluent. The hydrogen sulphide formed in the methanization process is so reduced to iron
sulphide (FeS) in the reactor and remains in the liquid phase.

2 Fe3+ + 3 S2-  2 FeS↓ + S

The stoichiometric dose to be applied is about 3.5 g of pure FeCl3 per g of S.

A calculation example is displayed hereafter:

Process parameter: - SO42- 480 mg/L

- Flow 16 m3/h
- Concentration of commercial FeCl3 41%
- Density of FeCl3 1 440 g/L
- Molar weight of FeCl3 161 g/mol
- Molar weight of Fe 56 g/mol

Quantity of sulphide produced in the reactor:

[Stotal] = 480 x 32 / 96 = 160 mg S/l of total sulphur components

It is assumed that the sulphates are completely converted and all the sulphur components (H2S,
HS- and S2-) react with FeCl3.

Quantity of ferric chloride for hydrogen sulphide removal:

160 x 3.5 = 560 mg of pure FeCl3 / litre effluent

560 / 0.41 = 1 366 mg of commercial FeCl3 / litre effluent
1366 / 1440 = 0.95 litre of commercial FeCl3 / m3 effluent
0.95 x 16 = 15.2 litre of commercial FeCl3 / h

Quantity of ferric chloride for granulation purpose:

BIOTHANE recommendation: 5 mg of Fe / litre effluent

5 x 161/56 = 14,4 mg pure FeCl3 / litre effluent

14,4 / 0,41 = 35,1 mg of commercial FeCl3 / litre effluent
35,1 / 1440 = 0,024 litre of commercial FeCl3 / m3 effluent
0,024 x 16 = 0,4 litre of commercial FeCl3 / h

 The amount of FeCl3 for H2S removal is 38 times higher than for granulation purpose.

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Notes:
 Due to competition reactions (for example precipitation of iron phosphate), the
dose to be applied is more important than the stoichiometry (determined
experimentally).
 Iron sulphide precipitation will lead to an increase in total sludge production. The
average mineral sludge production is 1.83 g of FeS per g of converted S.
 Iron sulphide precipitation will lead to a decrease of the VSS/TSS ratio.
 Take care not to modify the pH and/or alkalinity of the reactor due to FeCl 3
dosing.
 Hydrogen sulphide removal with ferric chloride is an expensive method due to
the chemical dosing which can reach high amounts. Instead of FeCl 3 dosing,
flash-aeration to remove H2S from anaerobic effluent and specific gas treatment
( Process note N° 3NP90 Treatment and utilisation of biogas) to remove H2S
from biogas can be applied.

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VI.TOXICITY & INHIBITION

The word “toxicity” usually creates a lot of fear, and it is a fact that unadapted methanogens are
sensible to several chemicals. However, thanks to scientific research and a huge experience on
full scale practice, most of the toxicity problems can be solved successfully.

A. Term explanation
The toxicity of a given compound depends on its concentration but also on the nature of the
micro-organisms exposed and the duration of the exposure. The effect of toxins is not always
the same in aerobic and anaerobic systems.

Depending on the chemical characteristics, anaerobic bacteria and especially methanogens can
be more sensitive to toxins than aerobic bacteria. The low growth rate of anaerobic bacteria
also means that recovery from a toxic situation resulting in partial or complete kill off of
bacteria is very slow.

Removal systems for toxins such as metals, cyanides, organic chlorides etc. must be installed
prior to anaerobic treatment. However it should be noted that anaerobic biomass can adapt to
certain toxins (H2S, Cu, CN-, -CHCl, complex organics etc.), after a certain time of exposure.
Actually, a compound which seems initially toxic can become degradable with adapted biomass.
Unfortunately it is difficult to predict whether acclimatisation will occur without experience,
only long term pilot testing will show (2-3 months until 6-12 months for difficult wastewaters
like PTA effluent).
Depending on the compound and its concentration, the toxic effects can be either bacteriostatic
or bactericidal:
 Bacteriostatic toxins inhibit growth and reproduction of the bacteria, but do not kill
them.
 Bactericidal toxins kill bacteria.
Usually, a same compound is bacteriostatic at low concentration and bactericidal at high
concentration.

Please refer to our intranet websites SOPHOS.DOC (https://veoliaeausophos.com/) and

(https://intranetsportal.veolia.net/ - “Digestion-methanization” community) for an exhaustive
data-bases of potential toxic compounds for anaerobic treatment.

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B. Organic acids
Organic acids either arrive directly in the anaerobic reactor with the substrate, or are formed in
the reactor due to bacterial activity. With a stable running anaerobic process, the incoming acid
load and the acid-degradation are in equilibrium thanks to methanogenic bacteria. As a
consequence the organic acid concentration remains relatively low (< 200 mg/L).

If the acid formation is much higher compared to the “acid removal capacity” of the
methanogenic bacteria, it leads to an accumulation of organic acids inhibiting the methanogenic
activity (bacteriostatic effect). As well as hydrogen sulphide (H2S), the inhibition is due to the
undissociated form of the organic acid (R-COOH).

Inhibition due to acetic acid depends on the pH value and the total acetic acid concentration.
This problematic becomes more and more significant when treating high concentrated and
easily acidifying wastewaters. In this case an accumulation of acetic acid can lead to an
inhibition. Moreover, the accumulation of organic acids involves a decrease of the pH in the
reactor, which will increase the inhibition effect.

In practice, if dealing with acid accumulation problems, following actions may be suggested:

 Check the acidification rate (ideal less than 30%, more problems above 50%).
 Decrease the OLR and/or SLR of the reactor.
 Increase the reactor pH by adding Ca(OH)2, Na2CO3, NaOH.

According to literature, 50% inhibition effect is reached, at pH 6,5 for:

 a propionic acid concentration of 150 mg/L (≈ 5 mg/L undissociated form).
 an acetic acid concentration of 1 000 mg/L (≈ 20 mg/L undissociated form).
The limits given by Biothane are more optimistic (especially for Memthane®):
 Maximum propionic acid concentration: 750 - 1000 mg/L.
 Maximum acetic acid concentration: 2 000 - 3 000 mg/L.
Higher concentrations seem to be possible with AnMBR technologies (return of experience in
progress).

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C. Nitrogen compounds
Nitrogen may be present in wastewater as different compounds:

 Oxidized forms: nitrite NO2- and nitrate NO3-.

 Reduced forms: ammonium NH4+.
 Complex molecules and macromolecules like aminoacids, proteins, biological cell
material etc...

1) Nitrite and nitrate

Both ions, nitrite and nitrate, may cause inhibitory effects on the methanogenic process. Nitrate
is toxic in the way that anaerobic degradation of organic compounds will take place only after
complete denitrification. Note that denitrification is made by heterotrophic bacteria which
consume organic compounds and reduce biogas production as well as methanogenic capacity.
In addition, the oxidised forms cause an increase of the redox potential, toxic to the
methanogenic process.

It is believed that the nitrite ion NO2- is more toxic than the nitrate ion NO3- but it is advised to
use for both ions limit of maximum 100 mg NO3- or NO2-/l in the influent of the anaerobic
system (= raw waste water feed + recirculated anaerobic effluent).
However, in practice, it is possible to treat higher concentrations through detoxification in a
buffer-and/or acidification tank. Biological denitrification process converts the “toxic” ions into
harmless nitrogen gas. The oxygen from the ions is liberated and used to oxidise organic carbon
compounds to CO2. A well designed denitrification process may be a cheap and relatively easy
method for COD-reduction.

2) Ammonium
As hydrogen sulphide (H2S) and organic acids, the undissociated form of ammonia (NH3) is more
toxic than the dissociated form (NH4+). The repartition of the two forms depends on the pH.
1
0,9
0,8
0,7
Fraction

0,6
NH3
0,5
NH4
0,4
0,3
0,2
0,1
0
7 7,5 8 8,5 9 9,5 10 10,5 11
pH

Figure 17: Proportion of ammonia forms as a function of pH.

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Methanogenesis is inhibited around 50-150 mg/L of free NH3. Ammonia NH4+ ions, which are
present in the influent, usually do not interfere with the methanogenic process until a
concentration from 1000 to 3000 mg/L NH4+, depending on the pH in the anaerobic reactor.

3) Complex molecules like proteins

In general, there’s no problem treating complex degradable nitrogen molecules like proteins
under anaerobic conditions. A temporary addition of nitrogen nutrients (in the free ammonia
form) may be required to start the hydrolysis reaction.

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D. Heavy metals and other inhibitors

Some metals are bacteriostatic and some bactericidal. Their toxicity is complex as it can
increase or decrease depending on which concentration and combination of metals are present
in the influent. Certain combinations of metals can be more toxic than the individual metals.

Some heavy metals – like mercury and silver – are toxic at very low concentrations because they
may bind to enzymes and change the structure of these proteins. In this way, they interfere
with the metabolic processes in the bacterial cell. Trace elements like manganese,
molybdenum, zinc, copper, cobalt and nickel are necessary for growth as they are constituents
of enzymes responsible for specific conversions. These components are required in
concentrations of several mmol/m3. At higher concentrations they become toxic.

In addition to the mechanism described above in which there is a structural change to enzymes,
a high concentration of a specific metal (not necessarily a heavy metal) may displace several
specific trace elements from their enzyme and thereby reduce metabolic activity.

The inhibitory effect of heavy metals will cause a decrease of the biogas production. Due to
inhibition of the methanogenic process, organic acid concentration will increase which will lead
to a pH drop. Thereby, the mobility of the heavy metals will be strengthened and the toxicity
will be increased.

In practice, wastewater often contains sulphate, which will be reduced to sulphide in the
methanogenic reactor. Fortunately, metal sulphides are very insoluble and precipitate in the
reactor (for ex. with Zn, Ni, Pb, Cd, Cu), this mechanism often counteracts metal toxicity. Only
chromium does not precipitate and must be considered separately.

Cleaning agents, fungicides, pesticides, disinfectants, lubricants used in the food industry. Most
of them are bactericidal. Also some solvents and hydrocarbons can be inhibitors. All these
compounds are sufficiently diluted in the wastewater but it is recommended to investigate
about their possible presence in an industrial project.

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Table 8 (Biothane) gives an overview about toxicity of heavy metals and different compounds. It
must be noted that in literature there is a lot of conflicting information about toxicity. The first
column gives the toxicity range found in literature. The toxic effect can be from disturbing
biological activity to total destruction of micro-organism. The second column, gives the toxic
threshold concentrations from Biothane (IC50: 50% inhibition or 50% loss of methanogenic
activity) resulting from their experience.

Toxic limit (mg/L) Toxicity IC50 (mg/L)

Compounds
Literature Biothane
Heavy metals
2+
Cu 50 – 150 75
2+
Zn 5 – 350 Unknown, never encountered in such high levels
2+
Ni 2 – 300 Unknown, never encountered in such high levels
2+
Pb 5 Unknown, never encountered in such high levels
3+
Cr 500 – 630 (adapted biomass: 1 000 – 2 500)
6+ 3+ 3+
Cr 1 – 200 More toxic than Cr , but reduced to Cr
Other compounds
Sodium 3 500 – 5 500 7 600 (adapted biomass: 10 000 – 15 000)
Potassium 2 500 – 6 000 6 100
Calcium 2 000 – 4 500 4 700
Magnesium 1 000 – 1 500 1 930 (adapted biomass: 3 000)
Aluminium 60 250
Iron 500 No real experience
-
Sulphide (H2S+HS ) 300 250
2-
Sulphate (SO4 ) 1 000 3 300
2-
Sulphite (SO3 ) 100 50
NH3 80 50
+ (1)
NH4 1 000 4 000 (within normal pH range 6.8 – 7.5)
-
NO3 100 100
-
NO2 100 100
-
Cl 8 000 Unknown if > 7 g/L (system in operation at 7 g/L)
Cationic and anionic Cationic: 100 20 – 50
detergent anionic: 500
(1) Toxicity is pH-dependent. Non-ionized form is toxic (free NH3).
(Concentrations at the inlet of the reactor taking into account the effluent recirculation).
Table 9: Toxicity overview of heavy metals and other compounds

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Table 9 shows the IC50 concentrations (ppm) for anaerobic and aerobic microorganisms for a
suite of chemicals (Blum& Speece-1991):

Aerobic
Toxicant Methanogens heterotrophs Nitrosomonas
Aromatics
Benzene and alkyl benzenes
Benzene 1200 520 13
Toluene 580 110 84
Xylene 250 1000 100
Ethylbenzene 160 130 96
Chlorinated benzenes
Chlorobenzene 270 310 0,71
1,2-Dichlorobenzene 150 910 47
1,3-Dichlorobenzene 260 720 93
1,4-Dichlorobenzene 86 330 86
1,2,3-Trichlorobenzene 24 96
1,2,4-Trichlorobenzene 120 7700 210
1,3,5-Trichlorobenzene 750 96
1,2,3,4-Tetrachlorobenzene 20 20
1,2,4,5-Tetrachlorobenzene 9,8
Hexachlorobenzene 4,6
2-Chlorotoluene 53
Alcohols
Benzyl-alcohol 2100 390
Methoxy benzene
Anisole 720
4-Chloroanisole 900
Aldhehydes
2-Furaldehyde 180
Nitriles
Benzonitrile 1100 470 32
m-Tolunitrile 230 290 0,88
Nitros
Nitrobenzene 13 370 0,92
2,6-Dinitrotoluene 7,9 180
Pentachloronitrobenzene 26
1-Nitronaphtalene 16 380
Other cyclics
Naphtalene 670 29
Benzidine 0,06
Phenol and alkyl phenols
Phenol 2100 1100 21
m-Cresol 890 440 0,78
p-Cresol 91 260 27
2,4-Dimethylphenol 71
3-Ethylphenol 140
4-Ethylphenol 240 14
Halogenated phenols
2-Chlorophenol 160 360 2,7
3-Chlorophenol 230 160 0,2

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4-Chlorophenol 270 98 0,73

2,3-Dichlorophenol 58 210 0,42
2,4-Dichlorophenol 63 0,79
2,5-Dichlorophenol 53 180 0,61
2,6-Dichlorophenol 150 410 8,1
3,4-Dichlorophenol 46
3,5-Dichlorophenol 14 3
2,3,4-Trichlorophenol 7,8 7,8 52
2,3,5-Trichlorophenol 1,8 3,9
2,3,6-Trichlorophenol 11 14 0,42
2,4,5-Trichlorophenol 4,6 23 3,9
2,4,6-Trichlorophenol 16 7,9
2,3,5,6-Tetrachlorophenol 0,13 1,5 1,3
Pentachlorophenol 0,04 0,06
2-Bromophenol 100 0,35
3-Bromophenol 140
4-Bromophenol 350 120 0,83
2,4,6-Tribromophenol 7,5 7,7
Pentabromophenol 0,02 0,27
Misc. substituted phenols
Catechol 1400
Resorcinol 1600 7,8
Hydroquinone 2800
2-Aminophenol 6,2 0,04 0,27
4-Aminophenol 25 0,07
2-Nitrophenol 12 11 11
3-Nitrophenol 18
4-Nitrophenol 4 160 2,6
2,4-Dinitrophenol 0,01
Aliphatics
Alkanes
Cyclohexane 150 29 97
Octane 2 45
Decane 0,35
Undecane 0,61
Dodecane 0,23
Pentadecane 0,09
Heptadecane 0,03
Nonadecane 0,01
Chlorinated alkanes
Chloromethane 50
Methylene chloride 7,2 320 1,2
Chloroform 0,9 640 0,48
Carbon tetrachloride 6,4 130 51
1,1-Dichloroethane 6,2 620 0,91
1,2-Dichloroethane 25 470 29
1,1,1-Trichloroethane 0,52 450 8,5
1,1,2-Trichloroethane 1,4 240 1,9
1,1,1,2-Tetrachloroethane 1,7 230 8,7
1,1,2,2-Tetrachloroethane 4,1 130 1,4
Pentachloroethane 11 150 7,9

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Hexachloroethane 22 32
1-Chloropropane 60 700 120
2-Chloropropane 620 440 110
1,2-Dichloropropane 180 43
1,3-Dichloropropane 18 210 4,8
1,2,3-Trichloropropane 0,63 290 30
1-Chlorobutane 110 230 120
1-Chloropentane 150 68 99
1,5-Dichloropentane 77 13
1-Chlorohexane 32 83 85
1-Chlorooctane 29 52 420
1-Chlorodecane 68 40
1,2-Dichloro-2-methylpropane 5,7
1-Chloro-2,2-dimethylpropane 5,9
Bromomethane 3,9
Bromodichloromethane 1,6
1,1,2-Trichlorotrifluoromethane 3,7
Chlorinated alkenes and alkynes
1,1-Dicholoroethylene 7,7
1,2-Dicholoroethylene 19
trans-1,2-Dicholoroethylene 48 1700 80
Trichloroethylene 13 130 0,81
Tetrachloroethylene 22 1900 110
1,3-Dichloropropane 0,57 120 0,67
5-Chloro-1-pentyne 44 86 0,59
Alcohols
Methanol 22000 20000 880
Ethanol 43000 24000 3900
1-Propanol 34000 9600 980
1-Butanol 11000 3900
1-Pentanol 4700 520
1-Hexanol 1500
1-Octanol 370 200 67
1-Decanol 41
1-Dodecanol 22 210 140
Chlorinatred alcohols
2,2-Dichloroethanol 18
2,2,2-Trichloroethanol 0,3 2
3-Chloro-1,2-propanediol 630
Ethers
Ethylether 17000
Isopropylether 4200 610
Ketones
Acetone 50000 16000 1200
2-Butanone 28000 11000 790
2-Hexanone 6100
4-Methyl-2-pentanone 9300 1100
1,4-Benzoquinone 33
Acrylates
Ethyl acrylate 130 47
Butyl acrylate 150 470 38

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Octyl acrylate 62
Carboxic acids
2-Chloropropionic acid 0,01 0,18 0,04
Trichloroacetic acid 0,001
Amines
1-Methylpyrrolidine 13000
Nitriles
Acetonitrile 28000 7500 73
Acrylonitrile 90 52 6
Sulfides
Carbon disulfide 340
Table 10: IC50 concentrations for anaerobic and aerobic microorganisms

For less common compounds a lab scale toxicity test can be proposed in some cases (VERI,
Biothane, VW STI, and other BUs from VWS can make this test).

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> TO KNOW MORE

Many technical documents are available in SOPHOS.DOC (https://veoliaeausophos.com)

Also a very complete e-library about anaerobic digestion is downloadable in the intranet portal
of Veolia Environment (https://intranetsportal.veolia.net/group/ve-corp/home) through an
To know more
inscription in the community called “Digestion – Methanization”.

Technical Department Experts for industrial methanisation

thierry.arnaud@veoliaeau.fr
emmanuel.cronier@veoliaeau.fr

Biothane experts:
barry.heffernan@veoliawater.com
frank.vanderzee@veoliawater.com

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