- Email: [email protected]
The electrolytic respirometer—I factors affecting oxygen uptake measurements
Water Research Vol. 10, pp. 1031 to 1040. Pergamon Press 1976. Printed in Great Britain.
THE ELECTROLYTIC RESPIROMETER--I FACTORS AFFECTING OXYGEN UPTAKE MEASUREMENTS JAMES C. YOUNG and E. ROBERTBAUMANN Department of Civil Engineering, Engineering Research Institute, Iowa State University, Ames, Iowa, U.S.A.
(Received 8 June 1976) Abstract--The electrolytic respirometer provides a more accurate and more complete measurement of biochemical oxygen demand (BOD) of wastewaters samples than normally is obtained by the standard dilution BOD method. Laboratory and field studies have shown that the electrolytic respirometer is a dependable instrument for making BOD measurements, is simple to operate, and provides a continuous readout of the oxygen uptake reaction. The major factors affecting oxygen uptake measurements with the electrolytic respirometer are the mixing rate and the concentration of oxygen demanding material in the sample. These system limitations can be overcome by using simple procedures such as increased mixing rate or enrichment of the oxygen in the air in contact with the sample. The precision of BOD measurement with the electrolytic respirometer was considerably greater than with the dilution method.
INTRODUCTION
The electrolytic respirometer was developed as a means of providing more accurate and more complete measurement of the biochemical oxygen demand (BOD) of wastewaters. Studies have shown that this system eliminates many technical problems encountered with other methods for determining oxygen demand and the rate at which it is exerted. Reviews of the design and application of electrolytic respirometers have been presented by Young, et al (1965), Montgomery et al. (1971) and Young and Baumann (1972). The purpose of Part 1 of this paper is to report the results of a comprehensive study designed to help determine the limitations of the system when used for measuring BOD and oxygen uptake rates. It should be emphasized that the term BOD is used in a broad sense to refer to any exertion or measurement of oxygen uptake by microorganisms in aqueous suspension.
lysis BOD measuring system is actually a large volume respirometer which provides semi-continuous and automatic registering and adjustment of the pressure change that is brought about within the reaction vessel because of oxygen consumption by microorganisms. OXYGEN ELECTRODE SWITCH
~
ELECTROLYSIS CELL
J-
ADAPTOR - CO2 ABSORBENT CONTAINER
%2
Description of the electrblytic respirometer
SOLUTION
The electrolytic respirometer consists of three major parts. A reaction vessel contains the sample and a magnetic stirring bar or other suitable mixing device (Fig. 1). An adaptor unit or alkali container holds potassium hydroxide or other basic solution to absorb metabolically produced carbon dioxide from the atmosphere above the sample. The critical part of the system is the electrolysis cell which contains a weak electrolyte such as sulfuric acid or sodium hydroxide. The electrolysis cell serves as a manometer to detect pressure changes and as an oxygen generator to maintain a constant partial pressure in the atmosphere within the sample container. The electrow.g. 10/11--~
HYDROGEN ELECTRODE
T
SAMPLE
I
~
STIRRINGMAGNET
Fig. 1. Schematic diagram showing the basic operation of the electrolytic respirometer. 1031
1032
JAMES C. YOUNG and E. ROBERT BAUMANN
Fig. 2. An operational 5-unit electrolytic respirometer as used in a constant temperature room.
Factors affectin 9 oxygen uptake in the electrolytic respirometer Factors which might affect the performance of the electrolytic respirometer in providing an accurate measure of oxygen requirements in any given sample include: (1) Capability of the system to transfer oxygen into solution across the air-liquid interface, as related to the oxygen production limits of the system, the mixing rate, the dissolved oxygen concentration in the sample, a n d the composition of gas above the sample; (2) substrate concentration and nutrient deficiency; (3) dilution; (4) buffer concentration; (5) sample size; and (6) size and nature of the inoculum. To understand the electrolytic respirometer more completely, laboratory studies were conducted to establish qualitatively a n d quantitatively how these factors influence oxygen uptake measurements. A more detailed description of those studies is given by Young and B a u m a n n (1972). EXPERIMENTAL STUDIES Laboratory studies were conducted using 5 or 6-unit electrolytic respirometer systems similar to that shown in Fig. 2. Test temperature was maintained at 20°C by keeping the units in a constant temperature room. The initial
temperature of the samples was adjusted to 20 _+ 0.5"C to minimize the effect of pressure changes within the vessel that otherwise would have occurred as a result of the sample temperature adjusting to the test temperature. A synthetic waste was used in most laboratory studies to provide a material which was consistent in composition from test to test. The synthetic substrate normally used was powdered Metrecal, a dietary food supplement manufactured by Mead Johnson & Co., Evansville, Indiana. A stock synthetic waste concentrate was made up for each test by diluting 8.50 g of powdered Metrecal to one liter to give a solution having 10,000mg/l ultimate BOD (BODL). Dilution was used to reduce this solution to the desired sample strength. A phosphate buffer which proved satisfactory for use with this synthetic waste was made by neutralizing 213.0 g/l of NaH2PO4' H20 (1.5 M) to pH 7.2 with potassium hydroxide. One milliliter of this solution per 50 mg/1 BODL maintained the sample pH between 6.7 and 7.3. Settled raw sewage or primary effluent from the Ames, Iowa, water pollution control plant was used as a seed source. Seed BOD was determined in each series of tests so that a correction could be made to reduce the measured BOD to the BOD of the synthetic substrate. Data were processed by computer to make seed corrections, to calculate a first-order BOD curve (including adjustments for lag time), and to plot each measured and calculated BOD curve for visual comparisons. A chemical nitrification inhibitor, 2 chloro-6-(trichloromethyl)pyridine (TCMP) (Hath Chemical Co., Ames, Iowa), was added to each sample in which inhibition of nitrification was desired I-Young (1973)].
The electrolytic respirometer--I
1000 mg/I BOD L O ½ - INCH VORTEX 1 - INCH VORTEX • 3 - INCH VORTEX • 5 - INCH VORTEX
E
d
o
ok~l~ v 0
--
1033
_ _
__aj
I
I
t
I
I
I
1
2
3
4
5
6
TIME, days
1200 1200 mg/I BODL
E
8
13" IINN(~HVO~TT~..~
800 --
4OO
__
• 5 - INCH VORTEX
_
0 1
_ ~
,
~ ~
/
AIR ABOVE SAMPLE
I
l
I
I
i
i
2
3
4
5
6
7
TIME; days
Fig. 3. Results of mixing-rate tests using air and oxygen-enriched air above the sample. RESULTS
Evaluation of factors affecting oxygen transfer Oxygen production rate. The equipment used in this study could supply oxygen at a rate of 42 mg/h, a rate far above any actually measured. Mixing rate. Essentially no difference in BOD curves (accumulated oxygen uptake versus time) was observed at vortex depths ranging from 1/2 to 5 in. (1.3 to 13 cm) with samples containing up to 500 mg/1 BODE. With BOD E concentrations ranging from 600 to 1000 rag/l, the rate of oxygen uptake decreased at the 1/2-in. (1.3 cm) vortex depth because of oxygen transfer rate limitations (Fig. 3). A vortex depth of about 3 in. (7.6 on) depth subsequently was used as a reference point for setting the stirring rate in any unit. Increasing the percentage of oxygen in the air above the sample to about 60% extended the range of waste concentrations over which BOD measurements could be made without encountering oxygen transfer limitations to at least 1,200 mg/l BODE (Fig. 3). This was accomplished easily by simply flushing the air space above the sample and in the electrolysis cell with pure oxygen just before assembling the unit. A 15-min adjustment period with the sample being stirred and with oxygen being produced in the electrolysis cell permitted an equilibrium to be established between the oxygen-enriched atmosphere and the dissolved oxygen concentration in the sample.
It should be emphasized here that the lag in oxygen uptake in the curves in Fig. 3 and subsequent figures is related to the amount of seed microorganisms added to each synthetic waste sample. This amount varied from test to test because of uncontrollable differences in seed samples, but in all cases was the same for replicate runs made on a given test date.
10.0
--
5.0
_
\ / I \ ~ ,.~ & \ ~
/
~
oo
I
~
800 -- BOD CURVE 110~0mg/I BOD| 750 mg/I BOD~"
500m~A Boo~
4 o6
0
100o.~ Boo, 750mg4 Boo, 5OOm~, ~
/~
I o t~
I
I
•
3__
•
•
0
i
o
~
-I
200 0
~"
10
20
30
40
50
60
70
80
TIME, hours
Fig. 4. Dissolved oxygen concentrations in the sample and associated BOD curves for four concentrations of synthetic waste.
1034
JAMESC. YOUNGand E. ROBERTBAUMANN
Dissolved oxygen in the sample. Measurements of dissolved oxygen(DO) concentration in the samples were made in a number of tests using the miniature Winkler method outlined in Standard Methods (1971). Separate 25-ml sample aliquots were transferred from reaction vessels equipped with a sampling port above the solution and adjustments in BOD were made for the volume of sample removed. The dissolved oxygen varied with time as shown in Fig. 4 when air was the gas above the sample. These results indicate that oxygen transfer was not a limiting factor for waste strengths less than about 750 mg/1 BODL when using air above the sample and when adequate stirring was provided. This corresponds reasonably well with the results of the stirring rate studies. The exact dissolved oxygen concentration below which oxygen becomes limiting is not known. Kalinske (1971) concluded from a survey of published work that DO concentrations from 0.5 to 35 mg/1 do not affect respiration rates as long as the cells remain dispersed. Samples mixed at lower rates may require higher minimum DO concentrations since flocculation occurs more readily than with rapid mixing. More accurate oxygen uptake measurements can be obtained with respirometers if the raw data are corrected for the rate of change in dissolved oxygen within the sample. Up to the point of minimum DO, the rate of change in DO should be added to the measured rate of oxygen uptake since this represents an amount of oxygen used but not measured. Similarly, the rate of increase in dissolved oxygen after the point of minimum DO should be subtracted from the measured rate of oxygen uptake. The net result of this correction is to introduce a plateau. With the quantities of oxygen transferred in the electrolytic respirometer this correction was a small part of the oxygen uptake reaction at all BOD concentrations. It should be emphasized that the need for this correction is the same with all respirometric and mano-
O & • •
100m
metric methods for measuring BOD. The maximum rate of depletion of the dissolved oxygen in the tests reported in Fig. 4 was about 0.7mg/I/h for the 500 mg/l BODL solution, as compared to a maximum oxygen uptake rate of 8 mg/l/h. Oxygen depletion curves reported for BOD measurements in Warburg respirometers for solutions having 300 mg/l BODL indicated a maximum oxygen uptake rate of about 5 mg/1/h of which 0.5 mg/l/h was attributable to removal of oxygen from solution [Bryant et al. (1967)]. The electrolytic and Warburg respirometers then appear to have similar oxygen transfer characteristics. The sample strength at which oxygen transfer limitations would exist would be increased considerably when using an oxygen-enriched atmosphere.
Gas composition above the sample The composition of the gas above the sample should remain about the same throughout a test period, otherwise there will be an accumulative error in the BOD measurement equal to the change in mass of oxygen. To determine whether changes occurred, the oxygen, nitrogen, and carbon dioxide in the gaseous atmosphere above the sample were measured periodically throughout the testing period when using samples of synthetic waste of various strengths. There was little change in gas composition for up to 13 days of testing for waste strengths up to 1000mg/l BODL when using air above the sample (Figs. 5 and 6). However. when the amount of oxygen in the atmosphere above the sample was increased to about 80% at the beginning of the test, the oxygen content gradually decreased, and the nitrogen content increased with the higher strength samples. With the 1000 mg/1 BODL samples (Fig. 5), much of the decrease in oxygen appeared to be a result of carbon dioxide accumulation in the system. No appreciable carbon dioxide accumulation occurred with the 600 mg/I BODt. samples (Fig. 6), but the percentage
GAS ANALYSIS BOD, % OF BODL(I,000 rag/l) OXYGEN, % NITROGEN, % CARBON DIOXIDE, %
d O m
80,
60
--
2 u
40
20,~ _
_
_ -
1
2
3
4
5
"0
I
2
3
4
5
TIME, clays
Fig. 5. Analysis of gas composition above 1000mg/1 BODL synthetic waste sample with (right) and without (left) oxygen enrichment. Carbon dioxide accumulation accounted for much of the decrease in oxygen content.
The electrolytic respirometer--I
1035
GAS ANALYSIS
I
o e~oo, % OF SOOL(600 ,~A)
Z
8
0 .
.
0
.
.
.
3
.
.
.
6
9
.
12
15
0
3
6
9
12
15
TIME, days
Fig. 6. Analysis of gas composition above 600 mg/1 BOD L synthetic waste sample with (right) and without (left) oxygen enrichment. The oxygen content in the oxygen-enriched sample decreased gradually over the 14-day test period. of gaseous oxygen decreased and the percentage of gaseous nitrogen increased. This pattern of changes in gas composition was more pronounced with the higher strength wastes. The magnitude of the change when starting with 60-80% oxygen above samples with 600-1000mg/1 BODL was about 5% in 5 days. This would be a significant error with low BOD samples, but oxygen enrichment is not needed for samples containing less than about 600 mg/1 BODL. The error would be less than 1% for samples having BOD's in excess of 1000 mg/1 and would be insignificant in measurements made for treatment plant control purposes. The cause of this decrease in oxygen content is not clear. Diffusion of oxygen through the electrolysis cell was ruled out since a corresponding diffusion of hydrogen into the unit through the cell would have been expected, and no detectable amount of hydrogen was observed in any gas sample analyzed. Also, similar diffusion would have occurred in low-strength samples and no such problem was observed.
Substrate concentration and nutrient deficiency When the concentration of substrate is high, as it may be in respirometers, the substrate or waste material itself may become a limiting nutrient, and waste utilization will reach a maximum value dependent only on the mass of microorganisms. This effect is demonstrated in Fig. 7 which shows the results of a series of dilutions in which all components of the sample, including the seed, were diluted equally. An oxygen enriched atmosphere (80~o+) was used to eliminate oxygen transfer problems. At BODL concentrations greater than about 1000 rag/l, the oxygen uptake rate remained essentially constant. Thimann (1963) has reported that with substrates such as glucose at concentrations above 40 mg/l (as glucose), the rate of substrate removal per unit of microorganism mass is constant. Buffer concentrations At the synthetic waste concentrations of 500 mg/l BODL, the shape of the BOD curve, the lag time,
SYNTHETIC WASTE
1200, -~E
100 mg/I 300 rag,/1 500 mg/l t000 mg/I 1500 mg/I
1
O ~ • • O
A~.41~ .~1~ I~r"
2
3
4
5
6
TIME, days
Fig. 7. B O D curves for low- and high-strength synthetic wastes show that the oxygen uptake rate
became essentially constani at a BOD concentration above 1000 rag/1.
1036
JAMES C. YOUNG a n d E. ROBERT BAUMANN
800 - -
AIR
400 k~ d
o
1
___L .............
J
I
_ J ......
k ......
OXYGEN-ENRICHED AIR
1
0 0
2
4
6
8
I0
12
14
TIME, cloys
Fig. 8. BOD curves using 500 mg/l BODL synthetic waste samples containing phosphate buffer concentrations ranging from 0.004 M to 0.02 M. and the BOD up to 14 days were not affected significantly by buffer concentrations ranging from about 0.004 M-O.02 M (Fig. 8). Oxygen enrichment did not affect the BOD curve at any buffer concentration. Similar results were observed using synthetic waste samples having BODL concentrations below 500 mg/1. Oxygen transfer rates became limiting when air was used with 750 mg/1 BODL samples (Fig. 9). The variation between samples was reduced considerably when using an oxygen-enriched atmosphere. Similar results were obtained with higher-strength synthetic waste samples. In all cases, there was no apparent relationship between the shape of the curve and the buffer concentration. That is, in one test the lower oxygen uptake rate might be associated with the lower buffer concentration while in a similar test the lower oxygen uptake rate was associated with the higher buffer concentration.
The recommended amount of buffer solution listed previously in this paper was satisfactory when measuring the BOD of synthetic wastes having a predictable BODL, or a standard buffer concentration of 0.02 M could have been used with all waste samples tested. However, with samples containing significant amounts of calcium or magnesium salts, it may not be possible to use a phosphate buffer; and more suitable buffers may have to be developed or the sample may have to be diluted. Fortunately, with domestic wastewaters, the natural buffering capacity generally is sufficient to control pH, and the addition of a buffer is not required. Sample size and barometric pressure The size of the sample used in the electrolytic respirometer normally should be sufficient to fill the reaction bottles as full as possible without causing
800 L
AIR
o 0
1
2
3
4
5
t
I
6
7
TIME, doys
Fig. 9. BOD curves using 750 mg/1 BODL synthetic waste samples containing phosphate buffer concentrations ranging from 0.006 M to 0.03 M.
The electrolytic respirometer--I
tion. This is demonstrated by results obtained when measuring the BOD of a 500 mg/1 BODL Metrecal solution (Fig. 10). With smaller sample volumes, or when measuring the BOD of such low-strength samples as secondary treatment plant effluent or stream or lake water, the sample should essentially fill the reaction vessel. Otherwise, corrections should be made.
the sample to overflow into the potassium hydroxide container at nominal-to-high stirring rates. As the sample volume is reduced, the error in the BOD measurements may increase because of the larger effect of barometric pressure changes. When the barometric pressure rises, the pressure inside the reaction vessel must also increase. This can only be accomplished by adding oxygen to the system. For barometric pressure increases, this addition is automatic and an equivalent amount of oxygen must be subtracted from the indicated demand to correct the error. When the barometric pressure decreases, the switching mechanism of the open-manometer electrolysis cell will not operate unless there is a BOD exerted which is at least equal to the pressure error. The correction for a barometric pressure decrease is then positive, correcting the BOD exertion which would have been indicated had the barometric pressure remained constant. The correction of barometric pressure changes is given by the following equation: Wo~ = 1.31Vo(AP)
Accuracy and precision Regardless of the method used for measuring BOD, the accuracy and precision of the measurement must be defined within acceptable limits. As yet, no standard exists by which to determine the accuracy of the BOD test; and the precision, or the ability to obtain reproducible test results, normally is emphasized. To obtain an indication of the precision possible with the electrolytic respirometer, analysis-of-variance tests were conducted using data from runs having adequate mixing or oxygen enrichment containing four' or more replicates. These analyses, summarized in Table 1, indicated that with a given sample strength the standard deviation did not change appreciably for any testing period of two days or more when sample BODL concentrations were below 750 mg/1 or when pure oxygen was added to the atmosphere above the sample. When oxygen transfer limitations became evident, as with the 750mg/1 BODL sample to which no oxygen was added initially to the atmosphere above the sample, the standard deviation and associated coefficient of variability (CV = standard deviation, S.D., as a percentage of the average) increased for the shorter test periods. In every case but two, the coefficient of variability of the 5-day BOD was less than or equal to 3.2~o. For samples with BODL values below 250 mg/1, a coefficient of variability of less than about 1~o was obtained after 5 days of testing. After two to three days of testing, the coefficient of variability in all test results
(3)
where Wo2 = oxygen required to correct the measured BOD, mg 02 V0 = volume of air space above sample, liters, and AP = barometric pressure change, millibars. For a 75-ml air volume above the sample, the correction is 0.98mg of 02 for a 10mb pressure change, which is a typical change occurring in any one week. The correction is not accumulative and is determined by the same function regardless of the time increment over which AP is measured. Thus, for daily BOD values, a correction needs to be made only to the daily accumulated BOD and not to the hourly subtotals. In most cases, the sample volumes greater than 500 ml will not require a barometric pressure correc-
SYNTHETICWASTE o 500 rag/l, 950ml 500 mg/I, 700ml • 500 rag/I, 500 ml BODL, volume
..tS o
o
I
]
I
I
I
l
I
400 - -
2OO
0
1037
I
l
I
SY/~n'METICWASTE o SO0 mg/'l, 950 ml ~ 500 ms~1, 750 rnl
f
• 500 rag/I, 500 ml BODL, voluml #
I
I
I
I
L
I
I
I
I
I
I
2
3
4
5
6
7
8
9
10
11
TIME, days
Fig. 10. BOD curves show that, when various volumes of sample in a 11. test bottle are used, the same results can be obtained using sample .volumes as low as 500 ml.
1038
JAMES C. YOUNG and E. ROBERT BAUMANN Table 1. Variability analyses for measured BOD of synthetic waste (Metreca1) samples. Day of test Sample
i
2
3
4
5
6
125 mg/1 BOD L n - 4 (5-25-71)
X* SD CV
25.50 1.23 4.82
62.3 1.44 2.32
80.7 1.56 1.93
90.8 i.ii 1.22
96.4 0.85 0.88
. . . . . . . . . . . .
250 m g / l BOD Air n ~ 4 L (5-25-71)
X SD CV
47.4 1.06 2.24
134,3 4.58 3.40
170.0 2.36 1.39
188.8 0.48 0.26
201.4 1.41 0.70
. . . . . . . . . . . .
250 m g / l BOD L 02 n " 4 (5-25-71)
X SD CV
44.5
125.7 3.02 2.40
162.2 2.11 1.30
188.8 0.43 0.23
199.4 1.54 0.77
. . . . . . . . . . . .
500 mg/l BOD Air n ~ 8 L (5-14-71)
X SD CV
101.5 17.2 16.9
264.1 13.5 5.1
331.1 16.4 4.9
373.9
500 m g / l BOD L O9 n ffi 7 (~-14-71)
~ SD CV
134.8 9.7 7.2
294.5 8.2 2.8
351.4 11.9 3.4
750 mE/1 BOD Air n = 7 L (4-24-71)
X SD CV
162.9 46.2 28.3
339.0 59.2 17.5
750 m8/l BOD L O9 n = 7 (~ -24-71)
X SD CV
265.5 21.2 8.0
1,000 m8/l BOD I00 m l seed L Air n = 8 (4-30-71)
X SD CV
1,000 mg/l BOD L I00 m l seed 02 n ffi 8 (4-30-71)
2.56 5.63
7+
391.9 11.8 3.0
410.8 12.2 3.0
428.4 11.9 2.8
379.0 9.9 2.6
396.4 8.7 2.2
413.9 10.4 2.5
429.7 14.3 3.3
468.2 54.0 11.5
524.6 47.7 9.1
592.7 13.6 2.3
. . . . . . . . . . . .
459.7 21.1 4.6
522.3 12.9 2.5
556.1 Ii.0 2.0
583.6 11.7 2.0
. . . . . . . . . . . .
235.9 50.5 21.4
493.6 55.1 11.2
658.8 94.4 14.3
733.9 20.1 2.7
783.8 10.7 1.4
809.8 17.0 2.1
----
X SD CV
340.9 33.9 I0.0
594.4 26.7 4.5
679.1 30. i 4.4
728.1 29.3 4.0
760.6 24.6 3.2
796,5 18.1 2.3
----
20 m 8 / l BOD L (12-14-68) n - 4
X SD CV
4.2 1.6 37.5
7.8 1.5 19.3
9.6 1.3 13.1
10.8 0.4 3.9
11.8 0.4 3.5
----
16,7 0.6 3.9
20 mg/l BOD L (1-8-69) n = 4
X SD CV
8.9 0.3 3.4
14.9 0.7 4.8
17.9 0.5 2.8
18.1 0.7 3.7
20.4 0.5 2.3
----
20.8 0.5 2.2
20 m 8 / l BOD L (2%) (4-24-69) n = 4
X SD CV
7.5 0.4 5.2
12.2 1.2 10.2
14.9 1.9 12.7
16.9 0.6 3.7
17.5 0.5 3.0
. . . . . . . . . . . .
20 m 8 / l BOD L (I£) (4-24-69) n = 4
X SD CV
7.8 0.05 0.6
12.1 0. ii 0.9
14.7 0.03 0.2
17.7 0.3 1.5
18.3 0.4 2.4
. . . . . . . . . . . .
5 mg/l BOD L (21) (4-29-69) n ffi4
X SD CV
2.6 0.5 19.0
3.2 0.4 12.8
4.1 0.4 9.9
4.8 0.5 10.4
5.0 0,3 5.7
. . . . . . . . . . . .
5 mg/l BOD L (i~) (4-29-69) n ~ 4
X SD CV
1.6 0.2 15.2
2.2 0.2 8.1
4.0 0.3 6.4
4.6 0.2 4.8
4.8 0.5 I0.I
. . . . . . . . . . . .
13.2 3.5
*
* X - average BOD, mg/l; SD = standard deviation, mg/l; CV = coefficient of variability, and n = number of samples.
was less than 3.5% for samples having BODL values below 500 mg/1. Initial electrolytic respirometer results were not encouraging when measuring the BOD of samples containing less than about 20rag/1 BODL until the sensitivity of the cell was recognized as a mechanical source of error in the system. The sensitivity could be increased by using a plexiglass tubing insert in the outer chamber of the electrolysis cell I-Young and Baumann (1972)]. This insert reduced the electrolyte volume change required to activate the power supply so that oxygen was added in smaller increments. The resulting precision with which low-level BOD determinations could be made was quite encouraging, with coefficients of variability of about 2 3% for BODL concentrations in the 1(~20 mg/l range (Table 1). For lower strength samples, the coefficient of variability increased but seldom was greater than 10%.
%;
SUMMARY AND DISCUSSION
As shown in Figs. 3 7. a high substrate concentration and a low oxygen content in the atmosphere within the sample container all caused variation between BOD curves even when mixing was adequate. The overall effect of these factors is summarized in Fig. 11. Here the maximum oxygen uptake rate, as measured by the maximum slope of measured BOD curves, is plotted against the BODL of each sample. Seed corrections, although a small fraction of the oxygen uptake at any one time, were made for all samples. These data show that the maximum rate of oxygen uptake increased somewhat linearly with increased BODL but that the scatter of data increased greatly with samples containing more than about 750 mg/1 BODL. Increasing the concentration of oxygen to
The electrolytic respirometer--I about 80~o in the air above the sample increased the oxygen transfer capability of the system to about 20mg/1/h at a BODL of 1200mg/1 from a rate of 12mg/l/h at a BODE of about 750mg/1. These numbers agree with those presented previously in connection with stirring rate studies. At BODE concentrations above about 1000 mg/1, substrate concentration no doubt was a factor in limiting the oxygen transfer rate whether or not the air above the sample was enriched with oxygen. The oxygen transfer limit of the electrolytic respirometer should not necessarily be considered in terms of the BOD s or BODE concentration beyond which oxygen transfer rate limitations occur. A more appropriate measure of the system capacity is the maximum rate of oxygen uptake which can be measured before oxygen transfer or substrate concentration begins to limit the oxygen uptake rates. For example, municipal wastewater having an oxygen uptake rate of 10 mg/1/h might have a BOD E twice that of an industrial wasteL water or synthetic wastes having the same uptake r a t e but containing a high percentage of soluble organic material which is rapidly synthesized and oxidized by bacterial cells. If high oxygen uptake rates are anticipated, as with high-strength wastes or mixed-liquor samples from activated sludge aeration tanks, the oxygen content of the air above the sample can be increased so that higher oxygen uptake rates can be measured accurately; or, the sample may be diluted so that t h e maximum oxygen uptake rate is below the system limit. Some electrolytic respirometer designs may provide higher oxygen transfer capabilities by providing more vigorous mixing, but this would tend to increase the complexity of the equipment design and operation [Montgomery et al., (1971)]. The significance of the above observation depends on application of the data. If kinetic studies are being made to determine substrate uptake characteristics of
20 - •
i
1039
microorganisms, then there is merit to tests conducted using full-strength wastes. If the objective of the test is to provide an estimate of total biodegradable material, then sample adjustments must be made so that oxygen transfer or substrate concentration limitations do not occur. If a BOD is being measured for waste treatment plant purposes, the test should be conducted at a waste concentration about the same as that occurring in the treatment process or stream receiving the wastewater. CONCLUSIONS
Laboratory studies have shown that the electrolytic respirometer is a dependable instrument for making BOD measurements. The system is simple to operate and provides a continuous readout of oxygen uptake. The major factor contributing to errors in oxygen uptake measurements are the mixing rate and the concentration of oxygen demanding organic material in the sample. Oxygen transfer limitations can be overcome by increased mixing or by enriching the air in contact with the sample with oxygen. The effect of high substrate concentration can be eliminated by diluting the sample. In the open manometer cell design described in this paper, a change in barometric pressure will change the volume of gas within the reaction vessel and cause more or less oxygen to be added than demanded by the sample. Although the effect of such barometric pressure changes is usually small, corrections can be made easily. Analyses of variance tests of replicate BOD tests indicated that, after two or three days of testing, the coefficient of variability was less than 3.5~ for samples having BODE values between 20 and 500 mg/1. Acknowledgements--This research work was supported in part by Federal Environmental Protection Agency
OXYGENUPTAKERATE o OXYGENADDED • NO OXYGENADDED '
15
•
10 5--
o
~
200
o
400
i
600 800 BODL, mg,/I
1000
1200
I
1400
Fig. 11. Oxygen uptake rate as a function of the substrate concentration increased until the substrate concentration was about 1000 mg/1 BODL. After this, oxygen uptake was variable.
1040
JAMES C. YOUNG and E. ROBERT BAUMANN
demonstration grant $800363 (formerly WP 16020 DUN) and the Engineering Research Institute of Iowa State University. Robert C. Kroner, chief (retired) of the Physical and Chemical Methods Section, EPA, Analytical Quality Control Laboratory, was project officer. Donald A. Haselhof, a graduate student in Sanitary Engineering at Iowa State University during the period of this study, helped with the data collection and analysis, part of which was included in his Master of Science thesis.
REFERENCES
Bryant J. O., Akers W. W. & Busch A. W. (1967) Limitations of oxygen transfer in the Warburg apparatus. Proc. Ind. Waste Conf. 22nd, Purdue University Extensions Series 131, pp. 68~698. Kalinske A. F. (1971) Effect of dissolved oxygen and substrate concentration on the uptake of microbial suspensions. J. War. Pollut. Control Fed. 41, 73-80. Montgomery H. A. C., Oaten A. B. & Gardiner D. K.
(1971) An automatic respirometer Its construction and use. Effluent and War. Treat. J. 11, 23 31. Simpson J. R. and Nellist G. R. (1970) Development and use of a large-volume automatic respirometer. War. Pollut. Control 59(~605. Standard Methods for the Examination o] Water and Wastewater (1971) 13th Ed. American Public Health Association, New York, N.Y. Thimann K. V. (1963) The Lift, of Bacteria. p. 633. MacMillan, New York. Young J. C. & Baumann E. R. (1972) Demonstration of the electrolysis method for measuring biochemical oxygen demand. Enq. Res. Inst. Rep. No. 72153, Iowa State University and EPA Final Report for projects WPI6020 and S-800363. Young J. C. & Clark J. W. (1965) High temperature BOD's by electrolysis. Wat. Sewage Wrks. 112, 341-345. Young J. C.. Garner W. & Clark J. W. (1965) An improved apparatus for biochemical oxygen demand. Analyt. Chem. 37, 784. Young J. C. (1973) Chemical methods for nitrification control. J. Wat. PoUut. Control Fed. 33, 637-646.
THE ELECTROLYTIC RESPIROMETER--I FACTORS AFFECTING OXYGEN UPTAKE MEASUREMENTS JAMES C. YOUNG and E. ROBERTBAUMANN Department of Civil Engineering, Engineering Research Institute, Iowa State University, Ames, Iowa, U.S.A.
(Received 8 June 1976) Abstract--The electrolytic respirometer provides a more accurate and more complete measurement of biochemical oxygen demand (BOD) of wastewaters samples than normally is obtained by the standard dilution BOD method. Laboratory and field studies have shown that the electrolytic respirometer is a dependable instrument for making BOD measurements, is simple to operate, and provides a continuous readout of the oxygen uptake reaction. The major factors affecting oxygen uptake measurements with the electrolytic respirometer are the mixing rate and the concentration of oxygen demanding material in the sample. These system limitations can be overcome by using simple procedures such as increased mixing rate or enrichment of the oxygen in the air in contact with the sample. The precision of BOD measurement with the electrolytic respirometer was considerably greater than with the dilution method.
INTRODUCTION
The electrolytic respirometer was developed as a means of providing more accurate and more complete measurement of the biochemical oxygen demand (BOD) of wastewaters. Studies have shown that this system eliminates many technical problems encountered with other methods for determining oxygen demand and the rate at which it is exerted. Reviews of the design and application of electrolytic respirometers have been presented by Young, et al (1965), Montgomery et al. (1971) and Young and Baumann (1972). The purpose of Part 1 of this paper is to report the results of a comprehensive study designed to help determine the limitations of the system when used for measuring BOD and oxygen uptake rates. It should be emphasized that the term BOD is used in a broad sense to refer to any exertion or measurement of oxygen uptake by microorganisms in aqueous suspension.
lysis BOD measuring system is actually a large volume respirometer which provides semi-continuous and automatic registering and adjustment of the pressure change that is brought about within the reaction vessel because of oxygen consumption by microorganisms. OXYGEN ELECTRODE SWITCH
~
ELECTROLYSIS CELL
J-
ADAPTOR - CO2 ABSORBENT CONTAINER
%2
Description of the electrblytic respirometer
SOLUTION
The electrolytic respirometer consists of three major parts. A reaction vessel contains the sample and a magnetic stirring bar or other suitable mixing device (Fig. 1). An adaptor unit or alkali container holds potassium hydroxide or other basic solution to absorb metabolically produced carbon dioxide from the atmosphere above the sample. The critical part of the system is the electrolysis cell which contains a weak electrolyte such as sulfuric acid or sodium hydroxide. The electrolysis cell serves as a manometer to detect pressure changes and as an oxygen generator to maintain a constant partial pressure in the atmosphere within the sample container. The electrow.g. 10/11--~
HYDROGEN ELECTRODE
T
SAMPLE
I
~
STIRRINGMAGNET
Fig. 1. Schematic diagram showing the basic operation of the electrolytic respirometer. 1031
1032
JAMES C. YOUNG and E. ROBERT BAUMANN
Fig. 2. An operational 5-unit electrolytic respirometer as used in a constant temperature room.
Factors affectin 9 oxygen uptake in the electrolytic respirometer Factors which might affect the performance of the electrolytic respirometer in providing an accurate measure of oxygen requirements in any given sample include: (1) Capability of the system to transfer oxygen into solution across the air-liquid interface, as related to the oxygen production limits of the system, the mixing rate, the dissolved oxygen concentration in the sample, a n d the composition of gas above the sample; (2) substrate concentration and nutrient deficiency; (3) dilution; (4) buffer concentration; (5) sample size; and (6) size and nature of the inoculum. To understand the electrolytic respirometer more completely, laboratory studies were conducted to establish qualitatively a n d quantitatively how these factors influence oxygen uptake measurements. A more detailed description of those studies is given by Young and B a u m a n n (1972). EXPERIMENTAL STUDIES Laboratory studies were conducted using 5 or 6-unit electrolytic respirometer systems similar to that shown in Fig. 2. Test temperature was maintained at 20°C by keeping the units in a constant temperature room. The initial
temperature of the samples was adjusted to 20 _+ 0.5"C to minimize the effect of pressure changes within the vessel that otherwise would have occurred as a result of the sample temperature adjusting to the test temperature. A synthetic waste was used in most laboratory studies to provide a material which was consistent in composition from test to test. The synthetic substrate normally used was powdered Metrecal, a dietary food supplement manufactured by Mead Johnson & Co., Evansville, Indiana. A stock synthetic waste concentrate was made up for each test by diluting 8.50 g of powdered Metrecal to one liter to give a solution having 10,000mg/l ultimate BOD (BODL). Dilution was used to reduce this solution to the desired sample strength. A phosphate buffer which proved satisfactory for use with this synthetic waste was made by neutralizing 213.0 g/l of NaH2PO4' H20 (1.5 M) to pH 7.2 with potassium hydroxide. One milliliter of this solution per 50 mg/1 BODL maintained the sample pH between 6.7 and 7.3. Settled raw sewage or primary effluent from the Ames, Iowa, water pollution control plant was used as a seed source. Seed BOD was determined in each series of tests so that a correction could be made to reduce the measured BOD to the BOD of the synthetic substrate. Data were processed by computer to make seed corrections, to calculate a first-order BOD curve (including adjustments for lag time), and to plot each measured and calculated BOD curve for visual comparisons. A chemical nitrification inhibitor, 2 chloro-6-(trichloromethyl)pyridine (TCMP) (Hath Chemical Co., Ames, Iowa), was added to each sample in which inhibition of nitrification was desired I-Young (1973)].
The electrolytic respirometer--I
1000 mg/I BOD L O ½ - INCH VORTEX 1 - INCH VORTEX • 3 - INCH VORTEX • 5 - INCH VORTEX
E
d
o
ok~l~ v 0
--
1033
_ _
__aj
I
I
t
I
I
I
1
2
3
4
5
6
TIME, days
1200 1200 mg/I BODL
E
8
13" IINN(~HVO~TT~..~
800 --
4OO
__
• 5 - INCH VORTEX
_
0 1
_ ~
,
~ ~
/
AIR ABOVE SAMPLE
I
l
I
I
i
i
2
3
4
5
6
7
TIME; days
Fig. 3. Results of mixing-rate tests using air and oxygen-enriched air above the sample. RESULTS
Evaluation of factors affecting oxygen transfer Oxygen production rate. The equipment used in this study could supply oxygen at a rate of 42 mg/h, a rate far above any actually measured. Mixing rate. Essentially no difference in BOD curves (accumulated oxygen uptake versus time) was observed at vortex depths ranging from 1/2 to 5 in. (1.3 to 13 cm) with samples containing up to 500 mg/1 BODE. With BOD E concentrations ranging from 600 to 1000 rag/l, the rate of oxygen uptake decreased at the 1/2-in. (1.3 cm) vortex depth because of oxygen transfer rate limitations (Fig. 3). A vortex depth of about 3 in. (7.6 on) depth subsequently was used as a reference point for setting the stirring rate in any unit. Increasing the percentage of oxygen in the air above the sample to about 60% extended the range of waste concentrations over which BOD measurements could be made without encountering oxygen transfer limitations to at least 1,200 mg/l BODE (Fig. 3). This was accomplished easily by simply flushing the air space above the sample and in the electrolysis cell with pure oxygen just before assembling the unit. A 15-min adjustment period with the sample being stirred and with oxygen being produced in the electrolysis cell permitted an equilibrium to be established between the oxygen-enriched atmosphere and the dissolved oxygen concentration in the sample.
It should be emphasized here that the lag in oxygen uptake in the curves in Fig. 3 and subsequent figures is related to the amount of seed microorganisms added to each synthetic waste sample. This amount varied from test to test because of uncontrollable differences in seed samples, but in all cases was the same for replicate runs made on a given test date.
10.0
--
5.0
_
\ / I \ ~ ,.~ & \ ~
/
~
oo
I
~
800 -- BOD CURVE 110~0mg/I BOD| 750 mg/I BOD~"
500m~A Boo~
4 o6
0
100o.~ Boo, 750mg4 Boo, 5OOm~, ~
/~
I o t~
I
I
•
3__
•
•
0
i
o
~
-I
200 0
~"
10
20
30
40
50
60
70
80
TIME, hours
Fig. 4. Dissolved oxygen concentrations in the sample and associated BOD curves for four concentrations of synthetic waste.
1034
JAMESC. YOUNGand E. ROBERTBAUMANN
Dissolved oxygen in the sample. Measurements of dissolved oxygen(DO) concentration in the samples were made in a number of tests using the miniature Winkler method outlined in Standard Methods (1971). Separate 25-ml sample aliquots were transferred from reaction vessels equipped with a sampling port above the solution and adjustments in BOD were made for the volume of sample removed. The dissolved oxygen varied with time as shown in Fig. 4 when air was the gas above the sample. These results indicate that oxygen transfer was not a limiting factor for waste strengths less than about 750 mg/1 BODL when using air above the sample and when adequate stirring was provided. This corresponds reasonably well with the results of the stirring rate studies. The exact dissolved oxygen concentration below which oxygen becomes limiting is not known. Kalinske (1971) concluded from a survey of published work that DO concentrations from 0.5 to 35 mg/1 do not affect respiration rates as long as the cells remain dispersed. Samples mixed at lower rates may require higher minimum DO concentrations since flocculation occurs more readily than with rapid mixing. More accurate oxygen uptake measurements can be obtained with respirometers if the raw data are corrected for the rate of change in dissolved oxygen within the sample. Up to the point of minimum DO, the rate of change in DO should be added to the measured rate of oxygen uptake since this represents an amount of oxygen used but not measured. Similarly, the rate of increase in dissolved oxygen after the point of minimum DO should be subtracted from the measured rate of oxygen uptake. The net result of this correction is to introduce a plateau. With the quantities of oxygen transferred in the electrolytic respirometer this correction was a small part of the oxygen uptake reaction at all BOD concentrations. It should be emphasized that the need for this correction is the same with all respirometric and mano-
O & • •
100m
metric methods for measuring BOD. The maximum rate of depletion of the dissolved oxygen in the tests reported in Fig. 4 was about 0.7mg/I/h for the 500 mg/l BODL solution, as compared to a maximum oxygen uptake rate of 8 mg/l/h. Oxygen depletion curves reported for BOD measurements in Warburg respirometers for solutions having 300 mg/l BODL indicated a maximum oxygen uptake rate of about 5 mg/1/h of which 0.5 mg/l/h was attributable to removal of oxygen from solution [Bryant et al. (1967)]. The electrolytic and Warburg respirometers then appear to have similar oxygen transfer characteristics. The sample strength at which oxygen transfer limitations would exist would be increased considerably when using an oxygen-enriched atmosphere.
Gas composition above the sample The composition of the gas above the sample should remain about the same throughout a test period, otherwise there will be an accumulative error in the BOD measurement equal to the change in mass of oxygen. To determine whether changes occurred, the oxygen, nitrogen, and carbon dioxide in the gaseous atmosphere above the sample were measured periodically throughout the testing period when using samples of synthetic waste of various strengths. There was little change in gas composition for up to 13 days of testing for waste strengths up to 1000mg/l BODL when using air above the sample (Figs. 5 and 6). However. when the amount of oxygen in the atmosphere above the sample was increased to about 80% at the beginning of the test, the oxygen content gradually decreased, and the nitrogen content increased with the higher strength samples. With the 1000 mg/1 BODL samples (Fig. 5), much of the decrease in oxygen appeared to be a result of carbon dioxide accumulation in the system. No appreciable carbon dioxide accumulation occurred with the 600 mg/I BODt. samples (Fig. 6), but the percentage
GAS ANALYSIS BOD, % OF BODL(I,000 rag/l) OXYGEN, % NITROGEN, % CARBON DIOXIDE, %
d O m
80,
60
--
2 u
40
20,~ _
_
_ -
1
2
3
4
5
"0
I
2
3
4
5
TIME, clays
Fig. 5. Analysis of gas composition above 1000mg/1 BODL synthetic waste sample with (right) and without (left) oxygen enrichment. Carbon dioxide accumulation accounted for much of the decrease in oxygen content.
The electrolytic respirometer--I
1035
GAS ANALYSIS
I
o e~oo, % OF SOOL(600 ,~A)
Z
8
0 .
.
0
.
.
.
3
.
.
.
6
9
.
12
15
0
3
6
9
12
15
TIME, days
Fig. 6. Analysis of gas composition above 600 mg/1 BOD L synthetic waste sample with (right) and without (left) oxygen enrichment. The oxygen content in the oxygen-enriched sample decreased gradually over the 14-day test period. of gaseous oxygen decreased and the percentage of gaseous nitrogen increased. This pattern of changes in gas composition was more pronounced with the higher strength wastes. The magnitude of the change when starting with 60-80% oxygen above samples with 600-1000mg/1 BODL was about 5% in 5 days. This would be a significant error with low BOD samples, but oxygen enrichment is not needed for samples containing less than about 600 mg/1 BODL. The error would be less than 1% for samples having BOD's in excess of 1000 mg/1 and would be insignificant in measurements made for treatment plant control purposes. The cause of this decrease in oxygen content is not clear. Diffusion of oxygen through the electrolysis cell was ruled out since a corresponding diffusion of hydrogen into the unit through the cell would have been expected, and no detectable amount of hydrogen was observed in any gas sample analyzed. Also, similar diffusion would have occurred in low-strength samples and no such problem was observed.
Substrate concentration and nutrient deficiency When the concentration of substrate is high, as it may be in respirometers, the substrate or waste material itself may become a limiting nutrient, and waste utilization will reach a maximum value dependent only on the mass of microorganisms. This effect is demonstrated in Fig. 7 which shows the results of a series of dilutions in which all components of the sample, including the seed, were diluted equally. An oxygen enriched atmosphere (80~o+) was used to eliminate oxygen transfer problems. At BODL concentrations greater than about 1000 rag/l, the oxygen uptake rate remained essentially constant. Thimann (1963) has reported that with substrates such as glucose at concentrations above 40 mg/l (as glucose), the rate of substrate removal per unit of microorganism mass is constant. Buffer concentrations At the synthetic waste concentrations of 500 mg/l BODL, the shape of the BOD curve, the lag time,
SYNTHETIC WASTE
1200, -~E
100 mg/I 300 rag,/1 500 mg/l t000 mg/I 1500 mg/I
1
O ~ • • O
A~.41~ .~1~ I~r"
2
3
4
5
6
TIME, days
Fig. 7. B O D curves for low- and high-strength synthetic wastes show that the oxygen uptake rate
became essentially constani at a BOD concentration above 1000 rag/1.
1036
JAMES C. YOUNG a n d E. ROBERT BAUMANN
800 - -
AIR
400 k~ d
o
1
___L .............
J
I
_ J ......
k ......
OXYGEN-ENRICHED AIR
1
0 0
2
4
6
8
I0
12
14
TIME, cloys
Fig. 8. BOD curves using 500 mg/l BODL synthetic waste samples containing phosphate buffer concentrations ranging from 0.004 M to 0.02 M. and the BOD up to 14 days were not affected significantly by buffer concentrations ranging from about 0.004 M-O.02 M (Fig. 8). Oxygen enrichment did not affect the BOD curve at any buffer concentration. Similar results were observed using synthetic waste samples having BODL concentrations below 500 mg/1. Oxygen transfer rates became limiting when air was used with 750 mg/1 BODL samples (Fig. 9). The variation between samples was reduced considerably when using an oxygen-enriched atmosphere. Similar results were obtained with higher-strength synthetic waste samples. In all cases, there was no apparent relationship between the shape of the curve and the buffer concentration. That is, in one test the lower oxygen uptake rate might be associated with the lower buffer concentration while in a similar test the lower oxygen uptake rate was associated with the higher buffer concentration.
The recommended amount of buffer solution listed previously in this paper was satisfactory when measuring the BOD of synthetic wastes having a predictable BODL, or a standard buffer concentration of 0.02 M could have been used with all waste samples tested. However, with samples containing significant amounts of calcium or magnesium salts, it may not be possible to use a phosphate buffer; and more suitable buffers may have to be developed or the sample may have to be diluted. Fortunately, with domestic wastewaters, the natural buffering capacity generally is sufficient to control pH, and the addition of a buffer is not required. Sample size and barometric pressure The size of the sample used in the electrolytic respirometer normally should be sufficient to fill the reaction bottles as full as possible without causing
800 L
AIR
o 0
1
2
3
4
5
t
I
6
7
TIME, doys
Fig. 9. BOD curves using 750 mg/1 BODL synthetic waste samples containing phosphate buffer concentrations ranging from 0.006 M to 0.03 M.
The electrolytic respirometer--I
tion. This is demonstrated by results obtained when measuring the BOD of a 500 mg/1 BODL Metrecal solution (Fig. 10). With smaller sample volumes, or when measuring the BOD of such low-strength samples as secondary treatment plant effluent or stream or lake water, the sample should essentially fill the reaction vessel. Otherwise, corrections should be made.
the sample to overflow into the potassium hydroxide container at nominal-to-high stirring rates. As the sample volume is reduced, the error in the BOD measurements may increase because of the larger effect of barometric pressure changes. When the barometric pressure rises, the pressure inside the reaction vessel must also increase. This can only be accomplished by adding oxygen to the system. For barometric pressure increases, this addition is automatic and an equivalent amount of oxygen must be subtracted from the indicated demand to correct the error. When the barometric pressure decreases, the switching mechanism of the open-manometer electrolysis cell will not operate unless there is a BOD exerted which is at least equal to the pressure error. The correction for a barometric pressure decrease is then positive, correcting the BOD exertion which would have been indicated had the barometric pressure remained constant. The correction of barometric pressure changes is given by the following equation: Wo~ = 1.31Vo(AP)
Accuracy and precision Regardless of the method used for measuring BOD, the accuracy and precision of the measurement must be defined within acceptable limits. As yet, no standard exists by which to determine the accuracy of the BOD test; and the precision, or the ability to obtain reproducible test results, normally is emphasized. To obtain an indication of the precision possible with the electrolytic respirometer, analysis-of-variance tests were conducted using data from runs having adequate mixing or oxygen enrichment containing four' or more replicates. These analyses, summarized in Table 1, indicated that with a given sample strength the standard deviation did not change appreciably for any testing period of two days or more when sample BODL concentrations were below 750 mg/1 or when pure oxygen was added to the atmosphere above the sample. When oxygen transfer limitations became evident, as with the 750mg/1 BODL sample to which no oxygen was added initially to the atmosphere above the sample, the standard deviation and associated coefficient of variability (CV = standard deviation, S.D., as a percentage of the average) increased for the shorter test periods. In every case but two, the coefficient of variability of the 5-day BOD was less than or equal to 3.2~o. For samples with BODL values below 250 mg/1, a coefficient of variability of less than about 1~o was obtained after 5 days of testing. After two to three days of testing, the coefficient of variability in all test results
(3)
where Wo2 = oxygen required to correct the measured BOD, mg 02 V0 = volume of air space above sample, liters, and AP = barometric pressure change, millibars. For a 75-ml air volume above the sample, the correction is 0.98mg of 02 for a 10mb pressure change, which is a typical change occurring in any one week. The correction is not accumulative and is determined by the same function regardless of the time increment over which AP is measured. Thus, for daily BOD values, a correction needs to be made only to the daily accumulated BOD and not to the hourly subtotals. In most cases, the sample volumes greater than 500 ml will not require a barometric pressure correc-
SYNTHETICWASTE o 500 rag/l, 950ml 500 mg/I, 700ml • 500 rag/I, 500 ml BODL, volume
..tS o
o
I
]
I
I
I
l
I
400 - -
2OO
0
1037
I
l
I
SY/~n'METICWASTE o SO0 mg/'l, 950 ml ~ 500 ms~1, 750 rnl
f
• 500 rag/I, 500 ml BODL, voluml #
I
I
I
I
L
I
I
I
I
I
I
2
3
4
5
6
7
8
9
10
11
TIME, days
Fig. 10. BOD curves show that, when various volumes of sample in a 11. test bottle are used, the same results can be obtained using sample .volumes as low as 500 ml.
1038
JAMES C. YOUNG and E. ROBERT BAUMANN Table 1. Variability analyses for measured BOD of synthetic waste (Metreca1) samples. Day of test Sample
i
2
3
4
5
6
125 mg/1 BOD L n - 4 (5-25-71)
X* SD CV
25.50 1.23 4.82
62.3 1.44 2.32
80.7 1.56 1.93
90.8 i.ii 1.22
96.4 0.85 0.88
. . . . . . . . . . . .
250 m g / l BOD Air n ~ 4 L (5-25-71)
X SD CV
47.4 1.06 2.24
134,3 4.58 3.40
170.0 2.36 1.39
188.8 0.48 0.26
201.4 1.41 0.70
. . . . . . . . . . . .
250 m g / l BOD L 02 n " 4 (5-25-71)
X SD CV
44.5
125.7 3.02 2.40
162.2 2.11 1.30
188.8 0.43 0.23
199.4 1.54 0.77
. . . . . . . . . . . .
500 mg/l BOD Air n ~ 8 L (5-14-71)
X SD CV
101.5 17.2 16.9
264.1 13.5 5.1
331.1 16.4 4.9
373.9
500 m g / l BOD L O9 n ffi 7 (~-14-71)
~ SD CV
134.8 9.7 7.2
294.5 8.2 2.8
351.4 11.9 3.4
750 mE/1 BOD Air n = 7 L (4-24-71)
X SD CV
162.9 46.2 28.3
339.0 59.2 17.5
750 m8/l BOD L O9 n = 7 (~ -24-71)
X SD CV
265.5 21.2 8.0
1,000 m8/l BOD I00 m l seed L Air n = 8 (4-30-71)
X SD CV
1,000 mg/l BOD L I00 m l seed 02 n ffi 8 (4-30-71)
2.56 5.63
7+
391.9 11.8 3.0
410.8 12.2 3.0
428.4 11.9 2.8
379.0 9.9 2.6
396.4 8.7 2.2
413.9 10.4 2.5
429.7 14.3 3.3
468.2 54.0 11.5
524.6 47.7 9.1
592.7 13.6 2.3
. . . . . . . . . . . .
459.7 21.1 4.6
522.3 12.9 2.5
556.1 Ii.0 2.0
583.6 11.7 2.0
. . . . . . . . . . . .
235.9 50.5 21.4
493.6 55.1 11.2
658.8 94.4 14.3
733.9 20.1 2.7
783.8 10.7 1.4
809.8 17.0 2.1
----
X SD CV
340.9 33.9 I0.0
594.4 26.7 4.5
679.1 30. i 4.4
728.1 29.3 4.0
760.6 24.6 3.2
796,5 18.1 2.3
----
20 m 8 / l BOD L (12-14-68) n - 4
X SD CV
4.2 1.6 37.5
7.8 1.5 19.3
9.6 1.3 13.1
10.8 0.4 3.9
11.8 0.4 3.5
----
16,7 0.6 3.9
20 mg/l BOD L (1-8-69) n = 4
X SD CV
8.9 0.3 3.4
14.9 0.7 4.8
17.9 0.5 2.8
18.1 0.7 3.7
20.4 0.5 2.3
----
20.8 0.5 2.2
20 m 8 / l BOD L (2%) (4-24-69) n = 4
X SD CV
7.5 0.4 5.2
12.2 1.2 10.2
14.9 1.9 12.7
16.9 0.6 3.7
17.5 0.5 3.0
. . . . . . . . . . . .
20 m 8 / l BOD L (I£) (4-24-69) n = 4
X SD CV
7.8 0.05 0.6
12.1 0. ii 0.9
14.7 0.03 0.2
17.7 0.3 1.5
18.3 0.4 2.4
. . . . . . . . . . . .
5 mg/l BOD L (21) (4-29-69) n ffi4
X SD CV
2.6 0.5 19.0
3.2 0.4 12.8
4.1 0.4 9.9
4.8 0.5 10.4
5.0 0,3 5.7
. . . . . . . . . . . .
5 mg/l BOD L (i~) (4-29-69) n ~ 4
X SD CV
1.6 0.2 15.2
2.2 0.2 8.1
4.0 0.3 6.4
4.6 0.2 4.8
4.8 0.5 I0.I
. . . . . . . . . . . .
13.2 3.5
*
* X - average BOD, mg/l; SD = standard deviation, mg/l; CV = coefficient of variability, and n = number of samples.
was less than 3.5% for samples having BODL values below 500 mg/1. Initial electrolytic respirometer results were not encouraging when measuring the BOD of samples containing less than about 20rag/1 BODL until the sensitivity of the cell was recognized as a mechanical source of error in the system. The sensitivity could be increased by using a plexiglass tubing insert in the outer chamber of the electrolysis cell I-Young and Baumann (1972)]. This insert reduced the electrolyte volume change required to activate the power supply so that oxygen was added in smaller increments. The resulting precision with which low-level BOD determinations could be made was quite encouraging, with coefficients of variability of about 2 3% for BODL concentrations in the 1(~20 mg/l range (Table 1). For lower strength samples, the coefficient of variability increased but seldom was greater than 10%.
%;
SUMMARY AND DISCUSSION
As shown in Figs. 3 7. a high substrate concentration and a low oxygen content in the atmosphere within the sample container all caused variation between BOD curves even when mixing was adequate. The overall effect of these factors is summarized in Fig. 11. Here the maximum oxygen uptake rate, as measured by the maximum slope of measured BOD curves, is plotted against the BODL of each sample. Seed corrections, although a small fraction of the oxygen uptake at any one time, were made for all samples. These data show that the maximum rate of oxygen uptake increased somewhat linearly with increased BODL but that the scatter of data increased greatly with samples containing more than about 750 mg/1 BODL. Increasing the concentration of oxygen to
The electrolytic respirometer--I about 80~o in the air above the sample increased the oxygen transfer capability of the system to about 20mg/1/h at a BODL of 1200mg/1 from a rate of 12mg/l/h at a BODE of about 750mg/1. These numbers agree with those presented previously in connection with stirring rate studies. At BODE concentrations above about 1000 mg/1, substrate concentration no doubt was a factor in limiting the oxygen transfer rate whether or not the air above the sample was enriched with oxygen. The oxygen transfer limit of the electrolytic respirometer should not necessarily be considered in terms of the BOD s or BODE concentration beyond which oxygen transfer rate limitations occur. A more appropriate measure of the system capacity is the maximum rate of oxygen uptake which can be measured before oxygen transfer or substrate concentration begins to limit the oxygen uptake rates. For example, municipal wastewater having an oxygen uptake rate of 10 mg/1/h might have a BOD E twice that of an industrial wasteL water or synthetic wastes having the same uptake r a t e but containing a high percentage of soluble organic material which is rapidly synthesized and oxidized by bacterial cells. If high oxygen uptake rates are anticipated, as with high-strength wastes or mixed-liquor samples from activated sludge aeration tanks, the oxygen content of the air above the sample can be increased so that higher oxygen uptake rates can be measured accurately; or, the sample may be diluted so that t h e maximum oxygen uptake rate is below the system limit. Some electrolytic respirometer designs may provide higher oxygen transfer capabilities by providing more vigorous mixing, but this would tend to increase the complexity of the equipment design and operation [Montgomery et al., (1971)]. The significance of the above observation depends on application of the data. If kinetic studies are being made to determine substrate uptake characteristics of
20 - •
i
1039
microorganisms, then there is merit to tests conducted using full-strength wastes. If the objective of the test is to provide an estimate of total biodegradable material, then sample adjustments must be made so that oxygen transfer or substrate concentration limitations do not occur. If a BOD is being measured for waste treatment plant purposes, the test should be conducted at a waste concentration about the same as that occurring in the treatment process or stream receiving the wastewater. CONCLUSIONS
Laboratory studies have shown that the electrolytic respirometer is a dependable instrument for making BOD measurements. The system is simple to operate and provides a continuous readout of oxygen uptake. The major factor contributing to errors in oxygen uptake measurements are the mixing rate and the concentration of oxygen demanding organic material in the sample. Oxygen transfer limitations can be overcome by increased mixing or by enriching the air in contact with the sample with oxygen. The effect of high substrate concentration can be eliminated by diluting the sample. In the open manometer cell design described in this paper, a change in barometric pressure will change the volume of gas within the reaction vessel and cause more or less oxygen to be added than demanded by the sample. Although the effect of such barometric pressure changes is usually small, corrections can be made easily. Analyses of variance tests of replicate BOD tests indicated that, after two or three days of testing, the coefficient of variability was less than 3.5~ for samples having BODE values between 20 and 500 mg/1. Acknowledgements--This research work was supported in part by Federal Environmental Protection Agency
OXYGENUPTAKERATE o OXYGENADDED • NO OXYGENADDED '
15
•
10 5--
o
~
200
o
400
i
600 800 BODL, mg,/I
1000
1200
I
1400
Fig. 11. Oxygen uptake rate as a function of the substrate concentration increased until the substrate concentration was about 1000 mg/1 BODL. After this, oxygen uptake was variable.
1040
JAMES C. YOUNG and E. ROBERT BAUMANN
demonstration grant $800363 (formerly WP 16020 DUN) and the Engineering Research Institute of Iowa State University. Robert C. Kroner, chief (retired) of the Physical and Chemical Methods Section, EPA, Analytical Quality Control Laboratory, was project officer. Donald A. Haselhof, a graduate student in Sanitary Engineering at Iowa State University during the period of this study, helped with the data collection and analysis, part of which was included in his Master of Science thesis.
REFERENCES
Bryant J. O., Akers W. W. & Busch A. W. (1967) Limitations of oxygen transfer in the Warburg apparatus. Proc. Ind. Waste Conf. 22nd, Purdue University Extensions Series 131, pp. 68~698. Kalinske A. F. (1971) Effect of dissolved oxygen and substrate concentration on the uptake of microbial suspensions. J. War. Pollut. Control Fed. 41, 73-80. Montgomery H. A. C., Oaten A. B. & Gardiner D. K.
(1971) An automatic respirometer Its construction and use. Effluent and War. Treat. J. 11, 23 31. Simpson J. R. and Nellist G. R. (1970) Development and use of a large-volume automatic respirometer. War. Pollut. Control 59(~605. Standard Methods for the Examination o] Water and Wastewater (1971) 13th Ed. American Public Health Association, New York, N.Y. Thimann K. V. (1963) The Lift, of Bacteria. p. 633. MacMillan, New York. Young J. C. & Baumann E. R. (1972) Demonstration of the electrolysis method for measuring biochemical oxygen demand. Enq. Res. Inst. Rep. No. 72153, Iowa State University and EPA Final Report for projects WPI6020 and S-800363. Young J. C. & Clark J. W. (1965) High temperature BOD's by electrolysis. Wat. Sewage Wrks. 112, 341-345. Young J. C.. Garner W. & Clark J. W. (1965) An improved apparatus for biochemical oxygen demand. Analyt. Chem. 37, 784. Young J. C. (1973) Chemical methods for nitrification control. J. Wat. PoUut. Control Fed. 33, 637-646.