Aerobic Decomposition of Algae William J. Jewelll and P e r r y L. M c C a r t y Stanford University, Stanford, Calif. 94305
The rate and extent of decomposition of both axenic algal cultures and mixed cultures of algae, bacteria, and zooplankton are described. Culture ages ranged from a few days to seven months, and the growth media from a synthetic freshwater to seawater. Algae and algal-related organic matter were composed of three fractions: a small fraction which respires within a few hours in the dark, a large fraction which decomposes slowly within a year, and a large refractory fraction which decomposes only a few percent per year. The refractory fraction varied from 12 to 87% (mean, 40%) with the higher values obtained for very young or very old cultures. The decomposition of the biodegradable portion of mixed cultures grown in the presence of bacteria and zooplankton followed first-order kinetics. The decay rate was a function of culture age and was higher for young cultures ( K ’ = 0.01 to 0.06 day-l) than for old cultures (0.01 to 0.03 day-’). The decomposition of pure cultures followed second-order kinetics during initial stages of decomposition, but subsequent decomposition could be described well with the first-order kinetics.
T
he increased rate of eutrophication of waters caused by man’s activities is a well-known problem. While a great deal of research has been devoted to determining the cause and extent of algal growth, relatively little has been conducted to determine the effect of subsequent algal decomposition on water quality. This paper presents a summary of some of the results of a study of the effects of aerobic algal decomposition on the oxygen, nitrogen, and phosphorus budgets of water (Jewell, 1968). The general objectives were to determine the rate and extent of decomposition of algae in terms of oxygen equivalents, and to formulate the results into mathematical expressions to allow better prediction of the kinetics of long-term algal decomposition.
Extent oj Algal Deconiposition The extent to which a given mass of algae will be biologically decomposed and the factors affecting it are of concern when evaluating the effect of algae on the oxygen budget of natural waters. There are some indications that algae are 100 biodegradable (Jannasch, 1964; Kuznetsov, 1968; Moss, 1968; Strickland, 1965). The experimental evidence for this has been obtained primarily from field studies. Moss (1968) reported that the annual oxygen deficit occurring in the hypolimnion of a pond approximately equaled that required to oxidize completely the annual contribution of phytoplankton material to the sediment. Others have indicated that a certain portion of algae is not readily susceptible to biological decomposition (McKinney, Present address: Dept. of Civil Engineering, University of Vermont, Burlington, Vt. 05401. To whom correspondence should be addressed.
1965; O’Connel and Thomas, 1965). Golueke and Oswald (1965), Golueke et al. (1957), Kleerekoper (1953), and Skopintzen and Broock (1940) have presented evidence indicating the presence of a nonbiodegradable portion varying between 33 and 76 % of the initially present organic material. The one view that algae are completely degradable and the other that a certain portion is resistant to biodegradation or “refractory” seem contradictory. However, degradation in lakes and oceans with residence times measured in decades and centuries cannot be equated with degradation occurring in laboratory vessels on time periods of a year or less. The purpose of this investigation was to obtain evidence not only on the extent of degradation of algae, but also on the rate at which degradation occurs in an attempt to put these different views in better perspective. Rate of Algal Decomposition. The rate at which algal material decomposes can be determined by measuring the use of oxygen during decomposition, with the results expressed as a first-order coefficient (Kbasee = milligrams of 0 2 used per milligram suspended solids per day). Such reported values for algae have generally varied between 0.01 to 0.08 day-l (Fitzgerald, 1964; Allen et al., 1964; Skopintzen and Broock, 1940; Varma and DiGiano, 1968; Wisniewski, 1958), including both oxygen usage by the decomposers which consume the algae as well as the respiration of the algae themselves. The above results are from measurements taken over a period of days. However, some reported respiration rates for axenic algal cultures have been based on measurements over a period of only minutes or hours. When grown with excess inorganic nutrients, axenic algal cultures often have initial oxygen utilization four to five times greater than the values for decomposers and algae indicated above (Allen et al., 1964; Cramer and Myers, 1949; Fitzgerald, 1964; Gibbs, 1962). After several hours in the dark, the initial high oxygen usage rates decrease to between 0.01 and 0.08 day-’. If the assumed initial respiration rate is 0.17 mg O$mg ss,lday (or 17% per day) for 10 hr and drops to 0.05 for five more days, the total quantity of cell material utilized represents 26 % of the total cell (assuming 1.42 mg COD equals 1 mg organic algal material). Thus, endogenous respiration does not account for the disappearance of more than 20 to 30% of the total cellular material of most algae. The information presently available emphasizes that the rate and extent of algal decomposition are only vaguely defined. The total quantity of oxygen required during aerobic decomposition of algae and the quantity of nutrients that can be regenerated will be reduced by the algal material that resists decomposition-i.e., the refractory material. Thus, the refractory fraction of algal-related organic material is a major factor influencing the effect of algal decomposition on water quality. Clarification of algal decay kinetics will provide information necessary to understand and avoid such serious consequences of algal decomposition as fish kills which result from deoxygenation of waters (Mackenthun et al., 1945). Volume 5, Number 10, October 1971
1023
Formulation of Decomposition Kinetics When algal material is first produced by photosynthesis, the organic portion is assumed to be composed of two fractions, a biodegradable fraction and a refractory fraction. The refractory organic material is defined as that algal-related material not biologically decomposed within a year by mixed cultures of microorganisms operating under noninhibitory conditions. Noninhibitory refers to conditions where the availability of organic carbon is the sole decomposer growth rate-limiting factor. In natural waters the biodegradable fraction may be consumed either by the endogenous respiration of the algae themselves, or by decomposers (bacteria, fungi, and microscopic animals). During the growth and decay of the decomposers, another refractory organic fraction will be formed from the remains of the decomposers themselves. Thus, after decomposition of algae is complete, the remaining refractory material will be composed of the refractory fraction of the algal cells and of the decomposers. The organic material produced through the photosynthetic activity of' algae plus that which results from the decomposition of this material (whether by algae themselves or by decomposer bacteria and animals) will be referred to as algalrelated organic material. A mathematical description of the rate of decomposition of algae in natural waters should consider the interactions of all major populations ranging from the bacteria to the nekton. However, individual treatment of each decomposing population would lead to an impractical mathematical expression. The following simplified development is believed to be sufficient for describing algal decay by microorganisms as occurs under most natural conditions. The change in algal mass with time at a given temperature is considered to be a function of two main variables: the algal mass and the decomposer population. If we assume that there were no outside sources of organic matter, the mass of the decomposer population present at any time will be a function of the quantity of algal material which they have previously decomposed. The active mass of decomposers in a heterogeneous system is difficult to measure. However, the decomposers probably decompose in the same way as the algae. That is, the rate of change of organic matter will depend on its mass as well as the amount already decomposed. This may be expressed as follows :
Total biodegradable organic mass, organic mass decomposed -f
(
If we assume the rate of decomposition is directly proportional to the biodegradable mass present and to that previously decomposed, the general decomposition function for an algal system can be written :
_dM_ dt
-k"MbMd
=
-k"Mb(Mi
- kf)
(2)
where M is the total organic mass at any time t , M b is the biodegradable organic mass at any time t , Md is the mass previously decomposed, M I is some initial biodegradable organic mass, and K" is a second-order decay rate coefficient. It is hypothesized that natural algal decomposition may assume two forms. First, when rapid growth of algae occurs, a high concentration of algal cells may result, with few de1024 Environmental Science & Technology
composers present. After a period of time the decomposers reach a significant population and the algae are then rapidly degraded. The second form of decomposition occurs when algae grow more slowly along with a population of decomposers. This is perhaps the more common case in the natural environment. Subsequent decomposition of this mixed population represents a limiting case to the first. That is, it represents the decomposition occurring in the first case after the decomposer mass has reached a maximum. This maximum probably occurs relatively early in the decomposition process since the decomposer mass in such a system represents only between 0.1 to 1 % of the total (Jorgensen, 1966; Odum, 1957; Teal, 1957; Ward et al., 1964). The change in total organic mass after this point is reached will be a function, primarily, of the total biodegradable organic mass only. This case can be described by first-order kinetics:
(3) where k' is a first-order decay rate coefficient. To integrate Equations 2 and 3, certain boundary conditions need to be defined. The decomposers represent only a small fraction of the total mass ( M ) ,so that the change in the total refractory organic material can be assumed to be negligible throughout the decomposition process. Thus, the nonbiodegradable material can be taken to equal fM0, wheref is the refractory fraction of the total algal-related material ( M o )plus a certain quantity which is proportional to the mass of decomposers present at time zero. Integration of Equation 2 then yields the following: (4) where
In similar fashion, the integration of Equation 3 gives:
or
M
=
(Mo - JMo)e-"''
+ fMo
(5b)
The relationship between Equations 4 and 5a is illustrated by reference to Figure 1. Here, calculated curves for Equation 4 are illustrated in which M 1was allowed to vary between 2 and 30% of M o . When Ml equals 2% of M o , the typical second order, or autocatalytic decay curve results. However, when M I equals 30% of Mo, the curve has much the same appearance as a first-order curve calculated from Equation 5a. In addition, at this point the curve is not significantly affected by changes in the value of M I . Thus, when the decomposer mass becomes established, the more easily handled first-order equation should be sufficient for predictive purposes. An experimental study was conducted to verify the applicability of Equations 4, 5a, and 5b and to evaluate the range of values for the coefficients f , k ' , and k" under a variety of conditions.
Methods A 5-gal sample of each water listed in Table I was filtered (glass fiber, GFjC 4.52-cm Whatman) to remove most par-
silver catalyst and mercuric sulfate for chloride complexing (Amer. Public Health Ass., 1965). A 20-ml sample was used in all COD analyses. The reagents were diluted for determination of low values of COD, and a standard deviation of less than 10% of the average was obtained at COD values as low as 20 mg/liter. The particulate COD was obtained by adding the glass-fiber filter pad directly to 20 ml of distilled water in the COD flask. Little error was introduced by doing this since the COD of the pad itself was low and consistent, and was accounted for by use of a blank. This test gives a direct measurement of the theoretical oxygen required to oxidize organic matter to carbon dioxide, water, and ammonia. The analytical values are within 95 t o 100% of the theoretical values for most biologically produced materials (El-Dib and Ramadan, 1966; U. S. Public Health Service, 1965). The theoretical ratio of COD/ISS varies from 1.1 for simple carbohydrates t o 2.9 for saturated long-chain fatty acids, with a n average for heterogeneous biological material of about 1.4. Comparisons between these and measured values were checked at times during the study. However, because of the numerous analyses, the limited culture size, and the relatively low organic concentration, it was not possible to determine the ISS concentration on all samples. All results in this paper will be expressed on a TSS basis. In most cases the ISS was greater than 95 % of the TSS, thus making the above comparisons valid. Nitrate nitrogen was measured with a modified brucine procedure (Jenkins and Medsker, 1964), nitrite nitrogen by the a-naphthylamine sulfanilic acid test (Amer. Public Health Ass., 1965), and organic nitrogen by microkjeldahl (Kirk, 1950). Total, total filtrate, and orthophosphorus of the filtrate were measured by use of dry combustion for total phosphorus and molybdate-stannous chloride for color development (Lee et al., 1965).
1 10
20
30
40
50
60
TIME, D A Y S Figure 1. Effect of varying the initial decomposer mass from 2 to 30 % of total initial mass as described by Equation 4 Variables other than t and Mi were held constant to determine the effect of decomposers on the kinetics of decomposition
ticulate material. After filtration, those with a significant soluble organic or ammonia concentration were aerated for several days t o allow oxidation of all reduced materials which might mask the algal interactions of interest. Then, a small portion of the particulate material removed by filtration was returned t o the sample in a n attempt to ensure that the biota would be close t o that in the sample when collected. The total nitrogen and phosphorus concentrations in each sample were adjusted to approximately 5 and 0.5 mg/liter, respectively, by addition of nitrates and phosphates. After treatment of the water samples as above, the concentrations of other nitrogen and phosphorus forms were less than 0.4 and 0.05 mg/liter, respectively. Eighteen liters of each prepared sample were placed in a 20-liter growth vessel and exposed t o diurnal fluorescent light (12 hr on and 12 off, 3200 lx a t the sample surface), while aerated with a n air-COr (1 %) mixture. At predetermined time periods after noticeable algal growth had occurred, samples were taken from the growth vessels, placed in the dark in decomposition vessels (ranging in volume from 1 to 4 liters), and aerated continuously with a similar air-C02 mixture. This aeration mixture was sufficient to maintain the p H between 6.8 and 7.3 in all samples except in the growth vessels when the suspended solids exceeded 800 mg/liter, usually after 400 days of growth. Lighting, temperature, and p H were held constant during any particular experiment. All experiments were conducted in constant temperature rooms and mostly at 20°C, although some studies at 4", 25", and 35°C were also completed. The change in organic mass was evaluated through measurements of total suspended solids (TSS) and ignitable suspended solids (ISS). Samples were filtered through Whatman glassfiber filters, GF/C, 4.54-cm diameter, and dried at 103°C for 1 hr t o determine TSS. The ISS was found as the loss in weight by combustion at 570°C for 10 min of the particulate material on the dried filter (Wyckoff, 1964). Dissolved organic carbon was determined in the filtrate with a Beckman carbonaceous analyzer. The oxygen-consuming potential was measured with the dichromate chemical oxygen demand test (COD) by use of a
Results Heterogeneous Cultures. Six samples were used initially in
Table I. California Water Samples U s e d in Heterogeneous Algal Growth and Decomposition Studies
Identification no. 1
2 3 4 5
6 7 8 9 10 11
12
Sample source San Pablo Bay (San Francisco estuary) tidal Sacramento River (Carquinas Bridge) nontidal Sacramento River Searsville Lake (Stanford) activated sludge effluent (Stanford Univ. pilot plant) synthetic river water Lake Tahoe activated sludge effluent (similar to no. 5) Folsom reservoir Rogers Creek (Big Basin park) Clear Lake (Konocti Bay) dear unpolluted brook (N. Calif.)
Type water estuarine water tidal river water slightly polluted fresh river water eutrophic lake treated effluent
oligotrophic lake treated effluent man-made reservoir intermittent stream eutrophic lake mountain stream
Volume 5, Number 10, October 1971 1025
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SEARSVILLE
LAKE, 4
12
.e PHOSPHORUS &-j
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1
200
300
I
TIME, o m
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40 0
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50 0
1
I
‘0
600
Figure 2. Laboratory growth of heteogeneous algal cultures in eutrophic lake water The total organic matter was measured by the COD test, and characteristics of the particulate matter were measured by the organic nitrogen and total phosphorus analyses. Circled numbers refer to points where samples were taken from the growing cultures and placed in the dark to decompose (branch samples as in Table 111)
long-term growth experiments (Table I). Within 12 days a noticeable growth of algae occurred in all units. The initial phytoplankton species were the more delicate flagellated forms, diatoms and green algae. As these species reached their stationary phase, new species of green algae usually became dominant. During this shift in species, a color change from bright green to light green or a yellowish color and then back to a dark green was sometimes observed. This fluctuation in color occurred several times and corresponded with plateaus in the growth curve. After a nutrient deficient environment developed, blue-green algae formed the final dominant group. In most samples, nitrogen fixation occurred as evidenced by a significant increase in the total nitrogen concentration. Loss of nitrogen from the system probably occurred after 400 days of growth because of a rise in the p H at this time due t o high algal concentrations. All samples had different types of algae with a minimum of four dominant species appearing in any one sample. Algal culture “age” is defined as the days of culture growth after the first noticeable growth appeared. To determine the effects of culture age on the variables studied, six branch samples were taken at periods which were intended to correspond with different physiological conditions of the algae. These periods ranged from 8 to 211 days after the first growth appeared. Algal culture age is synonymous with cellular nutri-
ent content, and thus the chemical composition of young cultures differed from that of old cultures. The growth in Searsville Lake water, as measured by total COD,organic nitrogen, and organic phosphorus, is representative of all long-term growth studies (Figure 2). The chemical characteristics of the various branch cultures at the beginning of decomposition were determined by use of the ratios COD/ TSS, COD/N, and COD/P(Table I1 includes representative values). The ratio of COD/TSS varied between 1.1 and 1.6 I
I
1
1
I
Table 11. Characteristics of Heterogeneous Algal and Decomposer Mass Synthesized “in Vitro” in a Eutrophic L a k e Water (Searsville L a k e ) at the Beginning of the D a r k Decomposition Phases
Culture COD, mg/liter Samage, suspended ple Branch days Total Solids 4 1 12 32 21 90 ... 4 2 16 4 3 24 , , . 200 4 4 31 . 300 250 4 5 59 530 450 4 6 211 1250 1000
Characteristics of suspended solids COD/TSS
1026 Environmental Science & Technology
COD/N coD/P
21
35
.. . . . .
...
. . .
1.15 1.15 1.28 1.32
48 47 64 46
450 900 1800
. . .
0
100
200 TIME,
300
DAYS
Figure 3. Change of total organic matter during dark decomposition (at 20°C) of photosynthetically produced organic matter (Figure 2), as measured by the total COD The organic matter is composed of mixtures of algal species initially present in Searsville Lake water, bacteria, and zooplankton
(mean 1.4) for all samples but did not change significantly with age. This theoretical use of oxygen during complete oxidation of the algae is within the range expected. The branch samples were placed in total darkness at 20°C during decomposition. The decomposition of the various Searsville Lake branch samples is representative of data obtained in this series of experiments (Figure 3). Wide fluctuations in decomposition pattern were expected because of the variability of microorganisms and water type. However, a consistent pattern was usually observed, and the curves were similar to those of firstorder decay, as postulated. It is apparent from these results that algal-related organic matter decomposes slowly, and a significant fraction of the initial organic material remains in the system after decomposition has apparently ceased or has become too slow to measure. The refractory fraction (COD remaining after stabilization/ initial COD) varied from 19 to 86% (average 4 4 7 3 and the decomposition rates for the branch samples as determined by first-order decay Equation 5a varied from 0.010 to 0.15 day-I (Table 111). The decomposition rates were determined by plotting the left side of Equation 5a vs. time of decomposition.
_I
,
100
I
1
1
I
I
I
I
I
I
I
I
LLJO
00
A L Q A L SYSTEM A Q E .
DAY)
Figure 4. Change of refractory fraction of heterogeneous mixtures of algae, bacteria, and zooplankton (Table I) with culture age-i.e., days of growth Decomposition temperature was 20°C
The slope of this line equals - k ' , and the line of best fit was estimated visually. Although not expected, a relatively good correlation was found between culture age and refractory fraction (Figure 4) and between culture age and first-order decay coefficient (Figure 5). The relatively high refractory fraction found in
Table 111. Refractory Organic Fractions and Decomposition Rates for Various Culture Ages of Heterogeneous Mixtures of Algae and Decomposers
Sample
1 1 1 1 1 2 2 2 2 2 3 3 3 3 3 3 4 4 4 4 4 4 5 5 5
5 5 5 6 6 6 6 6 6
Branch
1 2 4 5
6 1 2 4 5
6 1 2 3 4 5
6 1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6
Sample Number Refers to Identification Number (Table I) Period of Total COD, mg/liter Culture decornp., days Initial Final age, days
9 16 27 54 207 14 19 32 59 21 1 16 18 20 24 59 21 1 12 16 21 24 59 21 1 12 16 21 24 59 21 1 16 18 21 24 59 21 1
312 313 366 323 295 317 320 365 322 230 315 321 341 365 322 295 319 313 340 365 322 295 319 323 340 365 330 295 315 321 340 365 322 295
40 108 267 518 749 62 80 191 358 445 33 41 70 102 254 476 32 90 185 301 534 1250 48 22 5 250 286 259 1260 37 55 110 122 222 642
28 43 90 200 420 25 39 93 123 330 20 25 18 71 86 200 27 25 92 68 184 555
18 50 50 60 104 800 12 18 38 60 100 410
Refractory fraction, 1OO.f
70 40 34 38 56 40 48 49 34 74 60 61 26 70 34 42 86 27 50 23 34 44 37 22 19 21 40 64 33 34 35 49 45 64 Av. = 44
Decomposition rate ( k ' ) , day-1
0 100 0 034 0 019 0 010 0 011 0 150 0 034 0 010 0 010 0 025 0 140 0 052 0 040 0 067 0 014 0 021 0 080 0 034 0 054 0 032 0 014 0 021 0 028 0 041 0 026 0 021 0 030 0 015 0 055 0 016 0 042 0 057 0 049 0 011
Volume 5, Number 10, October 1971 1027
Y
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$0
a0
60
l
l
80
'
/
IbO
ALGAL CULTURE AGE,
I
l
I20
l
140
I
I
160
I
I
180
I
I
200
I
210
DAYS
Figure 5. Effect of algal culture age on the first order decay rate coefficient k' in Equation 5a at 20°C. These results were obtained from decomposition of branch cultures taken from long-term growth studies (samples 1-6, Table I)
most young cells was not expected. It seemed that biodegradability should decrease with age due to accumulation of the refractory fraction of old and decayed cells. There was some indication of such a buildup, but this was offset by the high resistance of the young cells. The five data points which fall below the lower boundary line in Figure 4 belong t o sample 5, which was mainly composed of blue-green algae. The exceptionally low refractory fraction here may have been related to the production of a large quantity of degradable mucilage material. Periodic microscopic examination indicated that even after a year's decomposition in the dark, most young cells were still in "fair" condition and were bright green, while the older cells which had decomposed more completely were brownishyellow and were not distinguishable from other debris. Bacterial and microscopic animal numbers and motility were high in all samples during the early phases of decomposition and decreased rapidly as the biodegradable fraction was utilized. Little or no activity could be detected at the end of the study when the refractory fraction was estimated. The first-order decay rates reported here (0.01 to 0.15 day-') are based on biodegradable mass only-Le., mg On/mg biodegradable mass/day-whereas the decay rates reported by others (0.01 to 0.08 day-') are based on the total mass-Le., mg 02/mgss/day. If the average refractory fraction of the cul-
tures studied by others was 44% (from Table 111), then the rate of oxygen utilization based on the biodegradable material would vary between 0.018 and 0.14 day-'. On this basis the range of values measured in this study agree well with that reported by others. Equation 5a described the course of decomposition quite well over the entire year of decomposition, as indicated by a comparison of the calculated lines and the measured values in Figure 3. The average difference between any single measured point and the calculated value for that point was 12.6% with a standard deviation of =t5.9 %. Thus, Equation 5a appears t o give a sufficiently close correlation for most purposes. The decay rate coefficients shown in Figure 5 are remarkably close to one another considering the wide range in organic concentrations (50-fold range) and the varied algal communities which developed in the different samples. As expected, the highest decay rates were, in general, associated with the younger cultures. The unexpectedly high refractory fraction of the younger cultures required additional evaluation. One difficulty with the preceding study was that the COD of young cultures was too low (10 to 40 mg/liter COD) t o be sure of the accuracy of the analyses. For this reason, further studies were initiated with six more natural water samples (Table I). After the algal cultures began t o grow and while excess nutrients were still in solution, approximately 4-liter samples were taken from each culture, aeration was discontinued, and the algae were allowed to settle for several hours in the light. The higher concentration which resulted (greater than 80 mg/liter COD in most cases) allowed more reliable analytical results. Nine hundredml samples of the concentrated cells were placed in decomposition vessels in the dark at 20°C as in the previous study. F o r eight different cultures with ages ranging from 10 to 24 days, the refractory fraction varied between 45 and 8 7 z (mean, 64 %), thus giving further evidence for a high refractory fraction in cells from young cultures. Another decomposition experiment was designed to determine if incomplete decomposition in the older cultures was a result either of inefficient decomposer populations or insufficient nitrogen and phosphorus. The branch 6 samples, representing the oldest cultures for the first six samples studied, were mixed together after about one year of decomposition when decomposition seemed complete. Nitrate nitrogen (5 mg/liter as nitrogen) and orthophosphate (2 mg/liter as phosphorus) were added together with 1 ml of a mixture of the following: mixed liquor suspended solids from an activated sludge waste treatment facility; algal pond sediment containing cladocerans
Table IV. Refractory Fraction of Axenic and Unialgal Cultures at Different Temperatures Chemical oxygen demand Initial Final Decomposition Algal _. Total ss Total Period, culture (rng/liter) (mg/liter) days Temp., "C Sample age, days 43 80 92 70 20 Chlorella 10 30 80 92 35 70 10 Chlorella 34 230 244 79 35 16 Chlorella 177 360 395 43 93 20 Chlorella 115 360 395 43 25 93 Chlorella 64 395 360 43 35 93 Chlorella 23 75 83 90 20 15 Chlorella, 26 75 83 90 25 15 grown with 24 75 83 90 35 15 bacteria and zooplankton 1028 Environmental Science & Technology
ss
Undecomposed fraction
33 29 31 157 87 52 23 25 ...
46 32 14 45 29 16 28 32 29
100 X f
and other small crustacea; and acclimated algal decay systems that had been fed various types of algae for a period of six months. The subsequent decomposition which resulted was minor, since the COD was reduced from 390 to 375 mg/liter after 100 days. Thus, insufficient nitrogen, phosphorus, or decomposer population did not seem to be the cause of the high refractory fraction in old cultures. Axenic and Unialgal Cultures. Further experiments were conducted with axenic and unialgal cultures to help clarify the nature of decomposition. I n addition, the effect of temperature (4", 20", 25", and 35°C) on the rate and extent of decomposition was evaluated. Axenic cultures with varying susceptibility to decomposition were chosen: Chlorella pyrenoidosa and C. wlgaris, Scenedesmus, and Chlamydomonas were grown on synthetic river water and a unialgal culture of Nitzschia closterium was grown on heat-sterilized Monterey Bay seawater. The growth and decomposition apparatus was as used previously with added precautions to prevent bacterial contamination. Branch samples corresponding to young cultures (10 to 20 days) and older cultures (70 to 90 days) were taken of' each sample and were placed in the dark at the different temperatures and in both axenic and seeded conditions. The seed used was the same as that described earlier. Typical decomposition patterns resulted with the Chlorella cultures (Figure 6). Decomposition of all cultures proceeded rapidly when an acclimated seed was present. In most cases (except with Chlamydomonas) a lag phase preceeded the maximum rate of decomposition. At 20" and 35°C the amount of material respired by axenic algal cultures alone averaged 32 % (of the initial COD) less than that respired by the seeded cultures. Although the fraction of the organic material utilized by the algal cell itself was approximately equal to the fraction utilized by the decomposers, a consideration of the rate of decomposition indicates a sig100,
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50 25 0
ALOAL A O E
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I O DAYS
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I I
100
125
150
-
' 0
25
50
75
TIME,
DAYS
Figure 6. Comparison of the decomposition of Chlorella pyrenoidosa in axenic cultures and in axenically grown cultures inoculated with decomposers Branch samples Here taken from cultures growing under axenic conditions after 10 to 93 days and were placed in total darkness at 4 O , 20", 25", and 35 "C
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l
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1
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20
30
40
50
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TIME,
DAYS
Figure 7. Comparison of calculated (second-order equation, Equation 4) and observed results for young and old Chlorella cultures grown under axenic conditions and inoculated with decomposers at the beginning of the dark phase Curve fit and equation variables, k" and M I obtained by trial and error solution
nificant difference. The decomposition in the seeded algal cultures at 20" to 35°C always approached completion in 20 days ; whereas an equivalent quantity of organic matter would have been utilized in 250 days if the respiration in the axenic cultures continued at the same rate (Figure 6). The average undecomposed fraction for all of the seeded cultures was 27% (Table IV). This fraction was assumed to equal the refractory fraction since rates of decomposition had reached negligible values. Decomposition generally was more complete than the heterogeneous cultures previously studied. This difference could have been due to the accumulation of refractory bacterial and animal residues during heterogeneous growth. To test this possibility, new cultures of Chlorella and Chlamydomonas were grown in the presence of bacteria and microscopic animals and then were placed in the dark for decomposition. The results (Table IV) indicate that the presence of bacteria and animals during growth did not contribute a significant additional amount of refractory material. The noted differences in the refractory fraction might have been the result of a more active decomposer inoculum, optimum temperatures of decomposition, or differences in degradability of the algal species. No generalizations could be made about the effect of temperature on extent of decomposition. With some cultures (Chlorella and Scenedesmus), a decrease in refractory fraction with increasing temperature resulted, while in most cases, the refractory fraction was essentially the same at temperatures of 20°C or above. The decomposition curves for the pure cultures when seeded appeared to follow second-order kinetics, and a n attempt was made to fit the data with Equation 5b. Trial and error procedures were used because of the difficulty in defining M I . Comparison of the calculated and observed data for Chlorella cultures was representative of the close correlation obtained with this approach (Figure 7). Similar results were found for Scenedesmus and Nitzchia, but with Chlamydomonas the decomposition more nearly approximated first-order kinetics. The latter culture exhibited a much higher endogenous respiration rate, and also contained a large quantity of dissolved organic matter. These two factors resulted in a high initial rate of decomposition on inoculation with decomposers. A first-order curve was fitted to the data for the seeded pure cultures thus ignoring the lag phase which occurred in most cases. The average differences between all measured ( M V )and Volume 5, Number 10, October 1971
1029
calculated ( C V ) values [(CV - MV)/CV X 1001 was 1 5 . 2 z with a standard deviation of 9.7 The calculated first-order decay rate in general varied between 0.015 and 0.07 day-1 for decomposition at 20°C, which is within the range for heterogeneous decomposition. The first-order rates increased about 1.5 to 2.5 times for a 10°C rise in temperature, as normally expected for biological reactions. The exception again was Chlamydomonas which had a decay rate of about 0.01 day-' at 20°C and this changed little with increasing temperature. In summary, the decomposition studies with axenic cultures indicated that bacteria and microfauna increased the rate and extent of algal decomposition to values which were in general in the same range as for heterogeneous cultures, once a n initial lag period required t o develop a decomposer population had occurred in the axenic cultures. With a few exceptions, the kinetics followed the same pattern as with heterogeneous cultures. The rate but not usually the extent of decomposition increased with increasing temperature.
z.
Discussion This laboratory investigation has shown that both heterogeneous and pure cultures of algae, with ages varying from a few days to over one-half year and grown in media ranging from freshwater to seawater, generally decomposed slowly and incompletely even when inoculated with a heterogeneous population of bacteria and microscopic animals. The mixtures of autotrophic and heterotrophic organisms that developed had a refractory portion of 12 to 86% that was not degraded within a year. Young cultures (less than 30 days old) exhibited this extreme range in refractory material, while older cultures had a refractory fraction range of 30 to 70%. The average refractory fraction for over 75 different algal cultures was 40%. Similar results were found in a study which followed this one for decomposition of algae under anaerobic conditions (Foree and McCarty, 1971). Possible reasons for a high refractory fraction in young cultures exists. Alexander and co-workers (Kuo and Alexander, 1967; Potgieter and Alexander, 1966) demonstrated that a controlling factor in the decomposition of certain fungi was the pigment, melanin. Also Jorgensen (1962) has shown that certain derivatives of chlorophyll (chlorophyllides) are antibacterial. Short-term (5-day) decomposition experiments with unialgal cultures (Chlorella, Scenedesmus, Lyngbya, and Uluthru) indicated that the minimum rate of decomposition was correlated with a chlorophyll content of 0.3 to 0.5% (dry weight). Although this indicated that the chlorophyll content may influence the refractory fraction in young cultures, no conclusive information was obtained. The fraction of refractory organics that was original algal material could not be separated from that which resulted from decomposer synthesis, but a n estimate of the respective fractions can be made. McCarty (1965), Kuntz and Forney (1959), Washington and Symons (1963), and Porges et al. (1953) have shown that the maximum growth of heterotrophic microorganisms from a given quantity of organic material is a function of the available energy. Under aerobic conditions, the mass of decomposers synthesized can be no more than about one half of the mass of organic matter consumed. The refractory fraction of heterotrophic microorganisms has been reported to be about 20% of the initial mass (Kuntz and Forney, 1959; Washington and Symons, 1963). Thus, in an algal culture where the initial organic mass is 100 mg/liter and the total refractory material is 40 mglliter, no more than 6 mglliter [(loo - 40) x 0.5 x 0.21 or less than 15 % of the total 1030 Environmental Science & Technology
refractory organics will be decomposer remains. Note that with respect to oxygen usage it does not matter whether the refractory organics are composed of algal or decomposer material. As long as the organic material remains in a reduced state, no oxygen is consumed. The high refractory fraction found for algal-related organic material is inconsistent with reports that algae are completely degraded within relatively short periods of time in the field (Kuzentsov, 1968; Ohle, 1962). It is recognized that laboratory experiments are of necessity artificially simplified, reproducible, and standardized, and differ from the natural water environment in at least four ways: Preselection of resistant species of algae unlike those abundant in natural situations may have occurred as a result of laboratory culture techniques; batch decomposition tests sometimes allow the development of' decomposer inhibitors (either bacterial or animal); the one-phase experimental system used in this study represents only one part of the two-phase sediment-water system which occurs in natural situations; and most chemical and physical conditions change continuously in the natural environment allowing numerous varieties of bacteria to function. Jannasch (1964) has indicated that for the above reasons quantitative data from laboratory experiments are of limited value to microbial ecologists. Thus, caution is advised in applying the results from this study directly to the natural environment. However, there is evidence that the laboratory results may apply to some extent to field conditions. If accumulation of inhibitory substances had been a factor affecting the laboratory results, then the degree of inhibition should have been a function of the initial concentration of algae. No such trend was indicated in the 74 experiments conducted with the organic mass varying over a 1000-fold range. Also, Foree and McCarty (1971) expanded the range of decomposition experimental variables to include anaerobic conditions and initial organic concentrations as high as 13,000 mglliter COD, and measured a similar average refractory fraction of 4475;. The bacterial and animal seed used was well acclimated to the decomposition of algal material, although perhaps different from organisms living in the field. In addition, there is considerable evidence to indicate that most forms of life do produce residues which are not readily degraded and tend to accumulate in the environment. The resistant nature of humus material in the soil is well-known (Stevenson, 1965). Accumulation of refractory materials in bottom muds of lakes and estuaries is also well documented. Strickland (1965) has noted that in all deep oceans, several hundred grams of particulate organic matter exist beneath each square meter of surface compared with a standing crop of phytoplankton of no more than two to three grams. The accumulated deposits of organic material on Earth have been estimated to be five to six orders of magnitude greater than produced each year by photosynthesis (Revelle and Suess, 1957), indicating some fraction of naturally produced organic matter, at least, is extremely resistant. These latter observations tend to indicate that organisms have indeed failed to complete their task. While the accumulation of refractive organic matter on Earth is significant, it would have to be three to four orders of magnitude higher if 40 of all the carbon which has been photosynthetically fixed were completely nondegradable. This leads to the conclusion that the refractory material as found in the study is, in fact, degradable, but the time span for complete degradation is many years or perhaps decades. For example, if 40% of the plankton is refractory and when deposited in the sediments decomposed at a rate of only 50%
every 10 years, then under steady-state conditions the organic matter in the sediments would be equivalent to less than six times the annual production. It is assumed that the annual production is the same each year and that the water body has been in existence at least 40 years. The total amount of refractory organics decomposed in the sediments would equal the contribution of refractory material to the sediments. Thus, the use of oxygen in the hypolimnion could be equal to the annual contribution of phytoplankton to the sediment but would be a result of the slow decomposition of accumulated organics from past decades and not from complete oxidation of the annual contribution. From this study, and from the results of others, algae and algal-derived organic matter are postulated to consist of three fractions. The first fraction consists of degradable storage products that disappear within a few hours after the organisms are placed in the dark. The oxygen demand of this fraction is probably insignificant in long-term considerations but its decomposition may be a significant factor affecting diurnal oxygen variations in natural waters. The second fraction is the biodegradable material evaluated in this study and represents about 30 to 70% of the mass of algae and algal-derived organic material. It is degraded at a fairly constant, but intermediate, first-order rate (0.01 to 0.06 day-’) so that it will be consumed within a year at normal temperatures. Because of its relatively slow rate of decomposition, it will significantly affect oxygen resources only in situations where it is present in high concentrations-e,g., in bottom sediments, the hypolimnion of reservoirs and lakes, or in ponds. The remaining fraction is the refractory material which decomposes slowly, probably at rates of no more than a few percent per year. Slow decomposition of the refractory fraction was found in this study with algal samples maintained in the dark for periods of up to 700 days. The refractory material may have a significant effect on the oxygen resources of natural waters only if it accumulates over many years, and only if the products of decomposition can diffuse up through overlying layers of sediment for subsequent aerobic oxidation. Because of the significance to the interpretation of natural phenomena, it is highly desirable that in situ studies be conducted to determine whether the quantity of refractory material formed under natural conditions corresponds with that measured in this laboratory study.
Acknowledgment This investigation was supported by Research Grant WP1037 and by Research Fellowship WP-33,633 from the Federal Water Pollution Control Administration, U. S. Dept. of the Interior. Mrs. Victoria Mongird’s assistance with the laboratory analyses is gratefully acknowledged. Literature Cited American Public Health Association, “Standard Methods for the Examination of Water and Wastewater,” 12th ed., APHA, AWWA, and WPCF, New York, N.Y., 1965,769 p. Allen, M. B., Fitzgerald, G. P., Rohlich, G. A., Water Pollut. Conrr. Fed. J. 36, 1049 (1964).
Cramer, M. L., Myers, J., Jr., Plant Physiol. 24,255 (1949). El-Dib, M. A., Ramadan, F. M., J. Amer. Civil Eng. Smit. Eng. Diu. 92, 97 (1966). Fitzgerald, G. P., Water Pollut. Contr. Fed. J. 36, 1524 (1964). Foree, E. G . , McCarty, P. L., Proc. 24th Industrial Waste Conference, Purdue Univ., Lafayette, Ind., in press, 1971. Gibbs, M., “Respiration” in “Physiology and Biochemistry of Algae,” R. A. Lewin, Ed., Academic Press, New York, N.Y., 1962, pp 61-86. Golueke, C. G., Oswald, W. J., Water Pollut. Contr. Fed. J . 37, 741 (1965). Golueke, C. G., Oswald, W. J., Gotaas, H. B., Appl. Microbiol. 5 , 4 7 (1957). Jannasch, H. W., Int. Ver. Theor. Angew. Limnol. Verh. 15, 562 (1964). Jenkins, D., Medsker, L., Anal. Chem. 36,610 (1964). Jewell, W. J., Ph.D. Thesis, Stanford University, Stanford, Calif., 1968 (Dissertation Abstracts No. 69-8201, p 4206B). Jgrgensen, C. B., “Biology of Suspension Feeding,” Pergamon Press, Elmsford, N.J., 1966, 357 pp. Jgrgensen, E. G., Physiol. Plant. 15,530 (1962). Kirk, P. L., Anal. Chem. 22, 354 (1950). Kleerekoper, H., J. Fish. Res. Bd. Can. 10, 283 (1953). Kuntz, R . R., Forney, C., Jr., Sewage Ind. Wastes 31, 819 (1959). Kuo, M. J., Alexander, M., J. Bacteriol. 94, 624 (1967). Kuznetsov, S. I., Limnol. Oceanogr. 13, 211 (1968). Lee, G. F., Clesceri, N. L., Fitzgerald, G. P., Inr. J . Air Water Pollut. 9, 715 (1965). Mackenthun, K. M., Herman, E. F., Bartsch, A . F., Trans. Amer. Fish. SOC.75, 175 (1945). McCarty, P. L., “Advances in Water Pollution Research,” Vol. 2, Pergamon Press, Elmsford, N.J., 1965, pp 169-187. McKinney, R. E., J. Amer. Civil Eng., San. Eng. Dia. 91, 77 (1965). Moss, B., New Phytol. 67, 49 (1968). O’Connel, R. L., Thomas, R. L., J. Amer. SOC.Civil Eng., Sun. Eng. Diu. 91, 1 , 92, 95 (1965). Odum, H. T., Ecol. Monogr. 27,55 (1957). Ohle, W., Kiel. Meeresforsch. (Ger) 18, 107 (1962). Porges, N., Jaswiez, L., Hoover, S. R., Appl. Microbiol. 1, 262 (1953). Potgieter, H. J., Alexander, M., J. Bacteriol. 91, 1529 (1966). Revelle, R., Suess, H. E., Tellus. 9, 18 (1957). Skopintzen, B. A., Broock, E. A., (In Russian), Mikrobiol. 9, 595 (1940). Stevenson, I. L., “Biochemistry of Soil” in “Chemistry of the Soil,” F. E. Bear, Ed., Reinhold, New York, N.Y., 1965, pp 242-291. Strickland, J. D. H. In., Riley, J. P., Skirrow, G., “Chemical Oceanography,” Vol. 1, Academic Press, New York, N.Y., 1965, pp 477-610. Teal, J. M., Ecol. Monogr. 27,283 (1957). U. S. Public Health Service, Robert A. Taft Sanitary Engineering Center, Cincinnati, Ohio, Pub. No. 999-WP-26, 1965, 75 PP. Varma, M. M., DiGiano, R., Water Pollut. Contr. Fed. J. 40. 613 (1968). ,- - Ward, C. H., Moyer, J. E., Vela, G . R., Deaelop. Ind. Microbioi. 6,213 (1964). Washington, D. R.’, Symons, J. M., Water Pollut. Contr. Fed. J. 34.283 (1963). Wisniewski, T. F.; Water Sewage Works 105,235, 300 (1958). Wyckoff, B. M., ibid. 111, 277 (1964).
Receiced for reuiew September 8 , 1970. Accepted April 26, 1971.
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