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Denitrification unit biocenosis
Wat. Res. Vol. 25, No. 12, pp. 1565-1573, 1991 Printed in Great Britain.All rights reserved
0043-1354/91 $3.00+0.00 Copyright © 1991 PergamonPress plc
DENITRIFICATION UNIT BIOCENOSIS A. GRABI/~ISKA=LONIEWSKA Institute of Environmental Engineering, Warsaw Technical University, Nowowiejska 20, 00 653 Warsaw, Poland (First received January 1990; accepted in revised form April 1991)
Al~traet--It has been found that during heterotrophic denitrification, biocenosis of the upfiow anaerobic sludge blanket (UASB) reactor is composed of bacteria, fungi, protozoa and rotifers. Among these groups of organisms close ecological relationships exist. The scheme of these relationships is proposed. Also described are quantity, taxonomy and biochemical features of denitrifying bacteria and fungi occurring in biocenoses during the processes carried out in the presence of methanol, glycerol, acetic and lactic acids. Key words--denitrification, carbon sources for denitrification, denitrifying biocenosis, yeasts and yeastlike microorganisms, ecology of wastewater purification process
NOMENCLATURE X = biomass concentration, as VSS (rag 1-1 ) Ox = biomass residence time (days) Bv = nitrate volumetric loading rate (rag NO3N l - I day -I ) B,~nt = Bv value beyond which the washing-out of biomass is observed (as above) Bx = nitrate loading rate per unit biomass (mg NO3-N mg- l day- i ) D = dilution rate (h -I ) /z = specific biomass growth rate (h- ~) E h = redox potential (mV) Optimal C: N ratio = mg C mg NO3-N removed-I--experimentally determined according to GrabifiskaLoniewska et al. (1975) INTRODUCTION The removal of nitrates from wastewaters can, depending on the technology employed (aerobic, anoxic and anaerobic methods), occur through their assimilation or dissimilation by microorganisms (Fig. 1). In the assimilation pathway nitrates are transformed under aerobic or anoxic conditions into a m m o n i u m nitrogen which is then incorporated into cellular protein. In general, it can be said that in this case nitrates are transformed into the biomass of bacteria, fungi, algae or higher plants. The intensity of these transformations, due to their anabolic nature, is rather low. Hence, the assimilation of nitrates is used in practice (waste stabilization ponds) only in the purification of wastewaters with low content of these compounds (maximally up to 20 mg NO3-N l-l). In the dissimilation pathway nitrates are reduced by bacteria to gaseous oxides (NO, N 2 0 ) or dinitrogen (denitrification). Being a catabolic transformation in anaerobic conditions it is much more efficient than the assimilation of nitrates. Denitrification has found widespread use in the removal of nitrates from wastewaters containing both high and low
concentrations of these compounds (reaching even 50 g NO3-N l-Z in wastewaters from the production of radioactive fuels). In the removal of nitrates in either of these systems the possibility of the development in the biocenoses of populations of microorganisms differentiated with respect to physiology should be taken into account. For example, it has been shown that in algae-based waste stabilization ponds approx. 60% nitrates are removed due to assimilation by algae and 40% by denitrification and sorption processes occurring in muddy sediments (Wr6bel, 1972). The purpose of this research was the quantitative and taxonomic characterization of the microorganisms forming the denitrification unit biocenosis and the determination of their role in the nitrate removal process. MATERIALS AND METHODS Operation of denitrifying unit The denitrification process was carried out in a modified UASB reactor using as a feed a synthetic medium containing 600 mg N O : N 1-~, essential biogens and as C-source one of the following compounds: methanol, glycerol, acetic or lactic acid. The concentration of the carbon compound in the feed gave optimal C:N ratios for the biocenoses equal to 1.17, 1.02, 1.26 and !.67, respectively. The process was carried out at different nitrate volumetric loading rates (Bv)--~ontrolled by dilution rate---D. Experimental arrangement, details of unit design and methods of calculating the process parameters have been given elsewhere (Grabifiska-Loniewska et al., 1985; Grabifiska-Loniewska, 1990). Chemical analyses Glycerol and acetic acid were assayed by the titration methods described by Bauer (1950). Lactic acid was determined speetrophotometrically at ,l = 570nm, using a 1.5% solution of p-hydroxydiphenyl in 5% NAOH for colour development (Zmarlicki, 1981). Samples with nitrates removed by ion exchange were used for this analysis. Other chemical analyses were carried out according to Hermanowicz et al. (1976).
1565
A. GRABII~ISKA-LONIEWSKA
1566
Nitrate metabolism in microorganisms
I
Assimilatory reduction
Bacteria,fungi,algae
+3 NO~ i nitrite NO~ nitrate
+1 -1 -3 =. [NOH] ----~-NH2OH--,,.- NH~ =. Amino acids nitroxyl hydroxyla- Jr ammonia (?) mine /
+5
Oxidation state of N
(higher plants,too)
l
INHoOH-R]
lNa R-type B]
Dissimilatory reduction Oxidation state of N
+5 NO3 nitrate
+3 == l
+2
==
NO2
l
nitrite
NO
nitricoxide
Na R-type A Bacteria •
fungi
r
only
r
Bacteria •
Nitrate respiration
+1 0 N2 0 =. N2 nitrous oxide I nitrogen
m
l .°.
I I
I
•
~
Denitrification
Abbreviations: enzymes: Na R-type B,assimilatory nitrate reductase; Na R-type A, dissimilatory (respiratory) nitrate reductase; NiR,nitrite reductase; NOR,nitric oxide reductase; N2OR,nitrous oxide reductase; NH 20H-R, hydroxylamine reductase.
Fig. 1. The pathways of nitrate metabolism in microorganisms. Biological analyses Microscopic observations of the biocenoses were made with a phase contrast microscope. The total numbers of bacteria and fungi were determined by the plate method. Samples to be plated out were treated for 15 min at 1000 rpm in a Unipan type 302 homogenizer. For cultivation of denitrifiers nitrate broth solidified with agar (2%) was used as the growth medium. Their count was based on visible gas production, nitrite detection and then their disappearance after the growth of picked colonies in nitrate broth, according to the method proposed by Gamble et al. (1970). The quantity of other saprophytic, non-denitrilying bacteria was calculated by subtracting the denitrifier count from the total bacteria count on the nitrate agar. The total number of fungi was determined on Martin's agar medium (Sl~vikovfi and Grabifiska-Loniewska, 1986/1988). Inoculated plates were incubated at 26°C and after 7 days bacterial and fungi species were recorded and isolated by the plate method. Denitrifying bacteria, yeasts and yeast-like strains were identified on the basis of their cell and culture morphology and physiological characters. Identification of moulds was based on descriptions of their asexual stage, including character of colonies and details of morphology of the spore-bearing structures (Webster, 1983). All isolated strains (with the exception of moulds) were tested for their ability to utilize methanol, glycerol, acetic and lactic acids as a sole C-source. The basal mineral medium used in these investigations was similar to Pichinoty et al.'s (1977) medium with nitrate as a N-source. Their capability to use ammonium sulphate as a N-source as well as to assimilate and dissimilate nitrates in the presence and absence of yeast extract was also tested. Yeasts and yeastlike strains were also checked for their ability to grow on a mineral medium with glucose but without any N-source. The methods of taxonomical and special physiological
studies of bacteria and yeasts are given in previous papers (Grabifiska-Loniewska, 1990; Sl~ivikov~i and GrabifiskaLoniewska, 1986/1988). RESULTS AND DISCUSSION Studies were performed at By ranges securing biocenosis growth characterized by the relationships: (i) ~ = 1/O2 > D and (ii) # = 1/6)x < D. In case (i), when By < Bvent, biomass concentration in the reactor (X) gradually increased. The values of Bveat, beyond which a sharp decrease in the biomass concentration in the reactor was observed, were comparable to "washing-out points". This showed that the flow rate was greater than the biocenosis growth rate (ii). The value of Bvent for the process carried out in the presence of methanol was: 1400 mg NO3-N 1-~ day-~; glycerol, 800mg NO3-N! ~; acetic acid, 250mg NO~-N 1- ~d a y - ~; and lactic acid, 600 mg NO3-N 1day -t (Fig. 2). In practice, the value of Bvcrit, at which maximal biomass concentration was attained, also determined the maximal admissible volumetric nitrate loading rate. Because, as a result of a further increase of By beyond Bvcrit, the unit nitrogen removal rate sharply decreased (Grabifiska-Loniewska, 1990). General characteristics o f the biocenoses The biocenoses consisted of bacteria, fungi, protozoa and rotifers. The structural matrix of the floes in the mixed liquor and biofilm fixed to the walls of the
Denitrification unit biocenosis Sign 1
•
C-source Methanol
9000
8000
1567
Equation X = - 2 . 7 x 1 0 " l ° ( B v ) 4 -3.5x10 "s (By) s + +4.4x10 "3 (Bv) 2 +4.96(Bv)
2
o
Glycerol
X = - t . 5 x l 0 " ~ ( B v ) 4 5.8x10 "s (Bv)3+ -1.3x10 -2 (By)2 +13(Bv)
3
x
Acetic acid
X = - 4 . 8 x 1 0 "e(Bv) 4 +8.1x10 "s (By)3+ -5.3x10 -2 (Bv) 2 +14(By)
4 ,',
Lactic acid
X = - 3 . 6 x 1 0 "8 (By) 4 +5.8x10 "s (By)3+ -4.4x10 "2 (By) 2 +22(Bv)
7000
6000
O')
5000
E
4000
3000
2000
1000
I 200
I
I 400
I
I ~1 600
3
I 800
e v
I
I 1000
I
I 1200
I
I 1400
I 1600
I
I 1800
(rag NO 3- N I1 day-1)
Fig. 2. Reactor biomass concentration (X) depending on the nitrate load (By) during the denitrification carried out in the presence of different C-sources. reactor was formed by bacteria and yeast cells, stuck together by slime. Hyphal fungi, represented by the genus Fusarium, grew in clumps in the mixed liquor. The shape, size and structure of the flocs depended on the type of hydrogen donor and the value of Bv. The flocs were particularly large (with some reaching a width of 396 #m and a length of 4140 #m) and branched, with a compact, granulated structure in biocenoses fed with acids. In the presence of glycerol the flocs were gelatinous. Flocs of biocenoses fed with methanol resembled those of standard activated sludge. As a rule, an increase in By value was accompanied by a greater dispersion of the flocs. This was particularly evident at Bv > Bvcnt (Figs 3 and 4). When the process was conducted at By < Bvcrit the number of denitrifiers and fungi per mg VSS was constant. Table 1 shows that biocenoses fed with compounds with a lower number of carbon atoms per molecule were characterized by a greater number of denitrifiers. However, the proportion of denitrifiers
was higher in biocenoses fed with alcohols than in those growing in the presence of organic acids. In the latter case a higher proportion of other saprophytic bacteria, not denitrifiers, was observed. These biocenoses were also characterized by a 10-fold increase in the number of fungi. This agrees with the nutritional requirements of fungi, which prefer organic acids over alcohols as a C-source (Lodder, 1970; Ko~kov~-Kr~itochvilov~i, 1982; Kreger-van Rij, 1984). These results also show that as regards both types of studied compounds, fungi were more abundant in biocenoses when a compound with a lower number of carbon atoms per mocelcule was used as a C-source. However, based on the proportion of these microorganisms in the biocenoses, lactic acid can be regarded as a better carbon substrate for them than acetic acid (0.21 and 0.15% of total number of microorganisms, respectively). When the process was carried out at Bv > B~erit the number of denitrifiers in all biocenoses dropped.
A. G~BZfiSKA-LONtEWSKA
1568
Methanol
ticularly evident in the biocenosis fed with lactic acid. Under these conditions, the proportion of fungi in biocenoses decreased, but only in those fed with alcohols (Table 1). From the ecological point of view, the occurrence of protozoa and rotifers in the biocenoses is of interest. Until now, the presence of these microorganisms in denitrifying biocenoses was not taken into account. Recently, the presence of microfauna in anoxic activated sludge was described by Wanner et al. (1987). The studies discussed in this paper show that Mastigota nd and Amoeba proteus were generally stable components of the biocenoses. Ciliated protozoa usually appeared at Bv, no higher than 330 mg NO3-N 1-1 day-l. Attached ciliates belonging to the genus Opercularia were observed in biocenoses fed with glycerol or acetic acid. Epistylis sp. was present in the biocenosis fed with glycerol. Free-swimming ciliates of the genus Glaucoma appeared in that fed with lactic acid. Rotifers were found in the biocenosis grown in the presence of acetic acid at By = 220 mg NO3-N 1-1 day -1. The reason for the disappearance of ciliates and rotifers from the biocenosis at Bv beyond 330 and 220mg NO3-Nl-~day -1 respectively, probably lies in the negative effect of nitrate concentration on their growth. At B v = 3 3 0 m g NO3-N 1-1 day -~ the hydraulic retention time was 43.5 h and thus the "washing out" of these organisms from the reactor could not have been responsible (Fig. 5).
E-E
108. 127.630 720 0.2110.17 1016
72. 54. 55. 57- 78. 450 475 228 380 323 0.12 0.1510.22 0.19 0.311
1975 374414320 4980 7050 5700
Glycerol
E 0~
:t.
~ Q
>E mE I max, txm
170484
170-1130 0.08 I 2440 (
0.01
120300
5296
0.14 0.27 0.40 4530 4070 3320
0.02
i .o i,,o i ,oo 1 o1, o0 0.03
0.04 0.08 0.09
0.12
Fig. 3. The size (on an average basis, without taking shape into consideration) and structure of biocenoses floes depending on operational parameters (D, X, By, Bx) in the processes carried out in the presence of methanol and glycerol as C-sources. Symbols: [], dense structure: D, loose structure. It should be noticed however that the decrease of the number of other saprophytic non-denitrifying bacteria at the same time was much smaller. Thus the percentage of these bacteria in the total number of microorganisms markedly increased. This was par-
Acetic acid
as below 170-900
85-360
52-230
40-270
o,o
,,,
io,° i o,, 1380
1200
1080
:'.'..'.:"
200
Lactic acid
..
-........ r
o
o o o
Size average lmm=10 p.m
:." ..'... • •
~ ~
•
• •
°
" •
. '
°
o o o
•
•
o
o o o
o o
o o
o o
o
1
max,
p. m BX x
i~ v
•
174-
223-
1800 4140 I 0,08 I 0.07 [ 3 2 8 0 4I3 0 0 220
0,01
330 0.02
o
"
"
356-2880 0.09 4790 440 0.03
° a
396-2880 0.13 5170 1670 0.04
172.
1440 0.31 2850 890 0.06
Fig. 4. The size (on an a v e r a g e b a s i s , without taking shape into consideration) and structure of biocenoses floes depending on the operational parameters (D, X, By, Bx) in the processes carried out in the presence of acetic and lactic a c i d s a s C-sources. Symbols: t':], dense structure; F~, loose structure.
Denitrification unit biocenosis
1569
Table 1. Numbers and proportions of bacteria and fungi in biocenosesdependingon C-sourceas wellas reactor (By)and biomass (Bx) nitrate loading Process parameters Nitrate loading Number per mg VSS Proportion in biocenoses (%) Bacteria Reactor (By) Biomass (Bx) "total" Denitrifiers Fungi Other C-source mg NOa-N1-~day-~ mg NO3-Nmg ~day ~ ( x 106) ( x 106) ( x 103) Denitrifiers bacteria Fungi Methanol 220-1330 0.124).22 400 384 27 95.8 4.1 0.006 1800 220-670
0.31 0.084).14
196 217
181 210
6.0 15.0
92.3 96.6
7.6 3.4
0.003 0.007
Acetic acid
1100 1330 220
0.27 0.40 0.16
201 156 386
196 129 330
15.1 1.6 580
97.5 82.6 85.4
2.4 17.3 14.5
0.007 0.001 0.15
Lactic acid
330 440 220-670
0.27 0.40 0.064).13
197 63 197
166 42 167
480 280 373
84.0 66.4 86.2
15.9 33.5 13.7
0.24 0.44 0.21
890 0.31 180 32 390 17.7 Comment: the characteristic values for the processes carried out at By~
82.2
0.22
Glycerol
Taxonomy of bacteria and fungi The bacterial flora consisted in the main of typical denitrifying species such as Pseudomonas stutzeri,
P. mendocina, P. aureofaciens, P. solanacearum, P. caryophylli, Alcaligenes denitrificans and Agrobacterium radiobacter (Leifson and Hugh, 1954; Bergey, 1974; Pichinoty et al., 1977). Denitrification was also found to be carried out by some bacteria, which had previously either not been known to belong to this physiological group (P. delafieldi, P. palleronii) or had been known to reduce nitrates to nitrites only (P. cepacia, P. diminuta, Proteus inconstans group A) (Bergey, 1974). Biocenoses fed with organic acids had a more diverse composition of denitrifying bacteria than those growing in the presence of alcohols. They usually contained 3-4 species, 2 or 3 of which could be considered as dominating. A typical species in the biocenosis fed with acetic acid was P. stutzeri and in that growing in the presence of lactic acid, P. diminuta. Those species which dominated during the process at Bv < Bvcrit were regarded as typical. When alcohols were used as a carbon source the bacterial flora was subject to greater selection. In the biocenosis fed with glycerol only varieties of sub-group A of Proteus inconstans were present. The biocenosis growing in the presence of methanol was occasionally dominated by P. stutzeri and Agrobacterium radiobacter but P. cepacia was a typical species for it (Fig. 6). The carbon source also influenced the species composition of the mycoflora. The main organisms were yeasts and yeast-like microorganisms, occasionally also hyphal fungi, represented by Fusarium sp., were present. A typical species for the biocenosis fed with methanol was Candida boidinii, and with acetic a c i d - Candidafamata. In biocenosis grown in the presence of lactic acid C. famata and Hansenula californica permanently dominated. Besides these organisms in these biocenoses, representatives of C. lambica, C.
tropicalis, C. inconspicua, C. maltosa, Rhodotorula WR 25/12--1
rubra and Geotrichum candidum were also found (Fig. 7). The taxonomy and morphological, cultural and physiological properties of bacterial and fungal strains isolated from the biocenoses have been described elsewhere by Grabifiska-Loniewska et al. 0986/1988, 1990).
Interactions between microorganisms Earlier studies (Grabifiska-Loniewska, 1990) have shown that most of the bacterial species isolated from biocenoses, in pure cultures carried out denitrification only in the presence of yeast extract or a mixture of organic compounds in the medium. During the course of the process in a reactor in which mineral medium was enriched only in the studied carbon compound, these substances promoting the occurrence of denitrification could be derived from autolysed cells of microorganisms in the biocenoses (bacteria, fungi, microfauna), or could be produced by some of these organisms (mainly by fungi). Yeasts and yeast-like microorganisms did not directly participate in denitrification. For their growth they utilized the compounds used as the source of carbon and hydrogen donor for denitrification, nitrates (assimilation pathway), a m m o n i u m nitrogen, amino acids (e.g. lysine, tryptophan) and vitamins. Most of these microorganisms grew in a mineral medium without any N source (GrabifiskaLoniewska and Sl/tvikov~, 1990). This may indicate their ability to fix N2 arising during denitrification. This fact has considerable ecological impact since it suggests that the growth of most fungal species in denitrification units occurs only in mixed culture with denitrifying bacteria. The microfauna of the biocenoses consisted of both saprotrophic and phagotrophic organisms. The former included Mastigota nd, the latter Amoeba proteus, free-swimming and attached ciliates and rotifers. Saprotrophic protozoa were particularly
1570
A. GRAm~SKA-LoNIEWSKA Methanol 2
2400
2300
x--
~E
1
g-o
_
Glycerol
"SE1 i ~ ¢.--
E
I g"
I Bx
I
o,o8
2 ~-
]
I
0.16
I 4~°3300
10.271
0.40
Acetic acid
13.351 55750
2 F I 8186
~-"
O
0.01
Lactic acid
9533 •
0
'ida • 3600
0.02
0.03
0.04 10.06
"0.08
0.09
Fig. 5. Animal microorganisms in biocenoses during the processes carried out with different C-sources and operational parameters (D, Bv, Bx). Symbols: II, Mastigota nd; g~, Amoeba proteus; [], Amoeba radiosa; [~, Testacea nd; D, Glaucoma sp.; I~, Opercularia sp.; I~, Epistylis sp.; ~, Rotifera nd.
numerous in the biocenoses in the periods immediately following change of Bv and at values By > Bv,~t. At the same time, a transient increase in the number of other saprotrophs, i.e. bacteria and fungi in the dispersed phase, was observed, due to the increased shredding of the floes and loosening of their structure. In turn the growth of phagotrophic protozoa and rotifers occurred, though only at By values lower than 330 mg NO 31- ~day- 1. The occurrence of animal microorganisms in the bioeenoses when the process was conducted at an Eh between - 100 and - 4 5 0 mV indicates their high capacity for adaptation. It can therefore be assumed that these organisms can utilize both oxygen and nitrates at electron aeceptors during respiration.
Based on the above characteristics of the nutritional requirements of different groups of microorganisms it can be said that in these biocenoses both positive and negative interactions take place. The positive interactions embrace competition and mutualism. The populations competing for food are saprotrophic protozoa from the Mastigota group, bacteria and fungi. Symbiotic relationships occur between denitrifying bacteria and fungi. Negative interactions include the predatory behaviour of phagotrophic protozoa towards bacteria and fungi and rotifers towards sapro and phagotrophie protozoa. Predatory protozoa may play a role in regulating the growth of bacteria and fungi and thus causing the "rejuvenation" of the population of these
Denitrification unit biocenosis Carbon sources
Typical species:
(Values of B v crit.)
Methanol
o m
1571
L 0.21 1016
1975
Glycerol
(14oo mg NOa-N I-1 day-l)
I
3744
I
4320
Ps. cepacia
I
7050
(800 mg NO 3-N I -1 day -1)
o
g
5700
Proteus inconstans group A, type
t~
~o
0.08 2440
_
_ _
.....
....
0.27 4070
0.40 3320
I I
Acetic acid (250 mg NO 3-N I -1 day -1)
Ps. stutzeri
~1380
1200
1080
200
(% 100 7 q 17
Lactic acid (600 mg NOa-N I -1 day -1)
8
Ps. diminuta
6O 4O 2
0.01
0.02
0.03
0.04
0.06
0.08
0.09
0.12
Fig. 6. Proportions of bacterial species in biocenoses fed with different C-compounds depending on the operational parameters (D, Bv, X, Bx) of the denitrification process. Symbols: [], Alcaligenes denitrificans; K], Agrobacterium radiobacter ; II, Ps. aureofaciens ; [~, Ps. caryophylli; [], Ps. cepacia ; [], Ps. delafieldii ; []~ Ps. diminuta; [], Ps. mendocina; [57, Ps. solanacearum; CA, Ps. stutzeri; [], Ps. palleroni; [7, Proteus inconstans g r o u p A, type a; I~, Proteus inconstans group A, type b.
microorganisms in the biocenoses. This is of importance in the maintenance of their appropriate physiological activity which in turn determines the intensity of biochemical processes in the reactor. A scheme of the ecological relationships between the individual groups of microorganisms populating the reactor is presented in Fig. 8. To sum up these observations it can be said that the role of the microfauna in denitrification unit biocenosis is much smaller than in aerobic activated sludge. Due to the presence of nitrates in the medium it contains no phagotrophic organisms at By > 330 mg NO3-N 1-~ day-l. Under such conditions they do not therefore play a particularly important
ecological role in regulating the growth of bacteria and fungi in the biocenoses. Hence in the scheme given in Fig. 8 when the process is conducted at By > 330mg NO3-N1-1 day -1, consumers of the trophic level III need not be taken into account. CONCLUSIONS
From the results obtained in this study, it is clear that during heterotrophic denitrification a specific biocenosis is formed. It is composed not only of denitrifiers but also of other saprophytic non-denitrifying bacteria, yeasts, yeast-like microorganisms and microfauna. These groups of microorganisms relate
Carbon sources ~o ~]
Methanol
1016
•
Jl
0.17 I 0.12 10.16
1975
3744
species:
Candida boidlnii
(1400 mg NO3-N I-1 day-l)
•
0.21
.. X
Typical
(Values of B v cril.)
4320
I
Acetic acid (250 mg NO 3.N I 1 day -1) o
LL _rk
o~
X
1380
1200
1080
Candida famata
200
(%) 100 8O 60 40 20
Lactic acid (600 mg NO3-N I -1 day -1) Candida famata Hansenula californica
lhk X
0.06 3280
0.07 4300
0.09 4790
J~v°l
220 0.01
330 0.02
440 0.03
::01
•
I
°,o i 0.06 ,oo i,,oo I 0.08
0.04
133011800 ] 0.09 0,12
Fig. 7. Proportions of fungal species in biocenoses fed with different C-compounds depending on the operational parameters (D, B,, X, B~) of the denitrification process. Symbols: rq, C. boidinii; r~, C. famata; [2], C. mahosa; [], C. tropicalis; k~, Geotrichum candidum; [], Hansenula californica; [~, Rhodotorula rubra; [~, Trichosporon cutaneum; II, Fusarium sp.
I
u,,,,z°r,
I I consu °r
Nitrate dissimilationdenitrification --I=-
,/
~N~,F. ~, ~F.do,F.~.o~c.~
assimilation
II
~N.l,xation
/
, iA L_
Ill
I
, Oen/t;ifIyinIgre~ra'~Ig~ . / ~ I
I
~o,.,..~o ........ ,
,
IA ~. . . . . . .
iA I
~ [
V [ [
/1/IA
Compounds released ,fromautolize/dcel~s I L
I
/ I L__J
Fig. 8. Ecological relationships between denitrification unit microorganisms (with organic compound as a hydrogen donor), according to Grabifiska-Loniewska. A, cell autolysis; U, utilization; C, consumption. Enzymes: NaR, nitrate reductase; NiR, nitrite reductase, NOR, nitric oxide reductase; N2OR, nitrous oxide reductase; I-III, trophic levels. 1572
Denitrification unit biocenosis to each other in strict ecological terms. Thus, the scope of biological studies in the control of denitrifying unit biocenosis should be similar to the standard activated sludge analyses. It has been found that besides denitrifiers, yeasts and yeast-like microorganisms played a major role in this biocenosis. They promoted the course of the denitrification process by the intensification of the biochemical activity of denitrifying bacteria. Therefore, the microbiological control studies of the denitrification process ought to include both the quantity of denitrifiers as well as yeasts and yeast-like microorganisms.
REFERENCES
Bauer K. H. (1950) Die Organische Analyse. Akad. Verlagsgesellschaft Geest und Portig. Bergey (1974) Bergey's Manual of Determinative Bacteriology, 8th edition. Williams & Wilkins, Baltimore, Md. Gamble T. N. Betlach M. R. and Tiedje J. M. (1977) Numerically dominant denitrifying bacteria from world soils. Appl. envir. Microbiol. 33, 926-939. Grabifiska-Loniewska A. (1990) Influence of different carbon compounds on the biocenoses formation during nitrogen removal process by denitrification method. Warsaw Technical University, Scientific Papers, No. 10, Warsaw (in Polish).
1573
Grabifiska-Loniewska A. and Sl~ivikovAE. (1990) Fungi in denitrification unit biocenosis. Wat. Res. 24, 565-572. Grabifiska-Loniewska A., Slomczyhski T. and Kafiska Z. (1985) Denitdfication studies with glycerol as a carbon source. Wat. Res. 19, 1471-1477. Hermanowicz W., Do~afiska W., Dojlido J. and Koziorowski B. (1976) Fizyczno-chemiczne Badanie Wody i ~ciek6w. Arkady, Warsaw. Ko~kov~-Kr~tocvilov~ A. (1982) Kvasink# a Kvasinokovit~ Mikroorganizmy. ALFA, SNTL, Bratislava, Prague. Kreger-van Rij N. J. W. (1984) The Yeasts, a Taxonomic Study. Elsevier, Amsterdam. Leifson E. and Hugh R. (1954) Alcaligenes denitrificans n. sp. J. gen. Microbiol, II, 512-513. Lodder J. (1970) The Yeasts, a Taxonomic Study. North Holland, Amsterdam. Pichinoty F., Mandel M. and Garcia J. L. (1977) Etude de six souches de Agrobacterium tumefaciens et A. radiobacter. Ann. Microbial. (Inst. Pasteur) 128A, 303-310. Sl~vikov~ E. and Grabifiska-Loniewska A. (1986/1988) The yeasts and yeast-like microorganisms in denitrification unit biocenosis. Acta Mycol. 22, 177-184. Wanner J., Chudoba J,, Kucman K. and Proske L. (1987) Control of activated sludge filamentous bulking--VII. Effect of anoxic conditions. Wat. Res. 21, 1447-1451. Webster J. (1983) Pilze. Springer, Berlin. Wr6bel S. (1972) Eliminacja mineralnych zwi~zk6w azoutu ze ~ciek6w przy udziale roglin ni~szych. Wiadomo~ci Ekologiczne 18, 179-203. Zmarlicki S. (1981) Cwiczenia z Analizy Mleka i Produkt6w Mlecznych. SGGW-AR, Warsaw.
0043-1354/91 $3.00+0.00 Copyright © 1991 PergamonPress plc
DENITRIFICATION UNIT BIOCENOSIS A. GRABI/~ISKA=LONIEWSKA Institute of Environmental Engineering, Warsaw Technical University, Nowowiejska 20, 00 653 Warsaw, Poland (First received January 1990; accepted in revised form April 1991)
Al~traet--It has been found that during heterotrophic denitrification, biocenosis of the upfiow anaerobic sludge blanket (UASB) reactor is composed of bacteria, fungi, protozoa and rotifers. Among these groups of organisms close ecological relationships exist. The scheme of these relationships is proposed. Also described are quantity, taxonomy and biochemical features of denitrifying bacteria and fungi occurring in biocenoses during the processes carried out in the presence of methanol, glycerol, acetic and lactic acids. Key words--denitrification, carbon sources for denitrification, denitrifying biocenosis, yeasts and yeastlike microorganisms, ecology of wastewater purification process
NOMENCLATURE X = biomass concentration, as VSS (rag 1-1 ) Ox = biomass residence time (days) Bv = nitrate volumetric loading rate (rag NO3N l - I day -I ) B,~nt = Bv value beyond which the washing-out of biomass is observed (as above) Bx = nitrate loading rate per unit biomass (mg NO3-N mg- l day- i ) D = dilution rate (h -I ) /z = specific biomass growth rate (h- ~) E h = redox potential (mV) Optimal C: N ratio = mg C mg NO3-N removed-I--experimentally determined according to GrabifiskaLoniewska et al. (1975) INTRODUCTION The removal of nitrates from wastewaters can, depending on the technology employed (aerobic, anoxic and anaerobic methods), occur through their assimilation or dissimilation by microorganisms (Fig. 1). In the assimilation pathway nitrates are transformed under aerobic or anoxic conditions into a m m o n i u m nitrogen which is then incorporated into cellular protein. In general, it can be said that in this case nitrates are transformed into the biomass of bacteria, fungi, algae or higher plants. The intensity of these transformations, due to their anabolic nature, is rather low. Hence, the assimilation of nitrates is used in practice (waste stabilization ponds) only in the purification of wastewaters with low content of these compounds (maximally up to 20 mg NO3-N l-l). In the dissimilation pathway nitrates are reduced by bacteria to gaseous oxides (NO, N 2 0 ) or dinitrogen (denitrification). Being a catabolic transformation in anaerobic conditions it is much more efficient than the assimilation of nitrates. Denitrification has found widespread use in the removal of nitrates from wastewaters containing both high and low
concentrations of these compounds (reaching even 50 g NO3-N l-Z in wastewaters from the production of radioactive fuels). In the removal of nitrates in either of these systems the possibility of the development in the biocenoses of populations of microorganisms differentiated with respect to physiology should be taken into account. For example, it has been shown that in algae-based waste stabilization ponds approx. 60% nitrates are removed due to assimilation by algae and 40% by denitrification and sorption processes occurring in muddy sediments (Wr6bel, 1972). The purpose of this research was the quantitative and taxonomic characterization of the microorganisms forming the denitrification unit biocenosis and the determination of their role in the nitrate removal process. MATERIALS AND METHODS Operation of denitrifying unit The denitrification process was carried out in a modified UASB reactor using as a feed a synthetic medium containing 600 mg N O : N 1-~, essential biogens and as C-source one of the following compounds: methanol, glycerol, acetic or lactic acid. The concentration of the carbon compound in the feed gave optimal C:N ratios for the biocenoses equal to 1.17, 1.02, 1.26 and !.67, respectively. The process was carried out at different nitrate volumetric loading rates (Bv)--~ontrolled by dilution rate---D. Experimental arrangement, details of unit design and methods of calculating the process parameters have been given elsewhere (Grabifiska-Loniewska et al., 1985; Grabifiska-Loniewska, 1990). Chemical analyses Glycerol and acetic acid were assayed by the titration methods described by Bauer (1950). Lactic acid was determined speetrophotometrically at ,l = 570nm, using a 1.5% solution of p-hydroxydiphenyl in 5% NAOH for colour development (Zmarlicki, 1981). Samples with nitrates removed by ion exchange were used for this analysis. Other chemical analyses were carried out according to Hermanowicz et al. (1976).
1565
A. GRABII~ISKA-LONIEWSKA
1566
Nitrate metabolism in microorganisms
I
Assimilatory reduction
Bacteria,fungi,algae
+3 NO~ i nitrite NO~ nitrate
+1 -1 -3 =. [NOH] ----~-NH2OH--,,.- NH~ =. Amino acids nitroxyl hydroxyla- Jr ammonia (?) mine /
+5
Oxidation state of N
(higher plants,too)
l
INHoOH-R]
lNa R-type B]
Dissimilatory reduction Oxidation state of N
+5 NO3 nitrate
+3 == l
+2
==
NO2
l
nitrite
NO
nitricoxide
Na R-type A Bacteria •
fungi
r
only
r
Bacteria •
Nitrate respiration
+1 0 N2 0 =. N2 nitrous oxide I nitrogen
m
l .°.
I I
I
•
~
Denitrification
Abbreviations: enzymes: Na R-type B,assimilatory nitrate reductase; Na R-type A, dissimilatory (respiratory) nitrate reductase; NiR,nitrite reductase; NOR,nitric oxide reductase; N2OR,nitrous oxide reductase; NH 20H-R, hydroxylamine reductase.
Fig. 1. The pathways of nitrate metabolism in microorganisms. Biological analyses Microscopic observations of the biocenoses were made with a phase contrast microscope. The total numbers of bacteria and fungi were determined by the plate method. Samples to be plated out were treated for 15 min at 1000 rpm in a Unipan type 302 homogenizer. For cultivation of denitrifiers nitrate broth solidified with agar (2%) was used as the growth medium. Their count was based on visible gas production, nitrite detection and then their disappearance after the growth of picked colonies in nitrate broth, according to the method proposed by Gamble et al. (1970). The quantity of other saprophytic, non-denitrilying bacteria was calculated by subtracting the denitrifier count from the total bacteria count on the nitrate agar. The total number of fungi was determined on Martin's agar medium (Sl~vikovfi and Grabifiska-Loniewska, 1986/1988). Inoculated plates were incubated at 26°C and after 7 days bacterial and fungi species were recorded and isolated by the plate method. Denitrifying bacteria, yeasts and yeast-like strains were identified on the basis of their cell and culture morphology and physiological characters. Identification of moulds was based on descriptions of their asexual stage, including character of colonies and details of morphology of the spore-bearing structures (Webster, 1983). All isolated strains (with the exception of moulds) were tested for their ability to utilize methanol, glycerol, acetic and lactic acids as a sole C-source. The basal mineral medium used in these investigations was similar to Pichinoty et al.'s (1977) medium with nitrate as a N-source. Their capability to use ammonium sulphate as a N-source as well as to assimilate and dissimilate nitrates in the presence and absence of yeast extract was also tested. Yeasts and yeastlike strains were also checked for their ability to grow on a mineral medium with glucose but without any N-source. The methods of taxonomical and special physiological
studies of bacteria and yeasts are given in previous papers (Grabifiska-Loniewska, 1990; Sl~ivikov~i and GrabifiskaLoniewska, 1986/1988). RESULTS AND DISCUSSION Studies were performed at By ranges securing biocenosis growth characterized by the relationships: (i) ~ = 1/O2 > D and (ii) # = 1/6)x < D. In case (i), when By < Bvent, biomass concentration in the reactor (X) gradually increased. The values of Bveat, beyond which a sharp decrease in the biomass concentration in the reactor was observed, were comparable to "washing-out points". This showed that the flow rate was greater than the biocenosis growth rate (ii). The value of Bvent for the process carried out in the presence of methanol was: 1400 mg NO3-N 1-~ day-~; glycerol, 800mg NO3-N! ~; acetic acid, 250mg NO~-N 1- ~d a y - ~; and lactic acid, 600 mg NO3-N 1day -t (Fig. 2). In practice, the value of Bvcrit, at which maximal biomass concentration was attained, also determined the maximal admissible volumetric nitrate loading rate. Because, as a result of a further increase of By beyond Bvcrit, the unit nitrogen removal rate sharply decreased (Grabifiska-Loniewska, 1990). General characteristics o f the biocenoses The biocenoses consisted of bacteria, fungi, protozoa and rotifers. The structural matrix of the floes in the mixed liquor and biofilm fixed to the walls of the
Denitrification unit biocenosis Sign 1
•
C-source Methanol
9000
8000
1567
Equation X = - 2 . 7 x 1 0 " l ° ( B v ) 4 -3.5x10 "s (By) s + +4.4x10 "3 (Bv) 2 +4.96(Bv)
2
o
Glycerol
X = - t . 5 x l 0 " ~ ( B v ) 4 5.8x10 "s (Bv)3+ -1.3x10 -2 (By)2 +13(Bv)
3
x
Acetic acid
X = - 4 . 8 x 1 0 "e(Bv) 4 +8.1x10 "s (By)3+ -5.3x10 -2 (Bv) 2 +14(By)
4 ,',
Lactic acid
X = - 3 . 6 x 1 0 "8 (By) 4 +5.8x10 "s (By)3+ -4.4x10 "2 (By) 2 +22(Bv)
7000
6000
O')
5000
E
4000
3000
2000
1000
I 200
I
I 400
I
I ~1 600
3
I 800
e v
I
I 1000
I
I 1200
I
I 1400
I 1600
I
I 1800
(rag NO 3- N I1 day-1)
Fig. 2. Reactor biomass concentration (X) depending on the nitrate load (By) during the denitrification carried out in the presence of different C-sources. reactor was formed by bacteria and yeast cells, stuck together by slime. Hyphal fungi, represented by the genus Fusarium, grew in clumps in the mixed liquor. The shape, size and structure of the flocs depended on the type of hydrogen donor and the value of Bv. The flocs were particularly large (with some reaching a width of 396 #m and a length of 4140 #m) and branched, with a compact, granulated structure in biocenoses fed with acids. In the presence of glycerol the flocs were gelatinous. Flocs of biocenoses fed with methanol resembled those of standard activated sludge. As a rule, an increase in By value was accompanied by a greater dispersion of the flocs. This was particularly evident at Bv > Bvcnt (Figs 3 and 4). When the process was conducted at By < Bvcrit the number of denitrifiers and fungi per mg VSS was constant. Table 1 shows that biocenoses fed with compounds with a lower number of carbon atoms per molecule were characterized by a greater number of denitrifiers. However, the proportion of denitrifiers
was higher in biocenoses fed with alcohols than in those growing in the presence of organic acids. In the latter case a higher proportion of other saprophytic bacteria, not denitrifiers, was observed. These biocenoses were also characterized by a 10-fold increase in the number of fungi. This agrees with the nutritional requirements of fungi, which prefer organic acids over alcohols as a C-source (Lodder, 1970; Ko~kov~-Kr~itochvilov~i, 1982; Kreger-van Rij, 1984). These results also show that as regards both types of studied compounds, fungi were more abundant in biocenoses when a compound with a lower number of carbon atoms per mocelcule was used as a C-source. However, based on the proportion of these microorganisms in the biocenoses, lactic acid can be regarded as a better carbon substrate for them than acetic acid (0.21 and 0.15% of total number of microorganisms, respectively). When the process was carried out at Bv > B~erit the number of denitrifiers in all biocenoses dropped.
A. G~BZfiSKA-LONtEWSKA
1568
Methanol
ticularly evident in the biocenosis fed with lactic acid. Under these conditions, the proportion of fungi in biocenoses decreased, but only in those fed with alcohols (Table 1). From the ecological point of view, the occurrence of protozoa and rotifers in the biocenoses is of interest. Until now, the presence of these microorganisms in denitrifying biocenoses was not taken into account. Recently, the presence of microfauna in anoxic activated sludge was described by Wanner et al. (1987). The studies discussed in this paper show that Mastigota nd and Amoeba proteus were generally stable components of the biocenoses. Ciliated protozoa usually appeared at Bv, no higher than 330 mg NO3-N 1-1 day-l. Attached ciliates belonging to the genus Opercularia were observed in biocenoses fed with glycerol or acetic acid. Epistylis sp. was present in the biocenosis fed with glycerol. Free-swimming ciliates of the genus Glaucoma appeared in that fed with lactic acid. Rotifers were found in the biocenosis grown in the presence of acetic acid at By = 220 mg NO3-N 1-1 day -1. The reason for the disappearance of ciliates and rotifers from the biocenosis at Bv beyond 330 and 220mg NO3-Nl-~day -1 respectively, probably lies in the negative effect of nitrate concentration on their growth. At B v = 3 3 0 m g NO3-N 1-1 day -~ the hydraulic retention time was 43.5 h and thus the "washing out" of these organisms from the reactor could not have been responsible (Fig. 5).
E-E
108. 127.630 720 0.2110.17 1016
72. 54. 55. 57- 78. 450 475 228 380 323 0.12 0.1510.22 0.19 0.311
1975 374414320 4980 7050 5700
Glycerol
E 0~
:t.
~ Q
>E mE I max, txm
170484
170-1130 0.08 I 2440 (
0.01
120300
5296
0.14 0.27 0.40 4530 4070 3320
0.02
i .o i,,o i ,oo 1 o1, o0 0.03
0.04 0.08 0.09
0.12
Fig. 3. The size (on an average basis, without taking shape into consideration) and structure of biocenoses floes depending on operational parameters (D, X, By, Bx) in the processes carried out in the presence of methanol and glycerol as C-sources. Symbols: [], dense structure: D, loose structure. It should be noticed however that the decrease of the number of other saprophytic non-denitrifying bacteria at the same time was much smaller. Thus the percentage of these bacteria in the total number of microorganisms markedly increased. This was par-
Acetic acid
as below 170-900
85-360
52-230
40-270
o,o
,,,
io,° i o,, 1380
1200
1080
:'.'..'.:"
200
Lactic acid
..
-........ r
o
o o o
Size average lmm=10 p.m
:." ..'... • •
~ ~
•
• •
°
" •
. '
°
o o o
•
•
o
o o o
o o
o o
o o
o
1
max,
p. m BX x
i~ v
•
174-
223-
1800 4140 I 0,08 I 0.07 [ 3 2 8 0 4I3 0 0 220
0,01
330 0.02
o
"
"
356-2880 0.09 4790 440 0.03
° a
396-2880 0.13 5170 1670 0.04
172.
1440 0.31 2850 890 0.06
Fig. 4. The size (on an a v e r a g e b a s i s , without taking shape into consideration) and structure of biocenoses floes depending on the operational parameters (D, X, By, Bx) in the processes carried out in the presence of acetic and lactic a c i d s a s C-sources. Symbols: t':], dense structure; F~, loose structure.
Denitrification unit biocenosis
1569
Table 1. Numbers and proportions of bacteria and fungi in biocenosesdependingon C-sourceas wellas reactor (By)and biomass (Bx) nitrate loading Process parameters Nitrate loading Number per mg VSS Proportion in biocenoses (%) Bacteria Reactor (By) Biomass (Bx) "total" Denitrifiers Fungi Other C-source mg NOa-N1-~day-~ mg NO3-Nmg ~day ~ ( x 106) ( x 106) ( x 103) Denitrifiers bacteria Fungi Methanol 220-1330 0.124).22 400 384 27 95.8 4.1 0.006 1800 220-670
0.31 0.084).14
196 217
181 210
6.0 15.0
92.3 96.6
7.6 3.4
0.003 0.007
Acetic acid
1100 1330 220
0.27 0.40 0.16
201 156 386
196 129 330
15.1 1.6 580
97.5 82.6 85.4
2.4 17.3 14.5
0.007 0.001 0.15
Lactic acid
330 440 220-670
0.27 0.40 0.064).13
197 63 197
166 42 167
480 280 373
84.0 66.4 86.2
15.9 33.5 13.7
0.24 0.44 0.21
890 0.31 180 32 390 17.7 Comment: the characteristic values for the processes carried out at By~
82.2
0.22
Glycerol
Taxonomy of bacteria and fungi The bacterial flora consisted in the main of typical denitrifying species such as Pseudomonas stutzeri,
P. mendocina, P. aureofaciens, P. solanacearum, P. caryophylli, Alcaligenes denitrificans and Agrobacterium radiobacter (Leifson and Hugh, 1954; Bergey, 1974; Pichinoty et al., 1977). Denitrification was also found to be carried out by some bacteria, which had previously either not been known to belong to this physiological group (P. delafieldi, P. palleronii) or had been known to reduce nitrates to nitrites only (P. cepacia, P. diminuta, Proteus inconstans group A) (Bergey, 1974). Biocenoses fed with organic acids had a more diverse composition of denitrifying bacteria than those growing in the presence of alcohols. They usually contained 3-4 species, 2 or 3 of which could be considered as dominating. A typical species in the biocenosis fed with acetic acid was P. stutzeri and in that growing in the presence of lactic acid, P. diminuta. Those species which dominated during the process at Bv < Bvcrit were regarded as typical. When alcohols were used as a carbon source the bacterial flora was subject to greater selection. In the biocenosis fed with glycerol only varieties of sub-group A of Proteus inconstans were present. The biocenosis growing in the presence of methanol was occasionally dominated by P. stutzeri and Agrobacterium radiobacter but P. cepacia was a typical species for it (Fig. 6). The carbon source also influenced the species composition of the mycoflora. The main organisms were yeasts and yeast-like microorganisms, occasionally also hyphal fungi, represented by Fusarium sp., were present. A typical species for the biocenosis fed with methanol was Candida boidinii, and with acetic a c i d - Candidafamata. In biocenosis grown in the presence of lactic acid C. famata and Hansenula californica permanently dominated. Besides these organisms in these biocenoses, representatives of C. lambica, C.
tropicalis, C. inconspicua, C. maltosa, Rhodotorula WR 25/12--1
rubra and Geotrichum candidum were also found (Fig. 7). The taxonomy and morphological, cultural and physiological properties of bacterial and fungal strains isolated from the biocenoses have been described elsewhere by Grabifiska-Loniewska et al. 0986/1988, 1990).
Interactions between microorganisms Earlier studies (Grabifiska-Loniewska, 1990) have shown that most of the bacterial species isolated from biocenoses, in pure cultures carried out denitrification only in the presence of yeast extract or a mixture of organic compounds in the medium. During the course of the process in a reactor in which mineral medium was enriched only in the studied carbon compound, these substances promoting the occurrence of denitrification could be derived from autolysed cells of microorganisms in the biocenoses (bacteria, fungi, microfauna), or could be produced by some of these organisms (mainly by fungi). Yeasts and yeast-like microorganisms did not directly participate in denitrification. For their growth they utilized the compounds used as the source of carbon and hydrogen donor for denitrification, nitrates (assimilation pathway), a m m o n i u m nitrogen, amino acids (e.g. lysine, tryptophan) and vitamins. Most of these microorganisms grew in a mineral medium without any N source (GrabifiskaLoniewska and Sl/tvikov~, 1990). This may indicate their ability to fix N2 arising during denitrification. This fact has considerable ecological impact since it suggests that the growth of most fungal species in denitrification units occurs only in mixed culture with denitrifying bacteria. The microfauna of the biocenoses consisted of both saprotrophic and phagotrophic organisms. The former included Mastigota nd, the latter Amoeba proteus, free-swimming and attached ciliates and rotifers. Saprotrophic protozoa were particularly
1570
A. GRAm~SKA-LoNIEWSKA Methanol 2
2400
2300
x--
~E
1
g-o
_
Glycerol
"SE1 i ~ ¢.--
E
I g"
I Bx
I
o,o8
2 ~-
]
I
0.16
I 4~°3300
10.271
0.40
Acetic acid
13.351 55750
2 F I 8186
~-"
O
0.01
Lactic acid
9533 •
0
'ida • 3600
0.02
0.03
0.04 10.06
"0.08
0.09
Fig. 5. Animal microorganisms in biocenoses during the processes carried out with different C-sources and operational parameters (D, Bv, Bx). Symbols: II, Mastigota nd; g~, Amoeba proteus; [], Amoeba radiosa; [~, Testacea nd; D, Glaucoma sp.; I~, Opercularia sp.; I~, Epistylis sp.; ~, Rotifera nd.
numerous in the biocenoses in the periods immediately following change of Bv and at values By > Bv,~t. At the same time, a transient increase in the number of other saprotrophs, i.e. bacteria and fungi in the dispersed phase, was observed, due to the increased shredding of the floes and loosening of their structure. In turn the growth of phagotrophic protozoa and rotifers occurred, though only at By values lower than 330 mg NO 31- ~day- 1. The occurrence of animal microorganisms in the bioeenoses when the process was conducted at an Eh between - 100 and - 4 5 0 mV indicates their high capacity for adaptation. It can therefore be assumed that these organisms can utilize both oxygen and nitrates at electron aeceptors during respiration.
Based on the above characteristics of the nutritional requirements of different groups of microorganisms it can be said that in these biocenoses both positive and negative interactions take place. The positive interactions embrace competition and mutualism. The populations competing for food are saprotrophic protozoa from the Mastigota group, bacteria and fungi. Symbiotic relationships occur between denitrifying bacteria and fungi. Negative interactions include the predatory behaviour of phagotrophic protozoa towards bacteria and fungi and rotifers towards sapro and phagotrophie protozoa. Predatory protozoa may play a role in regulating the growth of bacteria and fungi and thus causing the "rejuvenation" of the population of these
Denitrification unit biocenosis Carbon sources
Typical species:
(Values of B v crit.)
Methanol
o m
1571
L 0.21 1016
1975
Glycerol
(14oo mg NOa-N I-1 day-l)
I
3744
I
4320
Ps. cepacia
I
7050
(800 mg NO 3-N I -1 day -1)
o
g
5700
Proteus inconstans group A, type
t~
~o
0.08 2440
_
_ _
.....
....
0.27 4070
0.40 3320
I I
Acetic acid (250 mg NO 3-N I -1 day -1)
Ps. stutzeri
~1380
1200
1080
200
(% 100 7 q 17
Lactic acid (600 mg NOa-N I -1 day -1)
8
Ps. diminuta
6O 4O 2
0.01
0.02
0.03
0.04
0.06
0.08
0.09
0.12
Fig. 6. Proportions of bacterial species in biocenoses fed with different C-compounds depending on the operational parameters (D, Bv, X, Bx) of the denitrification process. Symbols: [], Alcaligenes denitrificans; K], Agrobacterium radiobacter ; II, Ps. aureofaciens ; [~, Ps. caryophylli; [], Ps. cepacia ; [], Ps. delafieldii ; []~ Ps. diminuta; [], Ps. mendocina; [57, Ps. solanacearum; CA, Ps. stutzeri; [], Ps. palleroni; [7, Proteus inconstans g r o u p A, type a; I~, Proteus inconstans group A, type b.
microorganisms in the biocenoses. This is of importance in the maintenance of their appropriate physiological activity which in turn determines the intensity of biochemical processes in the reactor. A scheme of the ecological relationships between the individual groups of microorganisms populating the reactor is presented in Fig. 8. To sum up these observations it can be said that the role of the microfauna in denitrification unit biocenosis is much smaller than in aerobic activated sludge. Due to the presence of nitrates in the medium it contains no phagotrophic organisms at By > 330 mg NO3-N 1-~ day-l. Under such conditions they do not therefore play a particularly important
ecological role in regulating the growth of bacteria and fungi in the biocenoses. Hence in the scheme given in Fig. 8 when the process is conducted at By > 330mg NO3-N1-1 day -1, consumers of the trophic level III need not be taken into account. CONCLUSIONS
From the results obtained in this study, it is clear that during heterotrophic denitrification a specific biocenosis is formed. It is composed not only of denitrifiers but also of other saprophytic non-denitrifying bacteria, yeasts, yeast-like microorganisms and microfauna. These groups of microorganisms relate
Carbon sources ~o ~]
Methanol
1016
•
Jl
0.17 I 0.12 10.16
1975
3744
species:
Candida boidlnii
(1400 mg NO3-N I-1 day-l)
•
0.21
.. X
Typical
(Values of B v cril.)
4320
I
Acetic acid (250 mg NO 3.N I 1 day -1) o
LL _rk
o~
X
1380
1200
1080
Candida famata
200
(%) 100 8O 60 40 20
Lactic acid (600 mg NO3-N I -1 day -1) Candida famata Hansenula californica
lhk X
0.06 3280
0.07 4300
0.09 4790
J~v°l
220 0.01
330 0.02
440 0.03
::01
•
I
°,o i 0.06 ,oo i,,oo I 0.08
0.04
133011800 ] 0.09 0,12
Fig. 7. Proportions of fungal species in biocenoses fed with different C-compounds depending on the operational parameters (D, B,, X, B~) of the denitrification process. Symbols: rq, C. boidinii; r~, C. famata; [2], C. mahosa; [], C. tropicalis; k~, Geotrichum candidum; [], Hansenula californica; [~, Rhodotorula rubra; [~, Trichosporon cutaneum; II, Fusarium sp.
I
u,,,,z°r,
I I consu °r
Nitrate dissimilationdenitrification --I=-
,/
~N~,F. ~, ~F.do,F.~.o~c.~
assimilation
II
~N.l,xation
/
, iA L_
Ill
I
, Oen/t;ifIyinIgre~ra'~Ig~ . / ~ I
I
~o,.,..~o ........ ,
,
IA ~. . . . . . .
iA I
~ [
V [ [
/1/IA
Compounds released ,fromautolize/dcel~s I L
I
/ I L__J
Fig. 8. Ecological relationships between denitrification unit microorganisms (with organic compound as a hydrogen donor), according to Grabifiska-Loniewska. A, cell autolysis; U, utilization; C, consumption. Enzymes: NaR, nitrate reductase; NiR, nitrite reductase, NOR, nitric oxide reductase; N2OR, nitrous oxide reductase; I-III, trophic levels. 1572
Denitrification unit biocenosis to each other in strict ecological terms. Thus, the scope of biological studies in the control of denitrifying unit biocenosis should be similar to the standard activated sludge analyses. It has been found that besides denitrifiers, yeasts and yeast-like microorganisms played a major role in this biocenosis. They promoted the course of the denitrification process by the intensification of the biochemical activity of denitrifying bacteria. Therefore, the microbiological control studies of the denitrification process ought to include both the quantity of denitrifiers as well as yeasts and yeast-like microorganisms.
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