Anaerobic Decomposition of Algae Edward G . Foreel and Perry L. McCarty Department of Civil Engineering, Stanford University, Stanford, Calif. 94305
The major objective was to determine the rate and extent of algal degradation under simulated natural conditions. Decomposition of heterogeneous and unialgal cultures was studied under dark, anaerobic, constant-temperature laboratory conditions. Effects of high sulfate concentration, bacterial seedings, temperature, pH, and cell composition on the rate and extent of degradation were evaluated. After 200 days, decomposition of algal cultures was essentially complete, and the undecomposed particulate organic matter remaining was termed the “refractory organic fraction.” This fraction ranged from 20-60 of the ash-free dry weight for various cultures with an average of 4 0 z . The decomposition of the biodegradable organic fraction could be adequately described by first-order decay kinetics with a range for the decay constant k of 0.011-0.032/day with an average 0.022/day. The rate and extent of degradation were similar to those found by other investigators under aerobic decomposition conditions.
T
he discharge t o waterways of inorganic nutrients, such as nitrogen and phosphorus, provides conditions which enhance algal growth. The presence of excessive quantities of algae and their resulting decay can produce significant water quality problems. The decomposition of algal organic matter may vary in rate, extent, and manner, depending primarily upon the prevailing environmental conditions, and although algae can be stabilized to some degree by methane fermentation under the conditions normally employed for the digestion of domestic waste sludge (Golueke, Oswald, et a[., 1957), little research has been conducted regarding the processes which occur in a dark, anaerobic environment closely simulating natural conditions. Such an environment was chosen for this study of algal decomposition. In natural situations, algae enter dark-anaerobic environments primarily by gravitational sedimentation, where, due t o conditions unfavorable for growth, they die and decompose. Of primary significance t o water quality is the occurrence of this phenomenon in the lower zones of productive lakes, reservoirs, and domestic and industrial waste treatment lagoons. Experimental
The major objectives of this study were to determine the rate and extent of algal degradation under dark, anaerobic, constant-temperature conditions and to evaluate the effects Present address, Department of Civil Engineering, University of Kentucky, Lexington, Ky. 30506. 842
Environmental Science & Technology
of important biological and environmental parameters. Both algae collected from natural waters and algae grown in the laboratory were utilized in the decomposition studies. The algal cultures grown in the laboratory were housed in a constant-temperature room at 20” C., illuminated with “daylight” fluorescent bulbs which provided an intensity of about 1000 ft.-candles at the surface of the culture vessels, and aerated with a mixture of 9 8 x air and 2 z carbon dioxide. The basic growth medium used in the majority of the studies is described in Table I. Stoppered 9-liter glass bottles equipped with three ports for introduction and removal of samples under anaerobic conditions were used both to grow and to hold the decomposing cultures. Prior to decomposition, cultures were purged with nitrogen gas to remove dissolved oxygen then housed in dark cabinets in constant-temperature rooms. Samples for analysis were removed from a side port of the bottle while the contents were stirred vigorously with a large magnetic stirrer. The cultures studied are described in Table 11. The cultures designated as “Pond Algae 1” and “Pond Algae 2” were collected from a pond at Los Gatos, Calif., on two different days and were both composed primarily of a filamentous alga. Inocula for the unialgal cultures (Cultures 31-116 and 160) were obtained from “The Culture Collection of Algae at Indiana University” (Starr, 1964), and the numbers in parentheses identify the various cultures in the collection. The inoculum for the “Heterogeneous” cultures was originally obtained from a pond in Kentucky and each was comprised of a mixture of many species of b3th green and blue-green algae. The algae present at the time of collection of the natural water samples (Cultures 162-165) were utilized as inocula and no additional organisms were added. The algae for Cultures 1-11 were collected from a pond and resuspended in synthetic media for the decomposition studies. The remaining cultures were grown in the laboratory and allowed to decompose in the same media in which they were grown with the exception of alterations in alkalinity which provided additional buffering capacity and the addition of seeding material which provided bacterial populations in some of the experiments. The period of growth indicated far the laboratory cultures (Cultures 31-173) is the total timz from the initial seeding of the media until the imposition of anae;obic conditions. Samples were removed from the decomposing cultures periodically and analyzed for chemical oxygen demand (COD) total and volatile suspended solids, pH, and various forms of nitrogen and phosphorus (Amer. P.H.A., 1965; Wyckoff, 1964). Selected samples were also analyzed for sulfates (Amer. P.H.A., 1965) and lipid content (Loehr and Rohlich, 1962). Gas analyses and individual volatile acid analyses were performed by gas chromatography (Young and McCarty, 1968).
COD was one of the major parameters used to evaluate the extent of decomposition of algal cultures. The dichromate reflux method with silver catalyst and mercuric sulfate for chloride complexing (Amer. P.H.A., 1965) gives t o within 95 t o 100% the theoretical quantity of oxygen required t o oxidize organics t o carbon dioxide, water, and ammonia. The theoretical ratio of COD/weight varies from 1.1 for simple carbohydrates to 2.9 for saturated long-chain fatty acids, with a n average for heterogeneous biological material of about 1.4. The COD also gives a measure of the theoretical quantity of methane which would be produced during anaerobic methane fermentation and of sulfide formed during decomposition by sulfate reduction (Lawrence, McCarty, et al., 1966). The production of 1 liter of methane gas (1 atm. and 20" C.) corresponds theoretically to a stabilization of 2.66 g. of COD, and the reduction of 1 g. of sulfate to sulfide corresponds t o the stabilization of 0.67 g. of COD. The abbreviated symbols used in the subsequent presentation and discussion of results are defined in the Nomenclature, including the means of determination of each parameter. The soluble and particulate fractions of the samples were separated by centrifuging for 15 min. at 20,000 X G. Table I11 presents a summary of the characteristics of the various cultures determined at the time of imposition of anaerobic conditions. Sa and Ma are measures of the quantity of particulate organic matter initially present, and z N a is a measure of the quantity of organic nitrogen initially present in the particulate material. The difference between M T aand Ma represents the quantity of soluble organics. The quantities of soluble nitrogen initially present were normally small and are not included in Table 111.
Table 11. Characteristics of Algal Cultures Used in Anaerobic Decomposition Studies. Growth
1 2 3 4 5
6 9 1O b llC 31 32 33 110 111 112 115 116 141 144 160 162 163 164 165 166 170 171 172 173
Results Methane Fermentation. The overall methane fermentation process is comprised of two principal steps (McCarty, 1964): conversion of complex organics to volatile organic acids, and fermentation of these acids to methane and carbon dioxide. Methane fermentation was found to be responsible for the stabilization of algal organics (measured as decreases in M T ) under anaerobic conditions in all the cultures studied except: (1) those with pH levels low enough to be toxic to the methane bacteria, (2) those from which all bacteria were excluded (axenic cultures). and (3) those with high initial csncentritions of sulfate. No significant stabilization occurred in cases (1) and
Table I. Chemical Composition of Synthetic Growth Medium Concentration Chemical (mg4.1 Comments
MgSO,. 7H.0 CaCI? NaHCO, KHC0:j K2HPO4 NHdCI Fe, B, Mn, M o
45.0 55.0 250 100
7 i
23.1 95.5 0.1 ) I
Zn, C u , Co, Ni, Cr, V
0.01
EDTA
2.0
KOH
1.0
'
1
I
200mg./1. alkalinity as CaCO.j 5 . 0 0 mg./l. P 25,O mg. :I. N added as 1 ml. trace element solution per liter of medium
period (days)
Number
(1
Pond Algae 1 Pond Algae 1 Pond Algae 1 Pond Algae 1 Pond Algae 1 Pond Algae 1 Pond Algae 2 Pond Algae 2 Pond Algae 2 Chlamydomonas ob. (219) Gloecystis max. (166) Chlorella p y . (26) Chlorella p y . (26) Anacystis ni. (625) Lyngbya sp. (622) Phormidiurn sp. (389) Anabaena sp. (B380) Heterogeneous Heterogeneous Chlorella p y . (26) Algae from Searsville Lake water Algae from San Joaquin River water Algae from Stockton Stab. Pond water Algae from agricultural drainage water Heterogeneous Heterogeneous Heterogeneous Heterogeneous Heterogeneous
. . . . . . . . . . . . . ... .. ..
34 34 34 49 49 49 49 49 29 29 48 48 48 48 48 54 30 30 30 30
Decomposition temperature 20' C. except as noted. Decomposition temperature 1 5 ' C. Decomposition temperature 25" C.
(2), and sulfate reduction was the mechanism responsible for stabilization in (3). The methane and carbon dioxide production and reduction in total C O D ( M T )are presented in Figure 1 for Culture 9 to illustrate the typical pattern observed. A precise calculation of the equivalence between the theoretical C O D of the methane produced and the measured reduction in M T in the culture over the entire period of decomposition could not be made because the voliime of the liquid in the vessel changed with time due to the removal of samples for analyses. However, the quantity of liquid volume change was small in comparison to the total liquid volume, and it was considered sufficient to take an effective liquid volume as being that at the time when the cumulative methane production was one half of the total. This liquid volume was 5720 ml. at 60 days conipared t o 6030 ml. at the beginning of decomposition. By use of this valde and the measured decrease in M 1 concentration of 2490 mg.#J.,the total measured C O D reduction over the 311-day period was 14.2 g. By use of the measured total volume of methane produced of 5600 ml. and the theoretical factor of 2.66-mg. C O D per ml. methane at 1 atm. pressure and 20" C. (the experimental conditions of gas measurement), the calculated C O D equivalent of the methane produced over the 311-day period was 14.9 g. Similarly close agreement was obtained for all of the cultures for which cumulative methane production was measured. Volume 4, Number 10, October 1970 843
Culture 1 2 3 4 5 6 9 10 11 31 32 33 110 111 112 115 116 141 144 160 162 163 164 165 166 170 171 172 173 Q
Table 111. Summary of Experimentally Determined Initial Characteristics of Algal Cultures Studied. so MTO Ma No 11,100 12,300 11,100 1.48 10,200 12,800 11,640 1.66 11,200 13,000 11,730 1.52 1,080 1,190 1,080 1.75 1,000 1,140 1,020 1.87 1,020 1,180 1,060 1.82 2,580 3,200 2,810 2.95 2,440 3,230 2,860 3.12 3,200 2,440 2,820 3.12 340 840 500 12.20 1,360 2,040 1,420 3.16 620 1,150 920 8.23 760 1,500 3.24 1,370 280 460 500 8.87 850 1,100 3.20 1,050 280 530 390 7.07 710 7.37 1,120 900 1,660 2,520 1.66 2,230 1,560 2,410 2.060 1.50 1,280 2,980 2,800 1 . 80 1,170 1,980 1,650 2.72 850 1,300 1,210 6.05 480 890 730 5.88 660 1,150 890 6.44 1,920 2,980 2,680 3.80 1,360 2,210 1,850 2.26 1,300 2,210 1,850 2.40 1,320 2,210 1,850 2.36 1,280 2,210 2.21 1,870
z
MolSo
1 .oo 1.14 1.05 1.00 1.02 1.04 1.09 1.17 1.16 1.47 1.04 1.48 1.80 1.64 1.24 1.39 1.27 1.34 1.32 2.19 1.41 1.43 1.52 1.35 1.40 1.36 1.42 1.40 1.46
Concentrations (mgJ1).
As shown in Figure 2 (using Culture 9 for illustration), only two volatile acids, acetic and propionic, were present i n significant quantities during the decomposition of a wide variety of algal cultures by methane fermentation. The typical pattern of variation consisted of an initial peak in acetic acid concentration lagged by a peak in propionic acid concentration. Measurable but relatively small concentrations of butyric, isobutyric, valeric, and isovaleric acids were also present during decomposition in most of the cultures. This pattern is similar t o that often observed for methane fermentation of complex wastes (McCarty, Jeris, et al., 1963).
Figure 1. Gas production and COD stabilization ( M T )cs. time for Culture 9
844 Environmental Science & Technologj
Sulfate Reduction cs. Methane Fermentation. To compare sulfate reduction and methane fermentation as mechanisms for the stabilization of algae under anaerobic conditions, a high sulfate concentration (2000 mq./l.) was introduced into Cultures 5 and 6, while 4, which was otherwise comparable, had a sulfate concentration of only 18 mg./l. Culture 5 was maintained with its natural biological population, while Cultures 4 and 6 were seeded with a small quantity of bottom deposits from a lake. In Culture 4, methane fermentation resulted in a stabilization of 75 % during 239 days of decomposition (Figure 3). In the two cultures with high sulfate concentration. no significant production of methane occurred, but sulfate reduction was responsible for a stabilization of 57% during the same 239-day period. The absence of data for Cultures 5 and 6 prior to day 140 (Figure 3) was due to interference of sulfides in the COD test which were subsequently removed by acidification and purging with nitrogen gas prior to C O D analysis. The lower stabilization in the sulfate-reduction cultures was found to be due primarily to the reduced utilization of lipids. These results suggest that in a natural anaerobic environment characterized by significant sulfate reduction, a greater portion of the resistant organic material may be comprised of lipids relative to those found in an environment characterized by active methane fermentation. Further studies would be required to confirm this. A striking difference was observed between the individual volatile acids formed during methane fermentation and sulfate reduction. As shown in Figure 4 only acetic acid was observed during sulfate reduction, while significant quantities of propionic acid as well were observed during methane fermenta-
tion. Based on the maximum acetic acid concentrations attained in the sulfate-reduction cultures, at least 60% of the total stabilization in these cultures resulted from the oxidation of acetic acid. At least three explanations are possible for the complete absence of propionic acid in the sulfate-reduction cultures: none was produced as a n intermediate during the degradation of the complex algal organics, it was produced and expelled from the bacterial cells, but was immediately utilized, or it was produced and underwent further conversion while still within the bacterial cells. Although none of these can be disproved from this study, the second possibility seems unlikely since not even a trace of propionic acid was observed by the sensitive chromatographic procedure used for analysis. A subsequent study with Culture 165 which was grown and allowed t o decompose in a sample of agricultural drainage water with a high sulfate concentration (1 535 mg./l.) gave similar results. Close agreement was obtained between the decrease in M T concentration and the theoretical C O D equivalent of the measured decrease in sulfate concentration. The long-term values for the three cultures are shown in Table IV. The C O D equivalents were calculated based on the following relationship :
I
+ s04*-
8H+ -k 8e-
Sulfate-reducting bacteria
4
+ 4H,O
S2-
(1)
indicating that the reduction of 1 mole of sulfate (96 8.) corresponds t o the oxidation of 8 equivalents of organic matter, or 64 g. on a COD or oxygen-equivalent basis. That only sulfate reduction and no methane fermentation occurred in the cultures with initially high sulfate concentrations suggest sulfate reduction is probably significant for the degradation of organics in anaerobic zones in oceans and brackish lakes since the initial sulfate concentrations used (1600 and 2000 mg./l.) were of comparable magnitude to those of seawater (about 2500 mg./l.). However, the absence of methane fermentation in this study was attributed to the toxicity of soluble sulfides to the methane bacteria (Lawrence, McCarty, et a/., 1966), and the accumulation of soluble sulfides may be considerably diminished in a parallel natural setting by diffusion, precipitation, etc. Effect of Bacterial Seeding. In cultures with wide variation in bacterial seeding, no significant differences were observed in either the rate or extent of reduction in particulate matter (Figure 5 ) or in the long-term extent of total C O D stabilization (Figure 6). There was, however, an increase in the rate of the total COD stabilization progressing from the culture 400
0-MEASURED M S C-TOTAL V O L A T I L E ACID COD A-ACETIC ACID COD *-PROPIONIC ACID COD .-BUTYRIC + ISOBUTYRIC ACID COD A - V A L E R I C t ISOVALERIC ACID COD
CULTURE
4,
- .
0 8
0
40
80
120
200
160
T I M E Iaoys)
5
400
-
CULTURE 5 . SULFATE REDJCTION
Y .J
3
~ -I20
J 203
160
TIME l d o y r i
Figure 2. Individual and total volatile acids COD and measured soluble COD ( M s ) concentrations during decomposition in Culture 9
93
8C
-
73
5
6c’
\
0
1=--€-.-& 40
I20 -1ME ! d o y i l
60
60
L?O
Figure 4. Comparison of volatile acids present during decomposition in comparable algal cultures undergoing methane fermentation and sulfate reduction C U L T U R E 4, M E T H A N E F E R M E N T A - I O N
3)
E
CULTURE 5 , S U L F A T E REDUCTIOh
:5 3 6- 4 3
L SULFATE
30
2c
REDUCTION
IO
0 4c
80
120
160
200
240
TIME (days)
Figure 3. Comparison of stabilization by methane fermentation and sulfate reduction during decomposition in comparable algal cultures
Table IV. Equivalence between Long-Term Values of Measured Decrease in COD ( M T )Concentration and Theoretical C O D Equivalent of Measured Decrease in Sulfate Concentration in High Sulfate Cultures 5
Decomposition time (days) Measured decrease in sulfate (mg.11.) COD equivalent of sulfate decrease (mg.11.) Measured decrease in Mr(mg./l.)
239 955 617 640
CUIt lure 6 165
239 900 600 617
230 885 590 565
Volume 4, Number 10, October 1970 845
3-CULTURE I, NO ADDITlONAL SEED C-CULTURE 2, LAKE BOTTOM DEPOSiT SEED A-CULTURE 3, DIGESTED SLUDGE SEED
i
80 c
2
60
L
-s I
40
1
:I 30
20
,
,
,,I
,
3 0
40
80
I20
200
I60
320
283
240
360
TIME 160111
Figure 5. Effects of bacterial seeding on particulate matter reduction ( M )in decomposing cultures of pond algae
I
Figure 6. Effects of bacterial seeding on COD stabilization ( M T )in decomposing cultures of pond algae
I
=
\ 30
CHLORELLA t
1 ,
43-
BbCTERlA
I
I
I
I
20
Figure 7. Effect of bacteria on COD stabilization ( M _ T ), in decomposing Chlorella ; culture
li
I
I I
~
0
C
2 0
4C
60
00 TIME ldoyi
I00
120
142
with no additional seed to the culture seeded with bottom deposits to the culture seeded with digested sludge (Figure 6). This indicates that methane-forming bacteria required for the stabilization of the algal organics were present in all of the cultures, but the initial total population levels were different. A subsequent study with a bacteria-free culture of Chlorella pyrenoidom was conducted to determine what, if any, role the bacteria might play in the conversion of particulate algal organics to soluble organics since no significant differences were observed in either the rate or extent of particulate matter reduction for the different seedings indicated above. Figure 7 indicates no significant stabilization occurred in the bacteria-free culture alone, while a relatively rapid stabilization was accomplished by the methane bacteria in a seeded aliquot from the same initial culture. However, the extent of particulate matter reduction in the bacteria-free culture was almost as great as in the seeded culture (Figure 8) indicating that the algae themselves were capable of accomplishing a significant portion of the cmversion from particulate to s d u ble organics. The rate of cmversim, however, was greater in the seeded culture, indicating that the bacteria had a significant influence on the degradation. Also, volatile acids analyses showed that the bacteria converted essentially all of the soluble organics released to volatile acids, while no volatile acids were produced in the bacteria-free culture. Additional reduction of particulate matter in the seeded culture probably resulted from the degradation of particulate lipids by methane fermentation as will be discussed later. Effect of Temperature. Three initially identical algal cultures were allowed to decompose at three different tempera846 Environmental Science & Technology
Figure 8. Effect of bacteria on oarticulate matter reducGon ( M ) in decomposing Chlorella culture
::I 00
20
40
60
80
100
I20
143
TiMEIdoyrI
tures: 15", 20°, and 25" C. While the rates of both particulate matter reduction and C O D stabilization increased with increasing temperature, the rate of stabilization was affected to a greater degree. The long-term extents of both reduction and stabilization, however, were not significantly different for the three temperatures. The increase in the rate of stabilization with temperature is attributed mainly t o increases in the rates of utilization of volatile acids by the methane bacteria (Figure 9). While the rate of utilization of acetic acid was affected by temperature, the rate of utilization of propionic acid was affected to a relatively greater degree. Effect of pH. Methane production and hence COD stabilization were not significantly affected at pH levels above 6.3, but almost no stabilization occurred at pH levels below 5.0. Reduction of particulate matter was not significantly affected at pH levels above 4.5, but was significantly inhibited at pH 3.2. Essentially all of the soluble organics produced at pH levels above 4.5 were converted to volatile acids, but only a negligible quantity of the soluble organics was converted to volatile acids at p H 3.2. Thus, the activity of the acid-forming as well as the methane-forming bacteria was seriously inhibited at p H 3.2. The effects of a low pH resulting after a few days of decomposition on C O D stabilization and particulate matter reduction in a decomposing culture are illustrated in Figure 10. Effect of Cell Composition. Unialgal cultures representing several common species of green and blue-green algae were grown and allowed to decompose. Differences were observed among the various cultures in both the rate and extent of
particulate matter reduction, but no significant trends were apparent. For example, blue-green algae were not consistently more nor less susceptible to degradation than green algae. The most significant effects on decomposition were associated with variations in lipid content. In cultures with low lipid content, the reduction in particulate matter was essentially independent of COD stabilization, while in cultures with high lipid contents reduction was dependent upon methane fermentation, and hence C O D stabilization. This is illustrated in Figure 11 for two cultures of Chlorella pyrenoidosa, one with a low lipid content (Culture 33-10z by weight), and the other with a high lipid content (Culture 160-6Ox). These results indicate the methane-producing bacteria were required for the degradation of the particulate lipids. Most fatty acids resulting from the hydrolysis of characteristic algal lipids are not soluble when excess calcium and (or) magnesium are present as was the case in this study. Due to the significance of chemical composition in relation to algal decomposition, equations were developed for estimating the protein, carbohydrate, and lipid contents. The assumptions utilized were that the sum of the protein, carbo-
rr-
‘I A-
M y , C U L T U R E 160 M , CULTURE 160 MT , CULTURE 33
v-
M
3 -
0-
80
, C U L T U R E 33
30
IO
,
,
, 1
0
40
80
I20
,
,
160
l
l
2
l
200
240
280
TIME I d o v s l
Figure 11. Effects of lipid content on COD stabilization ( M r ) and particulate matter reduction (M) in two decomposing cultures of Chlorella py. (26)
hydrate, and lipid fractions equals 100% of the ash-free dry weight and that these three constituents have the following empirical compositions (Milner, 1953): protein, C Z . . ~ ~ H ~ . I ~ 01.26N1.00; carbohydrate, (CH20)*; and lipid, ClsHaz02. The following equations result : y$r
XLi
=
=
6 . 2 5 ( z N ) = 625(N/S)
55.8(M/S) - 0.24(xPr)
%Cu = 100 - %Li -
4cc
,
25°C
,
Figure 9. Effects of temperature on the concentrations of individual volatile acids present in decomposing cultures of pond algae
20
-
3-
0
30
80
123
I60
203
243
-,ME Idoyi)
Figure 10. Effects of low pH on COD stabilization ( M T )and particulate matter reduction ( M ) in a decomposing culture on Lyngbya SP
-
y$r
-
59.8
(2) (3)
(4)
Equations 2-4 can be used to estimate the percentages of S present in an algal culture as proteins, lipids, and carbohydrates, respectively, based on measured values of S, M , and N . For this purpose it is also necessary to assume 100 % oxidation of organics in the COD test, No direct determinations of carbohydrate content were made for any of the cultures. Occasional analyses for the lipid content were made and protein content was routinely estimated from measurements of organic nitrogen. Directly measured values of the lipid content were compared with calculated values (Table V) and indicate reasonable agreement. Long-Term Extent of Degradation of Algal Organics and Composition of Refractory Material. The portions of the initial particulate matter ( M and 9 remaining after 200 days of decomposition were used as measures of the degradability of the algal organics. In general, no significant reduction in either M o r S occurred after this period of time. For example, decomposition was observed in Culture 3 for 613 days, the longest time of any culture studied, with the results shown in Table VI. A data summary is compiled in Table VI1 and includes all the cultures studied which met the following qualifications: decomposition observed for at least 200 days, decomposition temperature of 20” C., COD stabilization accomplished by methane fermentation, and pH remained above 5.5. Twenty cultures were studied which met these qualifications, including 5 cultures of algae collected from ponds, 8 unialgal cultures and 4 heterogeneous cultures grown in synthetic media, and 3 cultures grown in natural waters. The data indicate that after 200 days of decomposition an average of 41 % of the initial particulate matter remained undecomposed. From the mean nitrogen content and the mean ratio of MIS an estimated “average” composition for the refractory material was calculated: Z P r = 37.37& YJi = Volume 4, Number 10, October 1970 847
Discussion Table V. Comparison Between Measured and Calculated Lipid Contents Decomposition time Calculated Measured Culture (days) ZN MIS 7W 23 160 0 1.80 2.19 59.8 62.5 53.8 159 5.05 2.17 53.8 160 51.3 160 187 2.09 49.2 5.19 48.7 230 5.57 2.06 46.9 160 18.4 0 1.41 14.9 162 2.72 15.2 187 3.68 1.35 10.1 162 15.9 1.43 11.1 0 6.05 163 17.7 163 187 1.45 11.2 5.67 18.7 0 5.88 1.51 15.7 164 24.0 187 1.57 20.2 164 5.12 14.8 166 0 1.40 11.7 3.80 12.7 1.41 14.4 166 187 5.04 Table VI. Fractions of M and S Remaining in Culture 3 after Extended Periods of Decomposition Time M S remaining) (days) remaining) 200 22.9 19.6 613 20.4 17.9
(z
(z
14.0%, and %Ca = 48.7 %. The “average” initial composition was calculated in the same manner for the same group of cultures giving: y$r = 26.6%, %Li = ll.O%, and %Ca = 62.4%;;.These calculations indicate that the fractional composition of the refractory material was similar to that of the algae before decomposition. Kinetics of Particulate Matter Reduction. With all but three (these exceptions were cultures with high lipid contents in which particulate matter reduction was not first order as it was dependent upon COD stabilization by methane fermentation as indicated previously) of the cultures listed in Table VII, the rate of decay of particulate matter during decomposition could be described adequately by first-order decay kinetics as follows:
M
=
( M o - fMo)e-ki
+ ,fMo
(5)
where f, “the refractory organic fraction” of the algae, is defined as the fraction of the initial M remaining after 200 days of decomposition, M200/Mo. The k values determined by semilog plots (illustrated for 3 cultures in Figure 12) for 16 cultures had a mean value of 0.022 a standard deviation 0.007, and a range of 0.011-0.032/day. This range of values falls within the range (0.01-0.06/day) which was found (Jewel1 and McCarty, 1968) for the aerobic decomposition of algae with similar culture ages (29-54 days), suggesting that there may be no significant difference in the rate of decomposition of particulate algal organics under aerobic or anaerobic conditions. From values of k determined for the three cultures decomposing at 15”,20”, and 25” C., a value of 0.055j0C . was determined for the temperature coefficient, Ck, as defined by: k/k’
=
&k(T-T‘)
(6)
This value is comparable to those found for other biological systems. Thus, although the temperature study was rather limited in scope, the value found for Chcan be used over the range 15”-25” C. to provide a relatively good estimate of k at T given k’ at T‘. 848 Environmental Science & Technology
Anaerobic decomposition of algae will result when they settle to the bottom of stratified and eutrophic lakes and reservoirs or when they become covered with sediment which limits the diffusion of oxygen. There are two phases of anaerobic decomposition which are significant. In the first phase, the particulate organic matter is converted to soluble forms. This is brought about to some extent by the algae themselves in the absence of bacteria. However, the bacteria enhance the rate of solubilization and also effect the conversion of the soluble organics to short-chain fatty acids. Normally, acetic and propionic acids are the predominant acids formed, but in the presence of active sulfate reduction propionic acid may not be formed. The soluble organic acids may result in various water quality changes. As acids, they tend to lower the pH, affecting subsequent biological activity. The alkalinity of the surrounding water and the acid neutralizing capacity of many sediments may prevent the pH from dropping to biologically inhibiting values. In this case, the volatile acids may be fermented to methane gas or if sulfate is available, they will be oxidized to inorganic products by sulfate reducing microorganisms. The methane gas and sulfides formed can be transported by diffusion or dispersion to aerobic waters above where they may be oxidized, thus adversely affecting the oxygen resources of the overlying waters. The soluble organic acids may be transported in a similar manner and likewise affect the oxygen resources. The oxygen resources of a natural water will not be affected by anaerobic decomposition if methane or sulfides escape to the atmosphere. This is more likely with methane because of its low solubility than with sulfides, although the ebullition of hydrogen sulfide from highly anaerobic waters is well-known.
Table VII. Quantities and Characteristics of Refractory Material Remaining in 20 Algal Cultures after 200 Days of Decomposition M S MIS Culture remaining) zN (mg./mg.) 1 22.3 17.4 7.18 1.28 2 24.4 24.5 5.12 1.14 22.9 19.6 5.49 1.22 3 17.8 16.7 6.59 1.07 4 33.1 25.2 5.56 1.43 9 9.90 1.63 32.5 29.4 31 2.94 1.12 40.2 37.5 32 12.10 1.93 16.1 33 21.0 4.52 1.75 110 45.0 46.1 11.40 1.50 39.0 42.8 111 9.24 1.88 32.2 115 43.8 4.44 1.32 50.0 47.8 116 2.21 1.34 56.4 144 57.3 5.30 2.04 160 27.0 28.9 3.70 1.36 46.0 47.8 162 5.53 1.43 57.8 57.7 163 5.07 1.51 58.4 58.3 164 5.20 1.42 61 . O 59.9 166 3.70 1.57 64.3 57.6 172 3.87 1.62 60.3 54.7 173
(z
Mean Standard deviation Range
41.2
38.6
15.5 18-64
15.4 16-60
5.97 2.72 2.2-12.1
1.48 0.27 1.1-2.0
I””
r-
Acknowledgment This study was supported by research fellowships 1-F1-WP26, 177-01 and 5-F1-WP-26, 177-02 and by research grant WP-1037, all from the Federal Water Pollution Control Administration, United States Department of the Interior. Nomenclature
ck
=
f
= refractory organic fraction = M2m/Mo
k k’
temperature coefficient defined by Eq. 6
first-order decay coefficient, day-I decay coefficient at T’ M = particulate chemical oxygen demand concentration, calculated as M T Ms, mg./l. M remaining) = M expressed as a percentage of the initial M , calculated as 100(M/Mo) MZCQ = value of M after 200 days of decomposition = soluble chemical oxygen demand conMs centration, direct measurement, mg./l. MT = total chemical oxygen demand concentration, direct measurement, mg./l. MT(% remaining) = MT expressed as a percentage of the initial M T , calculated as 1 0 0 ( M ~ / MTJ MTO,MO,SO,%NO = initial values of M T , M , S, and %N at the beginning of decomposition N = particulate nitrogen concentration, calculated as difference between measured total and soluble Kjeldahl nitrogen concentrations, mg./l. = nitrogen content of particulate material Y8 expressed as a percentage of the total ash-free dry weight, calculated as lOO(N/S) %Pr, %Li, ZCa = protein, lipid, carbohydrate content expressed as a percentage of S = volatile suspended solids concentration, S direct measurement, mg.11. S (% remaining) = S expressed as a percentage of the initial S, calculated as 100(S/So) T = temperature, “C. T’ = temperature of determination of decay coefficient = =
(x
\
3 -
* 3-
40
80
I20
I60
ZCO
TIME ldoyil
Figure 12. Examples of plots used for the determination of the first-order decay constant, k Hydrogen sulfide also forms insoluble precipitates with many metals and may be held in the sediments in this form. Such precipitates may keep the concentration of soluble sulfides below that which inhibits methane fermentation so that the two anaerobic mechanisms may proceed simultaneously, even in waters with a high sulfate content. Long-chain fatty acids are not solubilized under most natural water conditions and must be converted biologically to shorter-chain fatty acids or other end-products to escape from anaerobic waters. Anaerobic conditions which result in excessive depression of pH may thus result in a buildup of lipid materials. Perhaps high soluble sulfide concentrations will also cause such a buildup as was observed in this study, although sulfate-reducing organisms capable of oxidizing these materials are probably present in the natural environment. One of the most significant aspects of this study was the observation that the rate and extent of conversion of the particulate matter of algae into soluble forms are essentially the same as those observed under aerobic conditions. This contradicts the general belief. Large accumulations of organic material such as coal and oil deposits have often been attributed to reduced decomposition because of anaerobic conditions. No support for this theory has been obtained in this study. It is as likely that the anaerobic conditions resulted from the large accumulations of organic materials, rather than the reverse. The decay of particulate to soluble COD in decomposing algal cultures can be described by first-order decay kinetics with an indicated mean value of 0.022/day and a relatively narrow range for the first-order decay rate, k , at 20” C. Application of the mathematical expression for the first-order decay requires a knowledge of the value of the refractory organic fraction of algae. An average refractory fraction of about 40 and a standard deviation of about 15 on either a particulate COD or volatile suspended solids basis were found for a wide range of algae. If characteristics such as the identity of the species and the organic composition of the algae under consideration are known, then the results can be used to make a better estimate of the refractory organic fraction of algae with similar characteristics.
x
Literature Cited American Public Health Association, “Standard Methods for the Examination of Water and Waste Water.” 12th ed.. New York, 1965. Golueke, C. G., Oswald, W. J., Gotaas, H. B., Appl. Microbiol. 5, 47-55 (1957). Jewell, W. J., McCarty, P. L., Technical Report No. 91, Department of Civil Engineering, Stanford University, Stanford, Calif., 1968. Lawrence, A. W., McCarty, P. L., Guerin, F. J. A., Int. J. Air Water Pollut. 10, 207-21 (1966). Loehr, R. C., Rohlich, G. A., Proceedings 17th Annual Purdue University Industrial Waste Conference, 21 5-32 (1962). McCarty, P. L., “Principles and Applications in Aquatic Microbiology,” Heukelekim, H., and Dondero, N.C., Ed., 314-43, Wiley, New York, 1964. McCarty, P. L., Jeris, J. S., Murdoch, W., J . Wuter Pollut. Contr. Fed. 35, 1501-15 (1963). Milner, H. W., “Algal Culture from Laboratory to Pilot Plant,” Burlew, J. S., Ed., 285-302, Carnegie Institution, Washington, D. C. 1953. Starr, R. C., Amer. J. Bot. 51, 1013-44 (1964). Wyckoff, B. M., Water Sewage Works 111, 277-80 (1964). Young, J. C., McCarty, P. L., Technical Report No. 87, Department of Civil Engineering, Stanford University, Stanford, Calif., 1968. Receiced for reuiew January 12, 1970. Accepted June I , 1970. Presented in part at the 24th Annual Purdue Industriul Waste Conference, May 1969. Volume 4, Number 10, October 1970 849
