1、Designation: D 5120 90 (Reapproved 2004)Standard Test Method forInhibition of Respiration in Microbial Cultures in theActivated Sludge Process1This standard is issued under the fixed designation D 5120; the number immediately following the designation indicates the year oforiginal adoption or, in th
2、e case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. Asuperscript epsilon (e) indicates an editorial change since the last revision or reapproval.1. Scope1.1 This test method covers a batch procedure that evaluatesthe impact of selected wastew
3、aters, materials, or specificcompounds on the respiration rate of an aqueous microbialculture, such as activated sludge.1.2 Alternative procedures for measurement of microbialactivity, such as adenosine 58 triphosphate (ATP), specificsubstrate utilization, etc. are not within the scope of this testm
4、ethod.1.3 The results obtained are based on comparisons in aspecific test series that examines a range of concentrations ofthe potentially inhibitory test candidate using batch methods ina laboratory. Results are completed in a short time frame (a fewhours).1.4 The test results are specific to the m
5、icrobial culture used.Microbial culture from different wastewater treatment plantswill differ in kinds and numbers of organisms, and performancecapability. Thus, there is no basis for comparing results formicrobial cultures from different treatment facilities.1.5 This standard does not purport to ad
6、dress all of thesafety concerns, if any, associated with its use. It is theresponsibility of the user of this standard to establish appro-priate safety and health practices and determine the applica-bility of regulatory limitations prior to use.2. Referenced Documents2.1 ASTM Standards:2D 4478 Test
7、Methods for Oxygen Uptake3. Terminology3.1 Definitions:3.1.1 respiration ratethe quantitative consumption ofoxygen by an aqueous microbial system. The consumption isgenerally expressed as mg O2/L/h.3.1.2 EC50the concentration of the test candidate in thisprocedure (volume percent or mg/L) that resul
8、ts in a reductionof respiration rate to 50 % of that observed for the control.4. Summary of Test Method4.1 This test method utilizes respiration rate as the indicatorof microbial activity.4.2 A batch system that contains a microbial culture (re-turned activated sludge from the process or a culture m
9、ain-tained in the laboratory), selected nutrient dose, and a dilutionof a compound, substance, wastewater, etc. (test candidate) isprepared in a container in the laboratory. The batch system iscalled a “cell suspension.”4.3 The nutrient dose introduces a large excess of biode-gradable substrate ther
10、eby putting the culture at a high meta-bolic rate. Inhibition of respiration by the test candidate isobserved under these conditions.4.4 The prepared cell suspension is aerated for a 2-h period.At the end of the period, the respiration rate is determinedusing a respirometric or an oxygen uptake tech
11、nique.4.5 A lower respiration rate for a cell suspension that hasreceived the test candidate compared to the respiration rate ofa control cell suspension indicates inhibition of respiration.5. Significance and Use5.1 The objectives of the respiration inhibition tests may bedefined by the interests o
12、f the user, but the test method isdesigned primarily for examination of the inhibition responsewith operating microbial systems such as an activated sludgeprocess treating domestic or industrial wastes.5.2 Different apparatus exist that facilitate continuous orcontinual measurement of respiration in
13、 microbial systems andeach may be used as the tool to observe respiration in this testmethod.5.3 Respirometry may utilize any apparatus and techniquethat will achieve the determination of respiration rate. Anumber of devices are presented in Appendix X1. Equivalencyin the experimental capability of
14、each device is not implied.The analyst should select the respirometric approach that bestsuits his needs.1This Test Method is under the jurisdiction of ASTM Committee D34 on WasteManagement and is the direct responsibility of Subcommittee D34.03.01 onThermal and Biological Treatment.Current edition
15、approved Sept. 28, 1990. Published November 1990.2For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at serviceastm.org. For Annual Book of ASTMStandards volume information, refer to the standards Document Summary page onthe ASTM website.1Copyright A
16、STM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.5.4 The inhibitory effect of a test candidate is identifiedmore completely by examining inhibition over a range ofconcentrations, such as determining the EC50. The use ofaerated containers permits
17、concurrent management of a seriesof cell suspensions. A respirometer for each cell suspensionmight also be used.6. Interferences6.1 This test method is most readily applied to substanceswhich, due to water solubility and low volatility, are likely toremain in the aqueous system.6.2 Results have been
18、 observed where cell suspensionscontaining the test candidate had a respiration rate greater thanthe blank, particularly at shorter aeration periods of the cellsuspensions (less than 1 h). Thus, a minimum aeration periodfor the cell suspensions before determinations of respirationrate is 2 h.6.2.1 O
19、ne reason for increased oxygen uptake rate in anexperimental cell suspension may be that severe physical orchemical reactions with the test candidate cause a fraction ofthe microbial culture to be lysed. The release of very readilybiodegradable soluble organic material from the lysed cellsmay suppor
20、t a higher oxygen uptake rate by the cell suspen-sion.6.2.2 An alternate reason for increased oxygen uptake rate isthat certain test candidates (2,4-dichlorophenol for example)may uncouple the transfer of electrons involved in the processcalled oxidative phosphorylation in which adenosine 58 triph-o
21、sphate (ATP) is formed by the phosphorylation of adenosine58 diphosphate (ADP). The result of the uncoupling is anincrease in the rate of oxygen consumption that is not relatedto substrate stabilization.6.2.3 A respiration rate by an experimental cell suspensionthat is greater than the respiration r
22、ate of the control representsmicrobial system damage. The degree of damage is notquantified by comparison of respiration rates for the testcandidate and the control. Whether the cause is due touncoupled electron transfer or lysis of cells can be determinedby comparing the filtered Dissolved Organic
23、Carbon (DOC) ofthe experimental cell suspension with the sum of the DOC ofthe control plus that added by the test candidate.Ahigher DOCrepresents cell lysis.6.3 Where industrial wastewaters in the sewer system arecontinually introducing inhibitory components to the collectivewastewaters, it may not
24、be feasible to utilize the returnedsludge from the process directly as the microbial culture. Themaintenance of a protected culture of organisms in the labo-ratory may be necessary.7. Apparatus7.1 Respirometer or an Oxygen probe An apparatuscapable of measuring the respiration rate or oxygen uptake
25、rateof the cell suspension.7.1.1 RespirometerA device that receives the cell suspen-sion, or an aliquot and provides a technique for measurementof oxygen utilization to be interpreted as respiration rate (seeAppendix X1).7.1.2 Dissolved Oxygen Probe and InstrumentationAnalternate device for the meas
26、urement of respiration rate asoxygen uptake rate.7.2 Culture TankIf it is deemed necessary to maintain amicrobial culture in the laboratory, the apparatus required is acontainer with adequate mixing and oxygen transfer. Thecontainer should hold at least four times the volume of culturethat might be
27、used in one day.7.2.1 The culture tank should be adequately mixed to insurethat the culture remains in suspension and that sufficientmechanical or bubble aeration occurs to maintain the desireddissolved oxygen (DO) concentration.NOTE 1Energy input should not be such that the biological floc issheare
28、d to sizes smaller than that which exists in the large-scale process.Mixing and aeration provided through diffused aeration in a laboratory-sized container may result in an excessive power input. Considercontrolling the power input per unit volume to approximately that whichexists in the large-scale
29、 process. For example, pure oxygen for aeration incombination with mechanical mixing may be utilized to achieve a balancebetween oxygen transfer and mixing. Determine the mixer power input bymeasuring the electrical power consumed at different operating speed, andadjust the mixer speed to achieve a
30、power input that is equivalent to thatwhich exists in the large-scale system.NOTE 2Cultures grown at low (0.5 to 2 mg/L) and high (5 mg/L)DO concentrations possess different kinetic capabilities. Thus, to maintaina laboratory culture with performance capabilities similar to those of thefull-scale cu
31、lture, the DO concentration should be maintained at the levelappropriate for the full-scale process. The probable explanation for thedifference in culture performance is that higher concentrations of oxygenpenetrate more completely through the floc particles.7.3 A pH Probe and Instrumentation.7.4 Di
32、ssolved Oxygen ProbeIf utilized, the followingapparatus is needed:7.4.1 Biochemical Oxygen Demand (BOD) bottles.7.4.2 Agitation Device, may be used with the dissolvedoxygen probe in the BOD bottle. The device must providecomplete mixing of the microbial culture in the BOD bottle.7.4.3 Magnetic Stirr
33、er and Magnetic Stirring Bar, alterna-tively, may be used to mix the BOD bottle.7.5 Beakers, 2-L size, (or other containers of suitable size).7.6 Clean, Oil-Free Air Supply, to provide cell suspensionmixing and aeration.7.7 Fritted Glass Diffusers or Pasteur-Pipets, as air diffus-ers.8. Reagents8.1
34、Microbial CultureThe microbial culture to be used isthe returned sludge from the full-scale facility. For thoseactivated sludge system where industrial contributions regu-larly cause microbial inhibition, direct use of the returnedsludge may be impractical. For those systems where microbialinhibitio
35、n is not a continuous problem, the returned sludge maybe used directly if, by observed system performance, it appearsto be healthy.8.1.1 A microbial culture may be maintained in the labora-tory. The culture should be maintained at the temperature of thefull-scale mixed liquor and approximately at th
36、e concentrationof the full-scale process returned sludge.8.1.2 If maintenance of a microbial culture is to be practicedin the laboratory, and if the inhibition tests are to be related toD 5120 90 (2004)2a specific activated sludge wastewater treatment process, theinitial microbial culture should be
37、taken from the processreturned sludge.8.1.2.1 Care should be taken to obtain the microbial culturewhen, by appearance and performance, the culture is consid-ered to be healthy.8.1.2.2 The microbial culture should be fed daily with theactual process wastewater if the wastewater is of suitablequality
38、and not inhibitory. Determine the wastewater quality byfollowing the procedure in Section 9. Prepare a control cellsuspension and a wastewater cell suspension. If the wastewatercell suspension does not show inhibition, it is suitable for useas feed material.8.1.2.3 If wastewater of good quality is n
39、ot available, asynthetic feed, such as Marlenes Mix (see 8.2) or other feedsimilar in character to the wastewater should be used (sucrosehas been used successfully with domestic wastewater activatedsludge). The feed application should not be excessive. Forexample, preferably it should be equal to ab
40、out one-half of thefood-to-microorganism ratio that exists in the full-scale pro-cess.8.1.2.4 When the full-scale system is considered to be ingood condition, replenish one third to one half of the volume ofthe microbial culture daily with the returned sludge from thefull-scale system. The replenish
41、ment will aid in maintaining amicrobial culture with approximately the same populationdynamics as the full-scale process.8.1.2.5 Replenishment and feeding should be done at theend of a work day so that the culture will have an overnightperiod to complete the synthesis of substrate. Replace anywater
42、that has evaporated over night by adding distilled ordeionized water.8.2 Nutrient Dose Preparation (Marlenes Mix)A solu-tion of the following substances is prepared for use whenconducting inhibition studies. Store the prepared solution in arefrigerator at 4C. Warm the portion of the feed to be used
43、intests to the operating temperature of the test before use.Replace the solution after 14 days of storage or earlier if itbecomes odorous.3Ingredient Quantity (g)Bacto peptone332Beef extract322Ammonium chloride 11Sodium chloride 1.4Calcium chloride (CaCl22H2O) 0.8Potassium dihydrogen phosphate 3.5Po
44、tassium monohydrogen phosphate 4.5Distilled water make up to 1 L8.3 Stock Inhibitor SolutionDissolve 0.5 g of 3,5-dichlorophenol in 10 mL of 1N NaOH, dilute to 30 mL withdistilled water, add 1N H2SO4to the point of incipientprecipitation (approximately 8 mL of 1N H2SO4will berequired), bring the vol
45、ume to 950 mL with distilled water,adjust the pH to the range of 7 to 8, and bring the volume to 1L. The EC50of 3,5-dichlorophenol for relatively non-acclimatized microbial cultures from domestic wastewaterplants is about 10 to 30 mg/Lbut may be outside this range. Foracclimatized microbial cultures
46、, the EC50will be higher, andvalues 200 mg/L has been reported. The stock solution maybe used to check experimental technique and possibly thesusceptivity of a microbial culture.9. Procedure9.1 Prepare experimental and control cell suspensions sothat each is identical in its concentration of microbi
47、al cultureand Marlenes mix.9.1.1 When the test is related to an operating activatedsludge process, the microbial culture concentration, and theinitial pH, make sure that the temperature of the cell suspen-sion (throughout the experimental period) is the same as that inthe process mixed liquor. As an
48、 example of the control cellsuspension, the following table applies if a microbial concen-tration of 2000 mg/L is desired and the microbial cultureconcentration is 10 000 mg/L.ComponentPercent ofPreparation VolumeMicrobial Culture (at 10 000 mg/L) 20AMarlenes Mix 3.8Tap Water 76.2 or as required to
49、equal 100AIf the culture is not at 10 000 mg/L, adjust the volume percent to obtain thedesired microbial concentration in the cell suspension.9.1.2 Make sure that the concentration of readily biodegrad-able organic material in the nutrient dose (such as MarlenesMix) is high enough that an additional increment of biodegrad-able organic material will not result in a significant increase inthe rate of respiration. That is, during these tests the microbialculture is essentially saturated with substrate. Marlenes Mixhas a soluble Chemical Oxygen Demand (COD) concentratio
