ASTM D5120-90(1995)e1
(Test Method)Standard Test Method for Inhibition of Respiration in Microbial Cultures in the Activated Sludge Process
Standard Test Method for Inhibition of Respiration in Microbial Cultures in the Activated Sludge Process
SCOPE
1.1 This test method covers a batch procedure that evaluates the impact of selected wastewaters, materials, or specific compounds on the respiration rate of an aqueous microbial culture, such as activated sludge.
1.2 Alternative procedures for measurement of microbial activity, such as adenosine 5` triphosphate (ATP), specific substrate utilization, etc. are not within the scope of this test method.
1.3 The results obtained are based on comparisons in a specific test series that examines a range of concentrations of the potentially inhibitory test candidate using batch methods in a laboratory. Results are completed in a short time frame (a few hours).
1.4 The test results are specific to the microbial culture used. Microbial culture from different wastewater treatment plants will differ in kinds and numbers of organisms, and performance capability. Thus, there is no basis for comparing results for microbial cultures from different treatment facilities.
1.5 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.
General Information
Relations
Standards Content (Sample)
NOTICE: This standard has either been superceded and replaced by a new version or discontinued.
Contact ASTM International (www.astm.org) for the latest information.
e1
Designation: D 5120 – 90 (Reapproved 1995)
Standard Test Method for
Inhibition of Respiration in Microbial Cultures in the
Activated Sludge Process
This standard is issued under the fixed designation D 5120; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. A
superscript epsilon (e) indicates an editorial change since the last revision or reapproval.
e NOTE—Section 12 was added editorially in September 1995.
1. Scope 3.1.2 EC —the concentration of the test candidate in this
procedure (volume percent or mg/L) that results in a reduction
1.1 This test method covers a batch procedure that evaluates
of respiration rate to 50 % of that observed for the control.
the impact of selected wastewaters, materials, or specific
compounds on the respiration rate of an aqueous microbial
4. Summary of Test Method
culture, such as activated sludge.
4.1 This test method utilizes respiration rate as the indicator
1.2 Alternative procedures for measurement of microbial
of microbial activity.
activity, such as adenosine 58 triphosphate (ATP), specific
4.2 A batch system that contains a microbial culture (re-
substrate utilization, etc. are not within the scope of this test
turned activated sludge from the process or a culture main-
method.
tained in the laboratory), selected nutrient dose, and a dilution
1.3 The results obtained are based on comparisons in a
of a compound, substance, wastewater, etc. (test candidate) is
specific test series that examines a range of concentrations of
prepared in a container in the laboratory. The batch system is
the potentially inhibitory test candidate using batch methods in
called a “cell suspension.”
a laboratory. Results are completed in a short time frame (a few
4.3 The nutrient dose introduces a large excess of biode-
hours).
gradable substrate thereby putting the culture at a high meta-
1.4 The test results are specific to the microbial culture used.
bolic rate. Inhibition of respiration by the test candidate is
Microbial culture from different wastewater treatment plants
observed under these conditions.
will differ in kinds and numbers of organisms, and performance
4.4 The prepared cell suspension is aerated for a 2-h period.
capability. Thus, there is no basis for comparing results for
At the end of the period, the respiration rate is determined
microbial cultures from different treatment facilities.
using a respirometric or an oxygen uptake technique.
1.5 This standard does not purport to address all of the
4.5 A lower respiration rate for a cell suspension that has
safety concerns, if any, associated with its use. It is the
received the test candidate compared to the respiration rate of
responsibility of the user of this standard to establish appro-
a control cell suspension indicates inhibition of respiration.
priate safety and health practices and determine the applica-
bility of regulatory limitations prior to use.
5. Significance and Use
2. Referenced Documents 5.1 The objectives of the respiration inhibition tests may be
defined by the interests of the user, but the test method is
2.1 ASTM Standards:
2 designed primarily for examination of the inhibition response
D 4478 Test Methods for Oxygen Uptake
with operating microbial systems such as an activated sludge
3. Terminology process treating domestic or industrial wastes.
5.2 Different apparatus exist that facilitate continuous or
3.1 Definitions:
continual measurement of respiration in microbial systems and
3.1.1 respiration rate—the quantitative consumption of
each may be used as the tool to observe respiration in this test
oxygen by an aqueous microbial system. The consumption is
method.
generally expressed as mg O /L/h.
5.3 Respirometry may utilize any apparatus and technique
that will achieve the determination of respiration rate. A
This Test Method is under the jurisdiction of ASTM Committee D34 on Waste
number of devices are presented in Appendix X1. Equivalency
Management and is the direct responsibility of Subcommittee D34.07 on Municipal
in the experimental capability of each device is not implied.
Solid Waste.
The analyst should select the respirometric approach that best
Current edition approved Sept. 28, 1990. Published November 1990.
suits his needs.
Annual Book of ASTM Standards, Vol 11.02.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.
NOTICE: This standard has either been superceded and replaced by a new version or discontinued.
Contact ASTM International (www.astm.org) for the latest information.
D 5120
5.4 The inhibitory effect of a test candidate is identified 7.1.2 Dissolved Oxygen Probe and Instrumentation—An
more completely by examining inhibition over a range of alternate device for the measurement of respiration rate as
concentrations, such as determining the EC . The use of oxygen uptake rate.
aerated containers permits concurrent management of a series 7.2 Culture Tank—If it is deemed necessary to maintain a
of cell suspensions. A respirometer for each cell suspension microbial culture in the laboratory, the apparatus required is a
might also be used. container with adequate mixing and oxygen transfer. The
container should hold at least four times the volume of culture
6. Interferences that might be used in one day.
7.2.1 The culture tank should be adequately mixed to insure
6.1 This test method is most readily applied to substances
that the culture remains in suspension and that sufficient
which, due to water solubility and low volatility, are likely to
mechanical or bubble aeration occurs to maintain the desired
remain in the aqueous system.
dissolved oxygen (DO) concentration.
6.2 Results have been observed where cell suspensions
NOTE 1—Energy input should not be such that the biological floc is
containing the test candidate had a respiration rate greater than
sheared to sizes smaller than that which exists in the large-scale process.
the blank, particularly at shorter aeration periods of the cell
Mixing and aeration provided through diffused aeration in a laboratory-
suspensions (less than 1 h). Thus, a minimum aeration period
sized container may result in an excessive power input. Consider
for the cell suspensions before determinations of respiration
controlling the power input per unit volume to approximately that which
rate is 2 h.
exists in the large-scale process. For example, pure oxygen for aeration in
combination with mechanical mixing may be utilized to achieve a balance
6.2.1 One reason for increased oxygen uptake rate in an
between oxygen transfer and mixing. Determine the mixer power input by
experimental cell suspension may be that severe physical or
measuring the electrical power consumed at different operating speed, and
chemical reactions with the test candidate cause a fraction of
adjust the mixer speed to achieve a power input that is equivalent to that
the microbial culture to be lysed. The release of very readily
which exists in the large-scale system.
biodegradable soluble organic material from the lysed cells
NOTE 2—Cultures grown at low (0.5 to 2 mg/L) and high (>5 mg/L)
may support a higher oxygen uptake rate by the cell suspen-
DO concentrations possess different kinetic capabilities. Thus, to maintain
sion. a laboratory culture with performance capabilities similar to those of the
full-scale culture, the DO concentration should be maintained at the level
6.2.2 An alternate reason for increased oxygen uptake rate is
appropriate for the full-scale process. The probable explanation for the
that certain test candidates (2,4-dichlorophenol for example)
difference in culture performance is that higher concentrations of oxygen
may uncouple the transfer of electrons involved in the process
penetrate more completely through the floc particles.
called oxidative phosphorylation in which adenosine 58 triph-
7.3 A pH Probe and Instrumentation.
osphate (ATP) is formed by the phosphorylation of adenosine
7.4 Dissolved Oxygen Probe—If utilized, the following
58 diphosphate (ADP). The result of the uncoupling is an
apparatus is needed:
increase in the rate of oxygen consumption that is not related
7.4.1 Biochemical Oxygen Demand (BOD) bottles.
to substrate stabilization.
7.4.2 Agitation Device, may be used with the dissolved
6.2.3 A respiration rate by an experimental cell suspension
oxygen probe in the BOD bottle. The device must provide
that is greater than the respiration rate of the control represents
complete mixing of the microbial culture in the BOD bottle.
microbial system damage. The degree of damage is not
7.4.3 Magnetic Stirrer and Magnetic Stirring Bar, alterna-
quantified by comparison of respiration rates for the test
tively, may be used to mix the BOD bottle.
candidate and the control. Whether the cause is due to
7.5 Beakers, 2-L size, (or other containers of suitable size).
uncoupled electron transfer or lysis of cells can be determined
7.6 Clean, Oil-Free Air Supply, to provide cell suspension
by comparing the filtered Dissolved Organic Carbon (DOC) of
mixing and aeration.
the experimental cell suspension with the sum of the DOC of
7.7 Fritted Glass Diffusers or Pasteur-Pipets, as air diffus-
the control plus that added by the test candidate. A higher DOC
ers.
represents cell lysis.
6.3 Where industrial wastewaters in the sewer system are
8. Reagents
continually introducing inhibitory components to the collective
8.1 Microbial Culture—The microbial culture to be used is
wastewaters, it may not be feasible to utilize the returned
the returned sludge from the full-scale facility. For those
sludge from the process directly as the microbial culture. The
activated sludge system where industrial contributions regu-
maintenance of a protected culture of organisms in the labo-
larly cause microbial inhibition, direct use of the returned
ratory may be necessary.
sludge may be impractical. For those systems where microbial
inhibition is not a continuous problem, the returned sludge may
7. Apparatus
be used directly if, by observed system performance, it appears
7.1 Respirometer or an Oxygen probe— An apparatus
to be healthy.
capable of measuring the respiration rate or oxygen uptake rate
8.1.1 A microbial culture may be maintained in the labora-
of the cell suspension.
tory. The culture should be maintained at the temperature of the
7.1.1 Respirometer—A device that receives the cell suspen- full-scale mixed liquor and approximately at the concentration
sion, or an aliquot and provides a technique for measurement of the full-scale process returned sludge.
of oxygen utilization to be interpreted as respiration rate (see 8.1.2 If maintenance of a microbial culture is to be practiced
Appendix X1). in the laboratory, and if the inhibition tests are to be related to
NOTICE: This standard has either been superceded and replaced by a new version or discontinued.
Contact ASTM International (www.astm.org) for the latest information.
D 5120
a specific activated sludge wastewater treatment process, the plants is about 10 to 30 mg/L but may be outside this range. For
initial microbial culture should be taken from the process acclimatized microbial cultures, the EC will be higher, and
returned sludge. values <200 mg/L has been reported. The stock solution may
8.1.2.1 Care should be taken to obtain the microbial culture be used to check experimental technique and possibly the
when, by appearance and performance, the culture is consid- susceptivity of a microbial culture.
ered to be healthy.
9. Procedure
8.1.2.2 The microbial culture should be fed daily with the
9.1 Prepare experimental and control cell suspensions so
actual process wastewater if the wastewater is of suitable
that each is identical in its concentration of microbial culture
quality and not inhibitory. Determine the wastewater quality by
and Marlene’s mix.
following the procedure in Section 9. Prepare a control cell
9.1.1 When the test is related to an operating activated
suspension and a wastewater cell suspension. If the wastewater
sludge process, the microbial culture concentration, and the
cell suspension does not show inhibition, it is suitable for use
initial pH, make sure that the temperature of the cell suspen-
as feed material.
sion (throughout the experimental period) is the same as that in
8.1.2.3 If wastewater of good quality is not available, a
the process mixed liquor. As an example of the control cell
synthetic feed, such as Marlene’s Mix (see 8.2) or other feed
suspension, the following table applies if a microbial concen-
similar in character to the wastewater should be used (sucrose
tration of 2000 mg/L is desired and the microbial culture
has been used successfully with domestic wastewater activated
concentration is 10 000 mg/L.
sludge). The feed application should not be excessive. For
Percent of
example, preferably it should be equal to about one-half of the
Component Preparation Volume
food-to-microorganism ratio that exists in the full-scale pro-
A
Microbial Culture (at 10 000 mg/L) 20
cess.
Marlene’s Mix 3.8
Tap Water 76.2 or as required to equal 100
8.1.2.4 When the full-scale system is considered to be in
A
good condition, replenish one third to one half of the volume of
If the culture is not at 10 000 mg/L, adjust the volume percent to obtain the
the microbial culture daily with the returned sludge from the desired microbial concentration in the cell suspension.
9.1.2 Make sure that the concentration of readily biodegrad-
full-scale system. The replenishment will aid in maintaining a
able organic material in the nutrient dose (such as Marlene’s
microbial culture with approximately the same population
Mix) is high enough that an additional increment of biodegrad-
dynamics as the full-scale process.
able organic material will not result in a significant increase in
8.1.2.5 Replenishment and feeding should be done at the
the rate of respiration. That is, during these tests the microbial
end of a work day so that the culture will have an overnight
culture is essentially saturated with substrate. Marlene’s Mix
period to complete the synthesis of substrate. Replace any
has a soluble Chemical Oxygen Demand (COD) concentration
water that has evaporated over night by adding distilled or
of approximately 60 000 mg/L. The resulting COD introduced
deionized water.
to a cell suspension by the nutrient dose is about 2300 mg/L.
8.2 Nutrient Dose Preparation (Marlene’s Mix)—A solu-
9.1.2.1 If the analyst elects to use an alternative nutrient
tion of the following substances is prepared for use when
feed, it sho
...
Questions, Comments and Discussion
Ask us and Technical Secretary will try to provide an answer. You can facilitate discussion about the standard in here.