Microb Ecol (1992) 23:127-142
MICROBIAL ECOLOGY © Springer-Verlag New York Inc. 1992
Interactions of Bacteria and Microflagellates in Sequencing Batch Reactors Exhibiting Enhanced Mineralization of Toxic Organic Chemicals S. K. Schmidt, R. Smith, D. Sheker, T. F. Hess, J. Silverstein, and P. M. Radehaus Department of Environmental, Population, and Organismic Biology, University of Colorado, Boulder, Colorado 80309, USA Received: July 16, 1991; Revised: November 22, 1991
Abstract. Community level interactions were studied in non-axenic sequencing batch reactors (SBRs) being used to treat 2,4-dinitrophenol (DNP). Increasing the influent DNP concentrations from 1 to 10 #g ml-~ eliminated large predatory organisms such as rotifers and ciliated protozoa from the SBRs. Under steady-state conditions at a DNP concentration of 10 ug ml ~, supplemental additions of glucose enhanced DNP degradation and led to the establishment of a microbial community consisting of five species of bacteria and a variety of microflagellates. The bacteria and flagellates exhibited oscillating population dynamics in this system, possibly indicating predator-prey interactions between these two groups. Only two of the five bacteria isolated from this system could utilize glucose as a growth substrate, and one of these two species was the only organism that could mineralize DNP in the system. The other three bacteria could grow using metabolic by-products of one of the glucose-utilizing strains (Bacillus cereus) found in the reactors. Supplemental glucose additions increased the average size of bacterial floc particles to 172/zm, compared with 41 #m in SBRs not receiving glucose. It is theorized that the enhanced mineralization of DNP in this non-axenic system was attributable to increased community interactions resulting in increased bacterial flocculation in SBRs receiving supplemental glucose additions.
Introduction Biological systems for the treatment of toxic chemicals are now commonplace, but very little is known about the communities of microorganisms that develop in these systems. Most work in the field ofbiodegradation has involved studies of isolated organisms, or studies of samples from real systems that are then incubated under unrealistic conditions in the laboratory. In contrast, real treatOffprint requests to: S. K. Schmidt.
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ment systems are usually not axenic, contain a multitude of interacting organi s m s , a n d a r e u s u a l l y o p e r a t i n g a t s o m e s o r t o f s t e a d y - s t a t e c o n d i t i o n [ 14, 19, 37]. A k n o w l e d g e o f b i o l o g i c a l i n t e r a c t i o n s i n c o n t i n u o u s l y o p e r a t i n g s y s t e m s is n e c e s s a r y i n o r d e r to d e v e l o p s t r a t e g i e s for e n h a n c i n g b i o d e g r a d a t i o n o f c h e m i c a l s o n a l a r g e scale. I n t h i s s t u d y b i o l o g i c a l i n t e r a c t i o n s w e r e e x a m i n e d i n s e q u e n c i n g b a t c h reactors (SBRs) that were being used to treat water contaminated with 2,4-dinitrop h e n o l ( D N P ) . D N P w a s c h o s e n f o r s t u d y b e c a u s e i t is e x t r e m e l y toxic, a n d previous research has indicated that dinitrophenols may be resistant to degr a d a t i o n i n i n d u s t r i a l w a s t e s t r e a m s [1]. S B R s w e r e s t u d i e d b e c a u s e o f t h e i r p r o m i s e as a s y s t e m f o r t r e a t i n g t o x i c w a s t e s a n d b e c a u s e t h e y a r e r e l a t i v e l y i n e x p e n s i v e a n d e a s y to o p e r a t e [ 13, 14]. S B R s o p e r a t e as f i l l - a n d - d r a w r e a c t o r s [13], t h a t is, t h e a c t i v e b i o m a s s is r e c y c l e d a n d t r e a t e d w a t e r is d e c a n t e d a n d r e p l a c e d w i t h c o n t a m i n a t e d w a t e r d u r i n g e a c h cycle o f o p e r a t i o n . D u r i n g s t e a d y s t a t e o p e r a t i o n o f a n S B R t h e b i o m a s s l o s t t o d e a t h a n d w a s h o u t is r e p l a c e d b y g r o w t h o f t h e a c t i v e p o p u l a t i o n d u r i n g e a c h cycle. T h u s , S B R s s e l e c t f o r efficient m i c r o b i a l c o m m u n i t i e s l i k e c o n t i n u o u s c u l t u r e s y s t e m s s u c h a s c h e m o s t a t s . O n e a d v a n t a g e o f S B R s o v e r c h e m o s t a t s is t h a t S B R s select for o r ganisms capable of functioning in a changing environment, whereas chemostats select f o r o r g a n i s m s t h a t a r e b e s t a d a p t e d t o c o n s t a n t c o n d i t i o n s . B e c a u s e few natural or human-made systems exhibit constant conditions, SBRs are more appropriate model systems for studying the microbial ecology of toxic waste treatment than chemostats. T h e c o m m u n i t y l e v e l s t u d i e s d e s c r i b e d in t h i s p a p e r w e r e c o n d u c t e d u s i n g SBRs that had reached steady-state operation under non-axenic conditions. S t u d i e s w e r e c o n d u c t e d to d e t e r m i n e t h e m e c h a n i s m o f e n h a n c e d D N P m i n eralization and to quantify bacterial-protozoan and bacterial-bacterial interactions in SBRs exhibiting enhanced biodegradation of DNP.
Methods S BRs Design, operation, and a diagram of the SBRs used in this study have been given in detail elsewhere [ 14]. The SBRs contained four liters of inorganic salts solution [ 14] supplemented with DNP and either 100 /~g of glucose ml -~ (SBR 3) or no glucose (SBR 1). Every 48 hours the stirring and aeration mechanisms were automatically turned off and the suspended floc particles were allowed to settle to the bottom of the reactors. This settling period lasted 1 hour and 50 minutes and was followed by a draw step [13] that lasted 40 minutes and removed the top 3.5 liters of the reactor volume each cycle. After decanting, the reactors were automatically refilled with contaminated water consisting of inorganic salts solution [ 14] and DNP at the concentrations given below. This reactor operation scheme gave very efficient recycle of the active microbial biomass [14]. The bioreactors were run continuously, cycling every 48 hours, for over a year and were used to treat DNP at several concentrations. DNP was added to the reactors using micro-dispensing meter pumps with synchronous drives (Fluid Metering, Inc., Oyster Bay, NY). Between October of 1988 and May of 1989 the reactors were run with DNP input concentrations of 4 ug of DNP ml -~ (for 138 days) and then 10 tzg ofDNP m1-1 (for 82 days). In May of 1989 the reactors were switched to an influent DNP concentration of 1 t~g ml ~ to test their effectiveness at degrading lower concentrations of DNP. In September of 1989 the DNP concentration in both reactors was
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switched back to 10 #g ml ~and was kept at that level until all of the experiments reported in this paper were done.
Bacteria The SBRs were initially inoculated with a Janthinobacterium sp. and a previously undescribed Actinomycete, both capable of completely mineralizing DNP [14, 29]. After the reactors had run non-axenically for five months under constant conditions with a DNP concentration of 10 ug m1-1, studies were conducted to quantify and isolate DNP-degrading and other bacteria from the SBRs. The most probable number (MPN) of DNP-mineralizing microorganisms in the SBRs was determined using a modification of the method of Schmidt and Gier [28, 29]. Reactor contents were diluted in a series of 10-fold dilutions. Eight 1-ml aliquots of each dilution were placed in the wells of 24-well sterile tissue culture plates (Coming Glass Works, Coming, NY), which contained 1 ml of sterile inorganic salts solution and enough DNP to give a final concentration of 5 ug ml -~. The tissue culture plates were incubated in the dark for 28 days at 24 _+ 2°C before DNP mineralization was determined. Mineralization was assumed to have occurred if the yellow color imparted by DNP disappeared from a given well of a tissue culture plate. Bacteria from positive MPN wells were obtained in pure culture and identified as described below. Direct isolation procedures and enrichment techniques [29] were also employed to isolate DNP degraders from the SBRs. Besides DNP-degrading bacteria, a number of other bacteria were isolated from the reactors. Pure cultures of these bacteria were maintained on agar plates containing 3 g of trypticase soy broth (BBL, Cockeysville, MD) and 15 g of Bacto-Agar (Difco Laboratories, Detroit, MI) per liter ofdeionized water or on agar containing 10 ug o f D N P m1-1 and an inorganic salts solution described previously [28]. Identification of all bacteria was done using previously described techniques [ 12, 14, 23, 29]. Carbon and energy source utilization tests were done using the auxanographic method as described previously [23], and flagella were stained using the method of Heimbrook et al. [12]. In addition, fatty acid profiles were done on each strain by Microbial ID, Inc. (Newark, DE). To determine if isolates from SBR 3 could grow using metabolic by-products of the DNP and glucose utilizing strains from this bioreactor, axenic cultures of bacteria isolated from SBR 3 were grown to stationary phase and then were filtered through sterile 0.2-tim filters. The filtrates were then inoculated with washed cultures of each of the bacteria to be tested. Results from these trials were compared to results from control incubations that were carried out using inorganic nutrient solution that had not been previously used to grow other organisms. Total numbers of bacteria were counted using several methods. In experiments in which growth on metabolic by-products from other strains was tested, bacteria were counted directly using a calibrated Thoma counting chamber (0.02 m m depth; Fleischhacker KG, Schwerte, Germany). Total beterotrophic bacterial numbers were determined in samples from the SBRs by plate counts on 0.1-strength trypticase-soy agar. The diameter of bacterial flocs in the SBRs was measured using a sedimentation and light extinction method [6; J Silverstein, M Bowman, T Hess, SK Schmidt, B Howe, Env Eng Proc, July, 1990].
Protozoa Heterotrophic microflagellates and ciliates were counted by placing 20 ul of reactor fluid on a microscope slide, covering the droplet with a cover slip, and then counting all of the protozoa in a 4-mm-wide swath across the middle of the cover slip. Counts were made using a phase-contrast microscope at 250 x or 400 x magnification. This method allowed the differentiation of dead or encysted protozoa from active organisms and eliminated some methodological difficulties associated with protocols that require filtering of samples [el. 24]. An organism was considered to be active if it had visible flagella and was either swimming or moving its flagella. Encysted organisms were usually more refractive under phase contrast microscopy than were active organisms and did not have visible flagella. Triplicate counts were made at each sampling interval, and the mean and
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standard error of the mean (SEM) are reported in this paper. The sizes of the heterotrophic microflagellates were measured using a calibrated ocular micrometer.
C h e m i c a l Analyses The analytical procedures used in this study have been described in more detail by Schmidt et al. [30] and Hess et al. [14]. Samples (6 ml) were withdrawn from the center of each reactor at regular intervals using a syringe with a 15-cm needle that was inserted through a septnm in the reactor wall. The samples were immediately filtered through 0.2-~tm polycarbonate filters (Nuclepore, Pleasanton, CA) and acidified with 1 drop of concentrated H2804. DNP concentration was measured using a Varian DMS 100 spectrophotometer (Varian Associates, Palo Alto, CA) set at 260 nm. In experiments in which 14C-labelled DNP was used, 1-ml subsamples were added to 4-ml Omni scintillation vials (Wheaton Co., Millville, NJ) along with 2.5 ml of ScintiVerse II scintillation cocktail (Fisher Scientific, Pittsburgh, PA). The radioactivity was counted using a liquid scintillation counter (LKB Wallac, 1209 Rackbeta, Turku, Finland). To verify that DNP was mineralized, ~4COz from ~4C-labelled DNP and NOz- evolution were also monitored in some experiments [14]. The partitioning of carbon from glucose into biomass, CO2, and metabolic by-products in cultures of Bacillus cereus was determined using ~4C-labelledglucose as has been previously described [27, 30]. To verify that ~"C left in solution at the end of experiments was not 14C-labelled glucose, the anthrone reaction for total carbohydrates was used [11].
Chemicals [u-~4c] 2,4-dinitrophenol (10.2 mCi mmol % and [U-14C] glucose (296 mCi mmol -~) were purchased from Sigma Chemical Company (St. Louis, MO). Unlabelled reagent grade 2,4-dinitrophenol was purchased from Fluka Chemical Company (Ronkonkoma, NY), and unlabelled reagent grade glucose was obtained from Mallinckrodt, Inc. (Paris, KY). The purity of all compounds was greater than 99%.
Data Analyses Nonlinear regression analyses were performed on all curves of DNP degradation using the LMFIT computer program [ 14]. The data comparing the responses of different strains of bacteria to filtrates of other strains were analyzed using ANOVAs and Scheffr's Multiple Comparison Test.
Results T h e e x p e r i m e n t s r e p o r t e d in t h i s p a p e r w e r e d o n e o v e r a p e r i o d o f o n e y e a r a n d ar e a c o n t i n u a t i o n o f w o r k p r e v i o u s l y r e p o r t e d b y H e s s et al. [ 14]. A t t h e beginning o f this p e r i o d the SBRs were switched f r o m an influent D N P conc e n t r a t i o n o f 1 /zg m1-1 t o a c o n c e n t r a t i o n o f 10 /~g m1-1. T h e S B R s w e r e o p e r a t i n g u n d e r s t e a d y - s t a t e c o n d i t i o n s w h e n all o f t h e w o r k r e p o r t e d b e l o w w a s c o n d u c t e d . S B R 3 w a s r e c e i v i n g 100 #g o f g l u c o s e m1-1 i n a d d i t i o n t o 10 /~g o f D N P m l -~ at t h e b e g i n n i n g o f e a c h 4 8 - h o u r cycle. S B R 1 s e r v e d as a c o n t r o l a n d r e c e i v e d 10 #g o f D N P m l 1 b u t n o s u p p l e m e n t a l a d d i t i o n s o f glucose. F i g u r e 1 s h o w s t h e r e s u l t s o f t y p i c a l D N P r e m o v a l c u r v e s d u r i n g o n e c y cl e o f t h e s e t w o r e a c t o r s . T h e S B R t h a t w a s r e c e i v i n g s u p p l e m e n t a l g l u c o s e
Community Interactions I
I
131 I
I
I
10 8
O. Z 6
Fig. 1. Mineralization of DNP in SBR 1 and SBR 3 after each reactor had been running under steady-state conditions for several months. SBR 3 received 10 ~tg ofDNP mF' and 100 ~g of glucose ml -t, whereas SBR 1 received only 10/~g of DNP ml ~at the beginning of each 48hour cycle of operation.
I)
4
2
~+ Glucose(SBR3)
o 5
10
15
20
25
30
Hours
Table 1. Steady-state values for MPN estimates of DNP-mineralizing bacteria and total suspended solids in SBRs receiving 10 gg of DNP ml ~ and either no glucose (SBR 1) or 100 #g of glucose ml 1 (SBR 3)
SBR no.
Glucose concentration (#g ml-')
1
0
3
100
MPNs (cells ml ')
Suspended solids (gg ml -~)
Contents
Effluent
Contents
Effluent
1.1 _+ 0.3 x 105 1.1 _+ 0.3 x 106
5.4 + 3.3 x 104 5.0 -+ 3.5 x 104
20 -+ 1 812 +_ 10
3.5 -+ 0.2 13 _+ 0.5
a d d i t i o n s s h o w e d e n h a n c e d D N P r e m o v a l kinetics, w h i c h h a v e b e e n d e s c r i b e d in detail elsewhere [ 14]. P r e v i o u s w o r k [ 14] i n d i c a t e d t h a t the s t i m u l a t o r y effect o f glucose o n D N P m i n e r a l i z a t i o n m a y h a v e b e e n d u e to the i n c r e a s e d flocculation a n d therefore i n c r e a s e d recycling o f D N P - m i n e r a l i z i n g o r g a n i s m s in S B R s receiving glucose s u p p l e m e n t s . T o test this h y p o t h e s i s , floc size m e a s u r e m e n t s a n d several exp e r i m e n t s were p e r f o r m e d . D i a m e t e r s o f bacterial flocs in S B R 3 r a n g e d f r o m 40 to 300 # m ( m e a n = 172 ~m), w h e r e a s flocs in S B R 1 were m u c h smaller, r a n g i n g in size f r o m 32 to 4 8 / ~ m ( m e a n -- 41 #m). S B R 3 also h a d m u c h higher s u s p e n d e d solids c o n c e n t r a t i o n s t h a n S B R 1 (Table 1). I n a d d i t i o n , M P N e s t i m a t e s i n d i c a t e d t h a t there were a p p r o x i m a t e l y 10-fold m o r e D N P - m i n e r alizing o r g a n i s m s in S B R 3 t h a n in S B R 1, a n d the loss o f D N P - m i n e r a l i z e r s in the r e a c t o r effluent was p r o p o r t i o n a t e l y l o w e r in S B R 3 t h a n in S B R 1 (Table 1). A n e x p e r i m e n t was c o n d u c t e d to see if the d e l e t i o n o f glucose f r o m o n e cycle o f o p e r a t i o n o f S B R 3 w o u l d cause a significant s l o w - d o w n o f D N P m i n e r a l i z a t i o n in this r e a c t o r (Fig. 2). It was r e a s o n e d t h a t if glucose was n e e d e d to m a i n t a i n the h i g h rate o f D N P m i n e r a l i z a t i o n d u r i n g e a c h cycle o f S B R o p e r a t i o n t h e n the effect o f glucose was directly o n the g r o w t h rate o f D N P m i n e r a l i z i n g o r g a n i s m s . If, o n the o t h e r h a n d , the effect o f glucose w a s a result
132
S.K. Schmidt et al. I
I
I
I
I
10
I
I
~ Jt
=.
I
I
I
I
l
+ Glucose 12/4/89
Fig. 2. Mineralization of 10 ~g of DNP ml-l during three consecutive cycles of operation of SBR 3. The first and last cycles were run with supplemental glucose additions (100 lzg of glucose ml-~) and the middle cycle was run without the addition of glucose. Prior to this experiment, SBR 3 was at steady-state and had received glucose at a rate of 100 ug ml ' every 48 hours for three months.
- Glucose 12/6/89
8
~6 c~ 4
4
I
0 0
I 1
I
I 2
I
I I 3 Hours
I 4
I
"it 5
6
Table 2. Comparison of the model estimates from two models ofsubstrate mineralizationapplied to three consecutive cycles of SBR 3 Date
Model
#max (hours -~)
K1 (hours -~)
Ks (~g ml-~)
Xo (#g ml -~)
12/4/89 12/6/89 12/8/89
IV IV IV
0.63 + 0.03 0.68 _+ 0.03 0.72 + 0.02
0.085 + 0.006 0.093 + 0.007 0.070 +_ 0.004
NA~ NA NA
NA NA NA
12/4/89 12/6/89 12/8/89
Monod Monod Monod
0.30 _+_0.04 0.46 + 0.13 0.47 + 0.06
NA NA NA
0.9 + 0.3 2.1 + 0.9 1.7 + 0.4
4.6 _+ 0.7 3.3 + 1.1 2.5 _+ 0.4
a NA, not applicable o f its effect o n t h e b u i l d - u p a n d m a i n t e n a n c e o f D N P - m i n e r a l i z i n g o r g a n i s m s o v e r m a n y cycles, t h e n t h e d e l e t i o n o f glucose f r o m o n e cycle o f S B R 3 o p e r a t i o n s h o u l d n o t r e s u l t i n a s i g n i f i c a n t c h a n g e i n D N P m i n e r a l i z a t i o n kinetics. F i g u r e 2 s h o w s the r e s u l t s o f a n e x p e r i m e n t i n w h i c h t h e glucose feed was t u r n e d off i n the s e c o n d o f t h r e e c o n s e c u t i v e cycles o f S B R 3 o p e r a t i o n . T h e D N P m i n e r a l i z a t i o n c u r v e s were a l m o s t i d e n t i c a l i n all o f the cycles a n d n o n l i n e a r r e g r e s s i o n c u r v e fits y i e l d e d s i m i l a r p a r a m e t e r e s t i m a t e s for all t h r e e c u r v e s ( T a b l e 2). T h e s e d a t a l e n d s u p p o r t to t h e h y p o t h e s i s t h a t the role o f glucose is v i a its effect o n i n c r e a s e d f l o c c u l a t i o n r a t h e r t h a n i n c r e a s e d g r o w t h rate o f t h e D N P - m i n e r a l i z i n g b a c t e r i a d u r i n g a n y o n e cycle o f S B R 3 o p e r a t i o n . F u r t h e r a t t e m p t s at u n d e r s t a n d i n g t h e effects o f glucose o n D N P m i n e r a l i z a t i o n were f o c u s e d o n e x a m i n i n g m i c r o b i a l p o p u l a t i o n d y n a m i c s i n S B R s 1 a n d 3. D u r i n g o p e r a t i o n w i t h a n i n f l u e n t D N P c o n c e n t r a t i o n o f 1 ~tg m1-1, b o t h S B R s d e v e l o p e d d i v e r s e m i c r o b i a l c o m m u n i t i e s t h a t i n c l u d e d yeasts, flagellates, ciliates, a m o e b a e , rotifers, a n d a t least t e n species o f b a c t e r i a ( H o w e a n d S c h m i d t , u n p u b l i s h e d ) . A f t e r t h e r e a c t o r s were s w i t c h e d f r o m a D N P c o n c e n t r a t i o n o f 1 ~tg m1-1 to 10 ~g m1-1, a d e c l i n e i n t h e n u m b e r o f rotifers a n d ciliates w a s n o t e d , a n d after t h e r e a c t o r s were o p e r a t e d for s e v e n m o n t h s at a D N P c o n c e n t r a t i o n o f 10 #g m1-1, o n l y flagellated p r o t o z o a a n d o c c a s i o n a l l y a s m a l l c i l i a t e d p r o t o z o a n were o b s e r v e d .
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The flagellated protozoa observed in the SBRs at steady-state were heterotrophic microflagellates of the class Zoomastigophorea, orders Chrysomonadida and Kinetoplastida. These protozoa were spherical to ovoid in shape (diam = 4 to 8 gin), had two flagella, and resembled organisms in the genera M o n a s (Chrysomonadida) and Bodo (Kinetoplastida) [4, 7, 8]. These protozoa were either attached to bacterial floes or found free-swimming between floes. Attached protozoa used one flagellum to attach themselves to the floes and the other to create a feeding current. Fenchel [7] reports a similar feeding strategy for several members of the Kinetoplastida. Microflagellate population dynamics were cyclical during steady-state SBR operation (Figs. 3-5). Population peaks were consistently observed during the first 14 hours of operation and population lows were normally observed after about 30 hours into each cycle (Fig. 3). Increases in protozoan numbers corresponded with a decline in plate counts of heterotrophic bacteria (Fig. 4). As with earlier SBR cycles (Figs. 1 and 2) DNP was completely mineralized in SBR 3 by six hours into the reaction cycle (Fig. 5). It is also of note that glucose was completely mineralized in SBR 3 by 1 hour into the reaction cycle [Howe and Schmidt, unpublished]. Ciliated protozoa were occasionally observed, but their numbers were too few to be accurately quantified. Protozoan population densities were approximately 10-fold lower in SBR 1 than in SBR 3 (data not shown). Microscopic observations indicated that most of the bacteria in the reactors were in floc particles. These flocs contained mostly coccoid cells,but occasional filamentous bacteria were observed. Five bacterial colony types were consistently observed during plate count procedures (on D N P agar and trypticase soy agar) carried out during February and March, 1990. These bacteria were obtained in pure culture, and their characteristics are given in Table 3. Of these five bacterial species, only one (isolate D) could use D N P as a source of carbon and energy. This organism is most likely a descendent of the Actinomycete, which was initiallyinoculated into the reactors. This bacterium was similar to the original Actinomycete except that it could no longer utilize arabinose, fumarate, or 1-naphthol as growth substrates (Table 3). However, both organisms contain tuberculostearic acid and had almost identical fatty acid profiles. Given all of the characteristics of these two actinomycctes, they are most likely in the genus Rhodococcus [29]. The DNP-mineralizing strain of Janthinobacterium that was initially inoculated into the SBRs was never recovered aRer steady-state conditions were reached. Bacterial isolates B and C are probably very closely related to one another as indicated by the data given in Table 3 and the fact that their fatty acid profiles were almost identical. Strain A is a very fastidious organism that could not be identified either by standard criteria (Table 3) or by fatty acid profile analysis. Isolate E could not be characterized as to carbon source utilization because it grew prolifically in the auxanographic plates even in the absence of any added carbon source. However, this organism was positively identified as Bacillus cereus using fatty acid profile analysis. Three (isolates A, B, and C) of the five bacteria isolated from SBR 3 could not utilize glucose or DNP as a source of carbon and energy (Table 3). This is
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S.K. Schmidt et al.
!,
I
I
I
I
I
I
1 E
~o .
9
m
.~ m
6
~
3
m
I
I
I
I
I
I
5
10
15
20
25
30
Fig. 3. Population densities of heterotrophic microflagellates in SBR 3 during a typical 48-hour cycle after steady-state operation had been achieved.
Hours
A o o o
2O
1
I
I
I
I
0
I
0 ~o
*'16
3
o.
8
O~ x
~
4
0~
I/.
z
z
l
5
10
15
i
z
I
20
25
30
I
35
Fig. 4. Population densities of heterotrophic microflagellates (direct counts) and total heterotrophic bacteria (plate counts) in SBR 3 during a typical 48-hour cycle.
Hours
surprising because the only organic c o m p o u n d s fed into the reactors were D N P a n d glucose. T h i s o b s e r v a t i o n led to a series o f e x p e r i m e n t s to d e t e r m i n e if these three o r g a n i s m s could use m e t a b o l i c b y - p r o d u c t s o f the other two bacteria in the reactors. Figure 6 shows that all three bacteria could grow in the sterile filtrates o f B. cereus but not in the filtrates o f the R h o d o c o c c u s sp. Several e x p e r i m e n t s were p e r f o r m e d to d e t e r m i n e i f the B. cereus p r o d u c e d significant a m o u n t s o f m e t a b o l i c b y - p r o d u c t s as a result o f its growth on glucose. Cultures grown to stationary phase on 14C-glucose excreted on average 16% o f the 14Clabel into the external m e d i u m , whereas 33% a n d 51% o f the label was f o u n d in b i o m a s s a n d CO2, respectively. This is in contrast to c a r b o n partitioning in the R h o d o c o c c u s sp., which c o n v e r t e d greater t h a n 98% o f b o t h glucose a n d D N P to either b i o m a s s or CO2. At present, the nature o f the growth stimulating c o m p o u n d p r o d u c e d b y B. cereus is not known, b u t analyses showed that there was no m e a s u r a b l e glucose in the filtrates at the end o f each e x p e r i m e n t with this organism.
Community Interactions 2O
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MICROBIAL ECOLOGY © Springer-Verlag New York Inc. 1992
Interactions of Bacteria and Microflagellates in Sequencing Batch Reactors Exhibiting Enhanced Mineralization of Toxic Organic Chemicals S. K. Schmidt, R. Smith, D. Sheker, T. F. Hess, J. Silverstein, and P. M. Radehaus Department of Environmental, Population, and Organismic Biology, University of Colorado, Boulder, Colorado 80309, USA Received: July 16, 1991; Revised: November 22, 1991
Abstract. Community level interactions were studied in non-axenic sequencing batch reactors (SBRs) being used to treat 2,4-dinitrophenol (DNP). Increasing the influent DNP concentrations from 1 to 10 #g ml-~ eliminated large predatory organisms such as rotifers and ciliated protozoa from the SBRs. Under steady-state conditions at a DNP concentration of 10 ug ml ~, supplemental additions of glucose enhanced DNP degradation and led to the establishment of a microbial community consisting of five species of bacteria and a variety of microflagellates. The bacteria and flagellates exhibited oscillating population dynamics in this system, possibly indicating predator-prey interactions between these two groups. Only two of the five bacteria isolated from this system could utilize glucose as a growth substrate, and one of these two species was the only organism that could mineralize DNP in the system. The other three bacteria could grow using metabolic by-products of one of the glucose-utilizing strains (Bacillus cereus) found in the reactors. Supplemental glucose additions increased the average size of bacterial floc particles to 172/zm, compared with 41 #m in SBRs not receiving glucose. It is theorized that the enhanced mineralization of DNP in this non-axenic system was attributable to increased community interactions resulting in increased bacterial flocculation in SBRs receiving supplemental glucose additions.
Introduction Biological systems for the treatment of toxic chemicals are now commonplace, but very little is known about the communities of microorganisms that develop in these systems. Most work in the field ofbiodegradation has involved studies of isolated organisms, or studies of samples from real systems that are then incubated under unrealistic conditions in the laboratory. In contrast, real treatOffprint requests to: S. K. Schmidt.
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ment systems are usually not axenic, contain a multitude of interacting organi s m s , a n d a r e u s u a l l y o p e r a t i n g a t s o m e s o r t o f s t e a d y - s t a t e c o n d i t i o n [ 14, 19, 37]. A k n o w l e d g e o f b i o l o g i c a l i n t e r a c t i o n s i n c o n t i n u o u s l y o p e r a t i n g s y s t e m s is n e c e s s a r y i n o r d e r to d e v e l o p s t r a t e g i e s for e n h a n c i n g b i o d e g r a d a t i o n o f c h e m i c a l s o n a l a r g e scale. I n t h i s s t u d y b i o l o g i c a l i n t e r a c t i o n s w e r e e x a m i n e d i n s e q u e n c i n g b a t c h reactors (SBRs) that were being used to treat water contaminated with 2,4-dinitrop h e n o l ( D N P ) . D N P w a s c h o s e n f o r s t u d y b e c a u s e i t is e x t r e m e l y toxic, a n d previous research has indicated that dinitrophenols may be resistant to degr a d a t i o n i n i n d u s t r i a l w a s t e s t r e a m s [1]. S B R s w e r e s t u d i e d b e c a u s e o f t h e i r p r o m i s e as a s y s t e m f o r t r e a t i n g t o x i c w a s t e s a n d b e c a u s e t h e y a r e r e l a t i v e l y i n e x p e n s i v e a n d e a s y to o p e r a t e [ 13, 14]. S B R s o p e r a t e as f i l l - a n d - d r a w r e a c t o r s [13], t h a t is, t h e a c t i v e b i o m a s s is r e c y c l e d a n d t r e a t e d w a t e r is d e c a n t e d a n d r e p l a c e d w i t h c o n t a m i n a t e d w a t e r d u r i n g e a c h cycle o f o p e r a t i o n . D u r i n g s t e a d y s t a t e o p e r a t i o n o f a n S B R t h e b i o m a s s l o s t t o d e a t h a n d w a s h o u t is r e p l a c e d b y g r o w t h o f t h e a c t i v e p o p u l a t i o n d u r i n g e a c h cycle. T h u s , S B R s s e l e c t f o r efficient m i c r o b i a l c o m m u n i t i e s l i k e c o n t i n u o u s c u l t u r e s y s t e m s s u c h a s c h e m o s t a t s . O n e a d v a n t a g e o f S B R s o v e r c h e m o s t a t s is t h a t S B R s select for o r ganisms capable of functioning in a changing environment, whereas chemostats select f o r o r g a n i s m s t h a t a r e b e s t a d a p t e d t o c o n s t a n t c o n d i t i o n s . B e c a u s e few natural or human-made systems exhibit constant conditions, SBRs are more appropriate model systems for studying the microbial ecology of toxic waste treatment than chemostats. T h e c o m m u n i t y l e v e l s t u d i e s d e s c r i b e d in t h i s p a p e r w e r e c o n d u c t e d u s i n g SBRs that had reached steady-state operation under non-axenic conditions. S t u d i e s w e r e c o n d u c t e d to d e t e r m i n e t h e m e c h a n i s m o f e n h a n c e d D N P m i n eralization and to quantify bacterial-protozoan and bacterial-bacterial interactions in SBRs exhibiting enhanced biodegradation of DNP.
Methods S BRs Design, operation, and a diagram of the SBRs used in this study have been given in detail elsewhere [ 14]. The SBRs contained four liters of inorganic salts solution [ 14] supplemented with DNP and either 100 /~g of glucose ml -~ (SBR 3) or no glucose (SBR 1). Every 48 hours the stirring and aeration mechanisms were automatically turned off and the suspended floc particles were allowed to settle to the bottom of the reactors. This settling period lasted 1 hour and 50 minutes and was followed by a draw step [13] that lasted 40 minutes and removed the top 3.5 liters of the reactor volume each cycle. After decanting, the reactors were automatically refilled with contaminated water consisting of inorganic salts solution [ 14] and DNP at the concentrations given below. This reactor operation scheme gave very efficient recycle of the active microbial biomass [14]. The bioreactors were run continuously, cycling every 48 hours, for over a year and were used to treat DNP at several concentrations. DNP was added to the reactors using micro-dispensing meter pumps with synchronous drives (Fluid Metering, Inc., Oyster Bay, NY). Between October of 1988 and May of 1989 the reactors were run with DNP input concentrations of 4 ug of DNP ml -~ (for 138 days) and then 10 tzg ofDNP m1-1 (for 82 days). In May of 1989 the reactors were switched to an influent DNP concentration of 1 t~g ml ~ to test their effectiveness at degrading lower concentrations of DNP. In September of 1989 the DNP concentration in both reactors was
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switched back to 10 #g ml ~and was kept at that level until all of the experiments reported in this paper were done.
Bacteria The SBRs were initially inoculated with a Janthinobacterium sp. and a previously undescribed Actinomycete, both capable of completely mineralizing DNP [14, 29]. After the reactors had run non-axenically for five months under constant conditions with a DNP concentration of 10 ug m1-1, studies were conducted to quantify and isolate DNP-degrading and other bacteria from the SBRs. The most probable number (MPN) of DNP-mineralizing microorganisms in the SBRs was determined using a modification of the method of Schmidt and Gier [28, 29]. Reactor contents were diluted in a series of 10-fold dilutions. Eight 1-ml aliquots of each dilution were placed in the wells of 24-well sterile tissue culture plates (Coming Glass Works, Coming, NY), which contained 1 ml of sterile inorganic salts solution and enough DNP to give a final concentration of 5 ug ml -~. The tissue culture plates were incubated in the dark for 28 days at 24 _+ 2°C before DNP mineralization was determined. Mineralization was assumed to have occurred if the yellow color imparted by DNP disappeared from a given well of a tissue culture plate. Bacteria from positive MPN wells were obtained in pure culture and identified as described below. Direct isolation procedures and enrichment techniques [29] were also employed to isolate DNP degraders from the SBRs. Besides DNP-degrading bacteria, a number of other bacteria were isolated from the reactors. Pure cultures of these bacteria were maintained on agar plates containing 3 g of trypticase soy broth (BBL, Cockeysville, MD) and 15 g of Bacto-Agar (Difco Laboratories, Detroit, MI) per liter ofdeionized water or on agar containing 10 ug o f D N P m1-1 and an inorganic salts solution described previously [28]. Identification of all bacteria was done using previously described techniques [ 12, 14, 23, 29]. Carbon and energy source utilization tests were done using the auxanographic method as described previously [23], and flagella were stained using the method of Heimbrook et al. [12]. In addition, fatty acid profiles were done on each strain by Microbial ID, Inc. (Newark, DE). To determine if isolates from SBR 3 could grow using metabolic by-products of the DNP and glucose utilizing strains from this bioreactor, axenic cultures of bacteria isolated from SBR 3 were grown to stationary phase and then were filtered through sterile 0.2-tim filters. The filtrates were then inoculated with washed cultures of each of the bacteria to be tested. Results from these trials were compared to results from control incubations that were carried out using inorganic nutrient solution that had not been previously used to grow other organisms. Total numbers of bacteria were counted using several methods. In experiments in which growth on metabolic by-products from other strains was tested, bacteria were counted directly using a calibrated Thoma counting chamber (0.02 m m depth; Fleischhacker KG, Schwerte, Germany). Total beterotrophic bacterial numbers were determined in samples from the SBRs by plate counts on 0.1-strength trypticase-soy agar. The diameter of bacterial flocs in the SBRs was measured using a sedimentation and light extinction method [6; J Silverstein, M Bowman, T Hess, SK Schmidt, B Howe, Env Eng Proc, July, 1990].
Protozoa Heterotrophic microflagellates and ciliates were counted by placing 20 ul of reactor fluid on a microscope slide, covering the droplet with a cover slip, and then counting all of the protozoa in a 4-mm-wide swath across the middle of the cover slip. Counts were made using a phase-contrast microscope at 250 x or 400 x magnification. This method allowed the differentiation of dead or encysted protozoa from active organisms and eliminated some methodological difficulties associated with protocols that require filtering of samples [el. 24]. An organism was considered to be active if it had visible flagella and was either swimming or moving its flagella. Encysted organisms were usually more refractive under phase contrast microscopy than were active organisms and did not have visible flagella. Triplicate counts were made at each sampling interval, and the mean and
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standard error of the mean (SEM) are reported in this paper. The sizes of the heterotrophic microflagellates were measured using a calibrated ocular micrometer.
C h e m i c a l Analyses The analytical procedures used in this study have been described in more detail by Schmidt et al. [30] and Hess et al. [14]. Samples (6 ml) were withdrawn from the center of each reactor at regular intervals using a syringe with a 15-cm needle that was inserted through a septnm in the reactor wall. The samples were immediately filtered through 0.2-~tm polycarbonate filters (Nuclepore, Pleasanton, CA) and acidified with 1 drop of concentrated H2804. DNP concentration was measured using a Varian DMS 100 spectrophotometer (Varian Associates, Palo Alto, CA) set at 260 nm. In experiments in which 14C-labelled DNP was used, 1-ml subsamples were added to 4-ml Omni scintillation vials (Wheaton Co., Millville, NJ) along with 2.5 ml of ScintiVerse II scintillation cocktail (Fisher Scientific, Pittsburgh, PA). The radioactivity was counted using a liquid scintillation counter (LKB Wallac, 1209 Rackbeta, Turku, Finland). To verify that DNP was mineralized, ~4COz from ~4C-labelled DNP and NOz- evolution were also monitored in some experiments [14]. The partitioning of carbon from glucose into biomass, CO2, and metabolic by-products in cultures of Bacillus cereus was determined using ~4C-labelledglucose as has been previously described [27, 30]. To verify that ~"C left in solution at the end of experiments was not 14C-labelled glucose, the anthrone reaction for total carbohydrates was used [11].
Chemicals [u-~4c] 2,4-dinitrophenol (10.2 mCi mmol % and [U-14C] glucose (296 mCi mmol -~) were purchased from Sigma Chemical Company (St. Louis, MO). Unlabelled reagent grade 2,4-dinitrophenol was purchased from Fluka Chemical Company (Ronkonkoma, NY), and unlabelled reagent grade glucose was obtained from Mallinckrodt, Inc. (Paris, KY). The purity of all compounds was greater than 99%.
Data Analyses Nonlinear regression analyses were performed on all curves of DNP degradation using the LMFIT computer program [ 14]. The data comparing the responses of different strains of bacteria to filtrates of other strains were analyzed using ANOVAs and Scheffr's Multiple Comparison Test.
Results T h e e x p e r i m e n t s r e p o r t e d in t h i s p a p e r w e r e d o n e o v e r a p e r i o d o f o n e y e a r a n d ar e a c o n t i n u a t i o n o f w o r k p r e v i o u s l y r e p o r t e d b y H e s s et al. [ 14]. A t t h e beginning o f this p e r i o d the SBRs were switched f r o m an influent D N P conc e n t r a t i o n o f 1 /zg m1-1 t o a c o n c e n t r a t i o n o f 10 /~g m1-1. T h e S B R s w e r e o p e r a t i n g u n d e r s t e a d y - s t a t e c o n d i t i o n s w h e n all o f t h e w o r k r e p o r t e d b e l o w w a s c o n d u c t e d . S B R 3 w a s r e c e i v i n g 100 #g o f g l u c o s e m1-1 i n a d d i t i o n t o 10 /~g o f D N P m l -~ at t h e b e g i n n i n g o f e a c h 4 8 - h o u r cycle. S B R 1 s e r v e d as a c o n t r o l a n d r e c e i v e d 10 #g o f D N P m l 1 b u t n o s u p p l e m e n t a l a d d i t i o n s o f glucose. F i g u r e 1 s h o w s t h e r e s u l t s o f t y p i c a l D N P r e m o v a l c u r v e s d u r i n g o n e c y cl e o f t h e s e t w o r e a c t o r s . T h e S B R t h a t w a s r e c e i v i n g s u p p l e m e n t a l g l u c o s e
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I
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10 8
O. Z 6
Fig. 1. Mineralization of DNP in SBR 1 and SBR 3 after each reactor had been running under steady-state conditions for several months. SBR 3 received 10 ~tg ofDNP mF' and 100 ~g of glucose ml -t, whereas SBR 1 received only 10/~g of DNP ml ~at the beginning of each 48hour cycle of operation.
I)
4
2
~+ Glucose(SBR3)
o 5
10
15
20
25
30
Hours
Table 1. Steady-state values for MPN estimates of DNP-mineralizing bacteria and total suspended solids in SBRs receiving 10 gg of DNP ml ~ and either no glucose (SBR 1) or 100 #g of glucose ml 1 (SBR 3)
SBR no.
Glucose concentration (#g ml-')
1
0
3
100
MPNs (cells ml ')
Suspended solids (gg ml -~)
Contents
Effluent
Contents
Effluent
1.1 _+ 0.3 x 105 1.1 _+ 0.3 x 106
5.4 + 3.3 x 104 5.0 -+ 3.5 x 104
20 -+ 1 812 +_ 10
3.5 -+ 0.2 13 _+ 0.5
a d d i t i o n s s h o w e d e n h a n c e d D N P r e m o v a l kinetics, w h i c h h a v e b e e n d e s c r i b e d in detail elsewhere [ 14]. P r e v i o u s w o r k [ 14] i n d i c a t e d t h a t the s t i m u l a t o r y effect o f glucose o n D N P m i n e r a l i z a t i o n m a y h a v e b e e n d u e to the i n c r e a s e d flocculation a n d therefore i n c r e a s e d recycling o f D N P - m i n e r a l i z i n g o r g a n i s m s in S B R s receiving glucose s u p p l e m e n t s . T o test this h y p o t h e s i s , floc size m e a s u r e m e n t s a n d several exp e r i m e n t s were p e r f o r m e d . D i a m e t e r s o f bacterial flocs in S B R 3 r a n g e d f r o m 40 to 300 # m ( m e a n = 172 ~m), w h e r e a s flocs in S B R 1 were m u c h smaller, r a n g i n g in size f r o m 32 to 4 8 / ~ m ( m e a n -- 41 #m). S B R 3 also h a d m u c h higher s u s p e n d e d solids c o n c e n t r a t i o n s t h a n S B R 1 (Table 1). I n a d d i t i o n , M P N e s t i m a t e s i n d i c a t e d t h a t there were a p p r o x i m a t e l y 10-fold m o r e D N P - m i n e r alizing o r g a n i s m s in S B R 3 t h a n in S B R 1, a n d the loss o f D N P - m i n e r a l i z e r s in the r e a c t o r effluent was p r o p o r t i o n a t e l y l o w e r in S B R 3 t h a n in S B R 1 (Table 1). A n e x p e r i m e n t was c o n d u c t e d to see if the d e l e t i o n o f glucose f r o m o n e cycle o f o p e r a t i o n o f S B R 3 w o u l d cause a significant s l o w - d o w n o f D N P m i n e r a l i z a t i o n in this r e a c t o r (Fig. 2). It was r e a s o n e d t h a t if glucose was n e e d e d to m a i n t a i n the h i g h rate o f D N P m i n e r a l i z a t i o n d u r i n g e a c h cycle o f S B R o p e r a t i o n t h e n the effect o f glucose was directly o n the g r o w t h rate o f D N P m i n e r a l i z i n g o r g a n i s m s . If, o n the o t h e r h a n d , the effect o f glucose w a s a result
132
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I
I
I
I
10
I
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~ Jt
=.
I
I
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l
+ Glucose 12/4/89
Fig. 2. Mineralization of 10 ~g of DNP ml-l during three consecutive cycles of operation of SBR 3. The first and last cycles were run with supplemental glucose additions (100 lzg of glucose ml-~) and the middle cycle was run without the addition of glucose. Prior to this experiment, SBR 3 was at steady-state and had received glucose at a rate of 100 ug ml ' every 48 hours for three months.
- Glucose 12/6/89
8
~6 c~ 4
4
I
0 0
I 1
I
I 2
I
I I 3 Hours
I 4
I
"it 5
6
Table 2. Comparison of the model estimates from two models ofsubstrate mineralizationapplied to three consecutive cycles of SBR 3 Date
Model
#max (hours -~)
K1 (hours -~)
Ks (~g ml-~)
Xo (#g ml -~)
12/4/89 12/6/89 12/8/89
IV IV IV
0.63 + 0.03 0.68 _+ 0.03 0.72 + 0.02
0.085 + 0.006 0.093 + 0.007 0.070 +_ 0.004
NA~ NA NA
NA NA NA
12/4/89 12/6/89 12/8/89
Monod Monod Monod
0.30 _+_0.04 0.46 + 0.13 0.47 + 0.06
NA NA NA
0.9 + 0.3 2.1 + 0.9 1.7 + 0.4
4.6 _+ 0.7 3.3 + 1.1 2.5 _+ 0.4
a NA, not applicable o f its effect o n t h e b u i l d - u p a n d m a i n t e n a n c e o f D N P - m i n e r a l i z i n g o r g a n i s m s o v e r m a n y cycles, t h e n t h e d e l e t i o n o f glucose f r o m o n e cycle o f S B R 3 o p e r a t i o n s h o u l d n o t r e s u l t i n a s i g n i f i c a n t c h a n g e i n D N P m i n e r a l i z a t i o n kinetics. F i g u r e 2 s h o w s the r e s u l t s o f a n e x p e r i m e n t i n w h i c h t h e glucose feed was t u r n e d off i n the s e c o n d o f t h r e e c o n s e c u t i v e cycles o f S B R 3 o p e r a t i o n . T h e D N P m i n e r a l i z a t i o n c u r v e s were a l m o s t i d e n t i c a l i n all o f the cycles a n d n o n l i n e a r r e g r e s s i o n c u r v e fits y i e l d e d s i m i l a r p a r a m e t e r e s t i m a t e s for all t h r e e c u r v e s ( T a b l e 2). T h e s e d a t a l e n d s u p p o r t to t h e h y p o t h e s i s t h a t the role o f glucose is v i a its effect o n i n c r e a s e d f l o c c u l a t i o n r a t h e r t h a n i n c r e a s e d g r o w t h rate o f t h e D N P - m i n e r a l i z i n g b a c t e r i a d u r i n g a n y o n e cycle o f S B R 3 o p e r a t i o n . F u r t h e r a t t e m p t s at u n d e r s t a n d i n g t h e effects o f glucose o n D N P m i n e r a l i z a t i o n were f o c u s e d o n e x a m i n i n g m i c r o b i a l p o p u l a t i o n d y n a m i c s i n S B R s 1 a n d 3. D u r i n g o p e r a t i o n w i t h a n i n f l u e n t D N P c o n c e n t r a t i o n o f 1 ~tg m1-1, b o t h S B R s d e v e l o p e d d i v e r s e m i c r o b i a l c o m m u n i t i e s t h a t i n c l u d e d yeasts, flagellates, ciliates, a m o e b a e , rotifers, a n d a t least t e n species o f b a c t e r i a ( H o w e a n d S c h m i d t , u n p u b l i s h e d ) . A f t e r t h e r e a c t o r s were s w i t c h e d f r o m a D N P c o n c e n t r a t i o n o f 1 ~tg m1-1 to 10 ~g m1-1, a d e c l i n e i n t h e n u m b e r o f rotifers a n d ciliates w a s n o t e d , a n d after t h e r e a c t o r s were o p e r a t e d for s e v e n m o n t h s at a D N P c o n c e n t r a t i o n o f 10 #g m1-1, o n l y flagellated p r o t o z o a a n d o c c a s i o n a l l y a s m a l l c i l i a t e d p r o t o z o a n were o b s e r v e d .
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The flagellated protozoa observed in the SBRs at steady-state were heterotrophic microflagellates of the class Zoomastigophorea, orders Chrysomonadida and Kinetoplastida. These protozoa were spherical to ovoid in shape (diam = 4 to 8 gin), had two flagella, and resembled organisms in the genera M o n a s (Chrysomonadida) and Bodo (Kinetoplastida) [4, 7, 8]. These protozoa were either attached to bacterial floes or found free-swimming between floes. Attached protozoa used one flagellum to attach themselves to the floes and the other to create a feeding current. Fenchel [7] reports a similar feeding strategy for several members of the Kinetoplastida. Microflagellate population dynamics were cyclical during steady-state SBR operation (Figs. 3-5). Population peaks were consistently observed during the first 14 hours of operation and population lows were normally observed after about 30 hours into each cycle (Fig. 3). Increases in protozoan numbers corresponded with a decline in plate counts of heterotrophic bacteria (Fig. 4). As with earlier SBR cycles (Figs. 1 and 2) DNP was completely mineralized in SBR 3 by six hours into the reaction cycle (Fig. 5). It is also of note that glucose was completely mineralized in SBR 3 by 1 hour into the reaction cycle [Howe and Schmidt, unpublished]. Ciliated protozoa were occasionally observed, but their numbers were too few to be accurately quantified. Protozoan population densities were approximately 10-fold lower in SBR 1 than in SBR 3 (data not shown). Microscopic observations indicated that most of the bacteria in the reactors were in floc particles. These flocs contained mostly coccoid cells,but occasional filamentous bacteria were observed. Five bacterial colony types were consistently observed during plate count procedures (on D N P agar and trypticase soy agar) carried out during February and March, 1990. These bacteria were obtained in pure culture, and their characteristics are given in Table 3. Of these five bacterial species, only one (isolate D) could use D N P as a source of carbon and energy. This organism is most likely a descendent of the Actinomycete, which was initiallyinoculated into the reactors. This bacterium was similar to the original Actinomycete except that it could no longer utilize arabinose, fumarate, or 1-naphthol as growth substrates (Table 3). However, both organisms contain tuberculostearic acid and had almost identical fatty acid profiles. Given all of the characteristics of these two actinomycctes, they are most likely in the genus Rhodococcus [29]. The DNP-mineralizing strain of Janthinobacterium that was initially inoculated into the SBRs was never recovered aRer steady-state conditions were reached. Bacterial isolates B and C are probably very closely related to one another as indicated by the data given in Table 3 and the fact that their fatty acid profiles were almost identical. Strain A is a very fastidious organism that could not be identified either by standard criteria (Table 3) or by fatty acid profile analysis. Isolate E could not be characterized as to carbon source utilization because it grew prolifically in the auxanographic plates even in the absence of any added carbon source. However, this organism was positively identified as Bacillus cereus using fatty acid profile analysis. Three (isolates A, B, and C) of the five bacteria isolated from SBR 3 could not utilize glucose or DNP as a source of carbon and energy (Table 3). This is
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surprising because the only organic c o m p o u n d s fed into the reactors were D N P a n d glucose. T h i s o b s e r v a t i o n led to a series o f e x p e r i m e n t s to d e t e r m i n e if these three o r g a n i s m s could use m e t a b o l i c b y - p r o d u c t s o f the other two bacteria in the reactors. Figure 6 shows that all three bacteria could grow in the sterile filtrates o f B. cereus but not in the filtrates o f the R h o d o c o c c u s sp. Several e x p e r i m e n t s were p e r f o r m e d to d e t e r m i n e i f the B. cereus p r o d u c e d significant a m o u n t s o f m e t a b o l i c b y - p r o d u c t s as a result o f its growth on glucose. Cultures grown to stationary phase on 14C-glucose excreted on average 16% o f the 14Clabel into the external m e d i u m , whereas 33% a n d 51% o f the label was f o u n d in b i o m a s s a n d CO2, respectively. This is in contrast to c a r b o n partitioning in the R h o d o c o c c u s sp., which c o n v e r t e d greater t h a n 98% o f b o t h glucose a n d D N P to either b i o m a s s or CO2. At present, the nature o f the growth stimulating c o m p o u n d p r o d u c e d b y B. cereus is not known, b u t analyses showed that there was no m e a s u r a b l e glucose in the filtrates at the end o f each e x p e r i m e n t with this organism.
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