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Cultivation of nitrifying bacteria in the retentostat, a simple fermenter with internal biomass retention
ELSEVIER
FEMS Microbiology
Ecology
19 (1996) 47-52
Cultivation of nitrifying bacteria in the retentostat, a simple fermenter with internal biomass retention W. Tappe *, C. Tomaschewski,
S. Rittershaus, J. Groeneweg
F[~rschungs~entrzrm Jiilich GmbH, fnstitut fir Biotechnologie 3, 52425 Jiilich. Germany Received 9 May 1995; revised 20 October
1995: accepted 25 October
1995
Abstract The retentostat was developed for long-term continuous, axenic cultivation of microorganisms at those low growth rates which prevail in most natural habitats and which cannot be established properly in chemostats. How a microbial population approaches ‘zero-growth’ was studied in axenic cultures of Nitrosomonas europaea with complete biomass retention at 25°C and constant input of a nutrient solution containing ammonium (0.57 mM) as energy source. Since only cell-free filtrate left the reactor, biomass accumulated until a stable maximum of 2.7 X IO9 cells ml- ’ (398 mg I-’ dry matter) was reached after about 5 weeks. In this state, growth rate approached zero, and the ammonium input just met the substrate demand required for maintenance energy (1.43 pmol NH, - N mg dm- ’ h- ’ ). The potential of the retentostat for studying interactions between different microorganisms was demonstrated with a cascade of cultures of Nitrosomonas, Nitrobacter, and a denitrifying Pseudomonas. Thereby the ammonia was completely eliminated from artificial wastewater. Kqwords: Biomass retention: Maintenance
energy;
Nitrosomonas ewopaea; Nitrobacter winogradskyi; Bioreactor
1. Introduction Little is known about the physiology of bacteria growing at rates considerably lower than their potential growth rates [I] although extremely slow growth and starvation are predominantly the rule rather than the exception in natural microbial populations [2,3]. Chemostatic cultures are commonly used to study bacterial growth kinetics because in steady states they are homogeneous systems having constant inputs and outputs [4]. If one wants to cultivate bacteria at those substrate concentrations and growth rates
^ Corresponding (2461) 612492.
author.
0168.6496/96/$15.00
Tel: +49
(2461) 614824;
0 1996 Federation
SSDl 0168-6496(95)00079-X
of European
Fax:
+49
Microbiological
cascade
assumed to be predominant in many bacterial habitats, dilution rates have to be extremely low (< 0.05 h-‘). This then leads to inhomogeneities of nutrient distribution in time and space [5,6]. Furthermore, the application of chemostats is restricted to studies on growing cells, simply because the dilution rate is equivalent to the growth rate. Hence, a chemostat cannot be operated in a steady-state near or at zero growth. These shortcomings in the use of chemostats have been overcome by combining bioreactors with microfiltration units to ensure continuous separation and feedback of the bacterial biomass. Such recycling techniques have been successfully applied to study the maintenance energy demand and physiology of slowly grown bacteria [7-lo]. With the ‘reSocieties. All rights reserved
tentostat’. described in detail in this paper, a simplified system was realized by extending a filtration module to a small bioreactor (360 ml) which allows internal biomass retention. This apparatus was used to grow a pure culture of N. e~vopaecr with complete biomass retention for the direct determination of maintenance energy demand. Cell volume distributions within the non-growing population before and after a nutrient shift-up were measured and used to discuss the admissibility of this method.
2. Materials
and methods
2.1. Technical ,feature.s The principle of a stirred microfiltration cell was employed as the basis of construction for the bioreactor with internal biomass retention (Fig. I). A glass cylinder (borosilicate glass, diam. 90 mm) was pressed together by wing bolts between the base and
1
Fig. 1. Technical design of the retentostat (additional in- and outlets are not shown). 1. ball bearing: 2. baffle and in- or outlet: 3, glass cylinder; 4, stirrer: 5, filter membrane; 6. silicon rubber gasket: 7, steel grid: 8, filtrate outlet.
the cover made from stainless steel (DIN 1.457 1). A microfiltration membrane (Millipore, 0.2 km pore size, PVDF. diam. 90 mm) was positioned on a support membrane (Sartorius polyester fabric) and a stainless steel grid (IO mm mesh width, 0.5 mm diam. wires) at the base. Flat silicon rubber gaskets (1 mm thick) sealed the contacts between the glass cylinder, the grid with the membrane filter, and the steel bottom. One bore-hole in the base connected with an outlet outside was sufficient to collect the filtrate because the texture of the grid permitted a horizontal filtrate flow. The volume below the filter membrane amounted to about 3 ml. In the center of the upper compartment (360 ml working volume) a teflon-coated magnetic stirring bar suspended from the lid by an axis mounted on ball bearings was held just above the filter membrane to produce a cross-flow effect and prevent clogging of the filter membrane. It was driven by a magnetic stirrer driver at 600 rpm (Ikamag REO, equipped with an extra-strong magnet) positioned under the reactor. Furthermore, two baffles guaranteed thorough mixing, and additional bore-holes in the cover allowed the installation of fittings for sampling, feed supply and exhaust-gas outlet. Conductivity contacts triggered a peristaltic pump for filtrate removal and maintained the culture volume constant. The whole system. including all fittings and tube connections, was sterilized in an autoclave.
The system could be operated either as a chemostat or as a bioreactor with biomass retention. Any growth rate between p = D (no filtrate drawn off; operation as chemostat) and p + 0 (filtrate flow equivalent to the nutrient input; biomass completely retained and accumulated in the system) could be maintained. In order to permit the desired long-term operation without interruptions due to backflushing or changing the filter membrane, a filtrate flow far below the maximum filtration capacity of the system was used. Previous tests showed that maximum filtration rates of 250 to 500 ml hh’ for about 2 days with pure bacterial cultures (30-350 mg ll’ dry matter of Nitrosomorlus europaea. Nitrobacter bcinogrudsl\?i,
W. Tappe et al. / FEMS Microbiology
Pseudomonas jkorescens), and activated sludge (4000 mg l- ’ dry matter) were obtainable. A filtration rate of 50- 100 ml h-’ proved to be low enough to prevent the bacteria from clogging the membrane and permitted uninterrupted cultivation for up to several months. At these low filtration rates no increase in pressure was needed to obtain the filtrate. Therefore. all experiments with biomass retention were carried out at a hydraulic dilution rate of 0.1 h- ’ requiring maximum filtrate flows of 36 ml hh’. 2.2. I. Chemostatic operation Without the need for biomass retention the bioreactor could be operated as a simple chemostat. If no changes in operation mode were intended during the experiment the filter membrane was omitted and the filtrate outlet was closed. 2.2.2. Retention mode During operation in the retention mode the dilution rate of the feed and the growth rate of the bacteria were uncoupled. Depending on the ratio of the filtrate flow rate and the suspension removal rate any p between D and p + 0 could be selected. 2.2.3. Multi-stage operatiorz This simple device favours the performance of multi-stage experiments. Since every stage delivers a sterile filtrate, this can be used as feed for a culture in the following reactor. In this way, backfeeding and crossfeeding experiments could easily be carried out for the examination of mutual effects between different bacterial strains. 2.3. Bacterial
Ecology
I9
f 1996147-52
49
Nitrobacter winogradskyi (strain ‘Engel’, also obtained from H.-P. Koops) was grown on the same medium after oxidation of the NH, to NO; by Nitrosomonas. Pseudomonas j7uorescens (ATCC 174821 was grown on the same medium supplemented with 8.2 mM sodium acetate. The medium was supplied continuously at a rate of 36 ml h- ’ to give a dilution rate of 0.1 hh’, and the filtrate was removed at the same rate (complete biomass retention for the determination of maintenance energy demand) or with 9/ 10 of the input rate (partial biomass retention in the bioreactor cascade experiments). The bioreactors with the nitrifiers were aerated with 15 1 h-’ filtered air and the denitrifying culture was sparged with N, at the same rate. All experiments were carried out at 25 k 1°C. The nitrifying cultures were regularly checked for heterotrophic contaminations by incubating samples in tryptic soy broth (Merck) for two weeks at 30°C. 2.4. Analytical
methods
Ammonia, nitrite and nitrate were measured with a segmented-flow autoanalyzer (Skalar). Cell counts, cell volume distributions and mean cell volumes were determined with an electronic particle counter (Elzone PC 280; 18 pm orifice). In previous experiments, a linear correlation between biovolume. POC. and dry matter was found. Thus, only small samples (a maximum of 3 ml per day) needed to be taken for electronic particle counting to minimize disturbances in growth kinetics, and POC and dry matter were calculated from the measured biovolume. DOC was measured with a TOC-analyzer (Shimadzu TOC5000).
strains, media, and growth conditions
Nitrosomonas europaea (strain Nm 35, kindly supplied by H.-P. Koops. University of Hamburg) was grown in an inorganic medium containing (g 1-l): NH,Cl, 0.306: Na,HPO,. 12 H,O, 0.1; 0.05; CaCl, . 2H,O, 0.015; MgSO, . 7H20, NaHCO,, 1.13. trace elements ( pg 1-i 1: MnCl, . 4H,O, 99; ZnSO, 7H,O, 6.3; NiSO, .7H,O. 26.3; CoSO, .7H,0.2.8 1; CuSO, .5H,O, 4.99. Fe-EDTA, 1 ml of the stock solution (Titriplex III, 9.3 g 1-l and FeSO,, 6.95 g 1-l).
3. Results and conclusions One of the specific features of the retentostat as compared to a chemostat without biomass retention is that high cell densities at lowest growth rates can be attained. The apparatus is therefore appropriate for studying starvation phenomena and maintenance energy demand in continuous pure cultures of bacteria. Any low growth rate can be adjusted in spite of a constant dilution rate of the nutrients avoiding the
above mentioned problems in chemostats with homogeneity and low biomass concentrations at extremely low dilution rates. After preliminary tests had proven the reliability of the retentostat for extended axenic fermentations, an experiment with Nitrosomonas was started. A pure culture of N. europaea was continuously grown in a retentostat for 6 weeks with complete biomass retention. The low ammonia input concentration of 5.7 mM ensured energy-limited growth and excluded product inhibition (N0,/HN02) as found to occur in former chemostat experiments at higher input concentrations (30 mM NH,Cl). During the first 5 days after inoculation with 1.5 X 10’ cells ml-’ from a chemostatic preculture, growth was not limited by ammonia and the culture reached a p,,,,, of 0.05 hh’ while the mean cell volume increased from initially 0.32 pm’ up to 0.6 pm3. Subsequently, ammonia became growth-limiting and during the accumulation of biomass, the ratio of ammonia per cell and unit of time decreased continuously. The decrease in biomass-specific ammonia availability resulted in decreasing growth rates accompanied by a diminution in mean cell volume (Fig. 2). Finally, biomass concentration reached a stable maximum of 2.7 X 1O9 cells ml ’ equivalent to 180 mg 1~ ’ POC or 398 mg 1-l of dry matter. At this stage, energy generated from ammonia oxidation must have been used completely for meeting maintenance energy demand (1.43 pmol NH, -N mg drn-’ h-l). In order to verify that growth was limited by the spe-
10’0
Fig. 2. Cell concentration and mean cell volume of N. europarn during cultivation at a constant nutrient flow rate and complete biomass retention. The arrow indicates the shift-up in dilution rate from 0.1 hK’ to 0.2 h-‘.
.
0.000
/
0.20
0.25
/
0.30
I
I
0.35
0.40
mean
Fig. 3. Specific r*!xzea.
growth
cell
0.45
0.50
0.55
0
volume (pm31
rate and mean cell volume
of N. err-
cific ammonia availability, the input was shifted up in doubling the dilution rate from 0.1 hh’ to 0.2 hh ’ In this manner. the amount of ammonia provided per unit of time was doubled without changing the input concentration. An increase in cell concentration indicated that ammonia really was the growth limiting factor (Fig. 2). The specific substrate demand for maintenance determined for the non-growing bacteria was about 2-3 times lower than the values extrapolated from chemostat cultures of the same strain (C. Tomaschewski, thesis in preparation). This decrease in specific maintenance energy demand with decreasing growth rates has also been observed in other studies and is the subject of much debate [ 1, lo- 131. Pirt [l] accounted for a dormant population within a slowly or non-growing population giving rise to a lower m-value calculated for the whole population. Hence, especially for slowly grown bacteria, the homogeneity and activity of the whole population has to be analyzed. In order to test the homogeneity of Nitrosomonas at zero-growth in the retentostat we used the following procedure: The division rate obtained from the derivative of the increase in cell concentration with time correlated linearly with the mean cell volume (Fig. 3). Therefore, the mean cell volume could serve as an indicator for the actual growth rate and the cell volume distribution should reflect the homogeneity of the population. The narrow log-normal distribution of non-growing Nitrosomonas cells with a mean cell volume of 0.25 pm” already indicated a homogeneous population since faster growing subpopula-
W. Tappe et al. / FEMS Microbiology
the existence of a dormant fraction without substrate consumption. Amongst many further potential applications of the retentostat for studies related to microbial ecology one additional example shall be briefly presented: The complex biological processes of nitrification and denitrification were simulated with pure bacterial cultures by means of a bioreactor cascade (Fig. 5). Three retentostats were operated in a series. In the first one, Nitrosomonas europaea was grown on a medium containing ammonia (5.7 mM) and acetate (8.2 mM). The sterile filtrate of this culture was fed to the second retentostat and served as the substrate for a pure culture of Nitrobacter winogradskyi. Subsequently, the filtrate of the second stage was pumped into the third retentostat, where nitrate was denitrified by Pseudomonas sp. under anoxic conditions using acetate as a source of carbon and energy. Under steady state conditions, approximately 90% of the ammonia input was oxidized to NO, in the first stage by Nitrosomonas. Nitrobacter on its part oxidized nearly 95% of the NO, to NO,. Acetate uptake (measured as DOC) was not detectable in these two stages although Nitrobacter should be able to grow mixotrophically [ 141. In the third stage the concentrations of NH,, NO, and
-1 0.08
0.44
0.26
cell volume
0.62
02
(urn’)
Fig. 4. Cell volume distribution of N. ewopaea after incubation with 0.7 mM NH,Cl.
before and 12 h
tions would have led to an uneven distribution in cell volume classes. The reactivity of the non-growing cells was tested by incubating diluted samples (10’ cells ml-’ ) with 0.7 mM NH,Cl for 12 h to permit a growth response. From the almost perfect symmetrical shift of the cell volume from 0.25 to 0.31 pm3 after substrate addition (Fig. 41, we conclude that all cells were in an active state. Therefore, the lower m-values determined for the non-growing bacteria compared to the higher maintenance requirements of the chemostat cultures could not be explained with air in
51
Ecology 19 (1996147-52
air m
Nz in
gas out surplus biomass t
II [
ammonia + aceticacid
1
Fig. 5. Configuration of the bioreactor outflow of the different stages.
Nitrobacter
cascade and the concentrations
of ammonia,
Pseudomonas
-
nitrite and nitrate in the nutrient reservoir and the filtrate
52
W. Tuppe et al. / FEMS Microhiolog~
NO; ranged below the detection level (35pM) of the segmented flow analysis. Since acetate as the carbon source was given in excess, denitrification was limited by the NO; and NO, input from the second reactor. The ammonia not oxidized by Nitrosomonas and Nitrobacter was obviously assimilated by Pseudomonas during anoxic growth in the last reactor. These examples indicate the value of the retentostat for the experimental investigation of microbial interactions. In general, the same presuppositions are valid for the retentostat as for chemostats: The correct interpretation of the measurements depends on the system’s homogeneity. Therefore, the microorganisms should grow in suspension without flocculation and adhesion to the internal surfaces of the reactor walls and installations. However, if adhesion occurs, the error in calculations will be relatively smaller when based on the higher cell densities attainable in the retentostat as compared to the much lower densities in chemostatic cultures growing at the same growth rate.
References [I] Pirt. S.J. (1987) The energetics of microbes at low growth rateb: Maintenance energies and dormant organisms. .I. Ferment. Technol. 65. 173-177. [2] Poindexter, J.S. (1981) Oligotrophy. Fast and famine existence. Adv. Microb. Ecol. 5. 63-89. [3] Van Elsas, J.D. and van Overbeek, L.S. (1993) Bacterial responses to soil stimuli. In: Starvation in Bacteria (Kjelle-
Ecolog,v I9 (I 9%) 47-52
berg. S.. Ed.), pp. 55-79. Plenum Prehs. New York and London. 141Herbert. D.. Elsworth, R. and Telling. R.C. (1956) The continuous culture of bacteria. A theoretical and experimental sudy. J. Gen. Microbial. I-l, 601-631. [51 Hamford, G.S. and Humphrey. A.E. (1966) The effect of equipment scale and degree of mixing on continuous fermentation yield at low dilution rates. Biotech. Bioeng. 8. 85-96. [61 Bulthuix, B.A.. Koningstein, GM.. Stouthamer. A.H. and Van Veraeveld. H.W. (1989) A comparison between aerobic growth of Brrcillus /ichrr~ifwmi.s in continuous culture and partial-recycling fermentor. with contributions to the discussion on maintenance energy demand. Arch. Microbial. 152. 199-507. [71 Chesbro. W., Evans. W. and Eifert, R. (1979) Very slow growth of E. L.o/~.J. Bacterial. 139. 635-638. [Xl Matin, A.. Auger. E.A., Blum. P.H. and Schultz. J.E. (1989) Genetic basis of starvation survival in nondifferentiating bacteria. Ann. Rev. Microbial. 42. 293-3 16. [9] Beyeler. W., Rogers, P.L. and Fiechter. A. (1983) A simple technique for the direct determination of maintenance energy coefficient: an example with Z?./vonro~rc~.\ rnubi[is. Appl. Microbial. Biotechnol. 19. 277-280. [IO] Van Verseveld, H.W.. Chesbro. W.R.. Braster. M. and Stouthamer, A.H. (1983) Eubacteria have 3 growth mode?, keyed to nutrient tlow. Arch. Microbial. 1.17. 176- 184. [I I] Stouthamer, A.H. and Bettenhausen. C. (1973) Utilization of energy for growth and maintenance in continuous and batch cultures of microorganisms. Biochim. Biophys. Acta 301. 53-70. [I21 Pin, S.J. (1982) Maintenance energy: a general model for energylimited and energy-sufficient growth. Arch. Microbiol. 133. 300-301. [ 131Neijssel. O.M. and Tempest. D.W. (1976) The role of energy-spilling reactions in the growth of Klrhirlltr trero~rrws NCTC 318 in aerobic chemostat culture. Arch. Microbial. I IO.305-31 I. [ 141 Bock, E., Koopa, H.P. and Harms. H. (1986) Cell biology of nitrifying bacteria. In: Nitrification (Presser, J.I.. Ed.). 17-38. Special publications of the Society for General Microbiology. Vol. 30. IRL Press.
FEMS Microbiology
Ecology
19 (1996) 47-52
Cultivation of nitrifying bacteria in the retentostat, a simple fermenter with internal biomass retention W. Tappe *, C. Tomaschewski,
S. Rittershaus, J. Groeneweg
F[~rschungs~entrzrm Jiilich GmbH, fnstitut fir Biotechnologie 3, 52425 Jiilich. Germany Received 9 May 1995; revised 20 October
1995: accepted 25 October
1995
Abstract The retentostat was developed for long-term continuous, axenic cultivation of microorganisms at those low growth rates which prevail in most natural habitats and which cannot be established properly in chemostats. How a microbial population approaches ‘zero-growth’ was studied in axenic cultures of Nitrosomonas europaea with complete biomass retention at 25°C and constant input of a nutrient solution containing ammonium (0.57 mM) as energy source. Since only cell-free filtrate left the reactor, biomass accumulated until a stable maximum of 2.7 X IO9 cells ml- ’ (398 mg I-’ dry matter) was reached after about 5 weeks. In this state, growth rate approached zero, and the ammonium input just met the substrate demand required for maintenance energy (1.43 pmol NH, - N mg dm- ’ h- ’ ). The potential of the retentostat for studying interactions between different microorganisms was demonstrated with a cascade of cultures of Nitrosomonas, Nitrobacter, and a denitrifying Pseudomonas. Thereby the ammonia was completely eliminated from artificial wastewater. Kqwords: Biomass retention: Maintenance
energy;
Nitrosomonas ewopaea; Nitrobacter winogradskyi; Bioreactor
1. Introduction Little is known about the physiology of bacteria growing at rates considerably lower than their potential growth rates [I] although extremely slow growth and starvation are predominantly the rule rather than the exception in natural microbial populations [2,3]. Chemostatic cultures are commonly used to study bacterial growth kinetics because in steady states they are homogeneous systems having constant inputs and outputs [4]. If one wants to cultivate bacteria at those substrate concentrations and growth rates
^ Corresponding (2461) 612492.
author.
0168.6496/96/$15.00
Tel: +49
(2461) 614824;
0 1996 Federation
SSDl 0168-6496(95)00079-X
of European
Fax:
+49
Microbiological
cascade
assumed to be predominant in many bacterial habitats, dilution rates have to be extremely low (< 0.05 h-‘). This then leads to inhomogeneities of nutrient distribution in time and space [5,6]. Furthermore, the application of chemostats is restricted to studies on growing cells, simply because the dilution rate is equivalent to the growth rate. Hence, a chemostat cannot be operated in a steady-state near or at zero growth. These shortcomings in the use of chemostats have been overcome by combining bioreactors with microfiltration units to ensure continuous separation and feedback of the bacterial biomass. Such recycling techniques have been successfully applied to study the maintenance energy demand and physiology of slowly grown bacteria [7-lo]. With the ‘reSocieties. All rights reserved
tentostat’. described in detail in this paper, a simplified system was realized by extending a filtration module to a small bioreactor (360 ml) which allows internal biomass retention. This apparatus was used to grow a pure culture of N. e~vopaecr with complete biomass retention for the direct determination of maintenance energy demand. Cell volume distributions within the non-growing population before and after a nutrient shift-up were measured and used to discuss the admissibility of this method.
2. Materials
and methods
2.1. Technical ,feature.s The principle of a stirred microfiltration cell was employed as the basis of construction for the bioreactor with internal biomass retention (Fig. I). A glass cylinder (borosilicate glass, diam. 90 mm) was pressed together by wing bolts between the base and
1
Fig. 1. Technical design of the retentostat (additional in- and outlets are not shown). 1. ball bearing: 2. baffle and in- or outlet: 3, glass cylinder; 4, stirrer: 5, filter membrane; 6. silicon rubber gasket: 7, steel grid: 8, filtrate outlet.
the cover made from stainless steel (DIN 1.457 1). A microfiltration membrane (Millipore, 0.2 km pore size, PVDF. diam. 90 mm) was positioned on a support membrane (Sartorius polyester fabric) and a stainless steel grid (IO mm mesh width, 0.5 mm diam. wires) at the base. Flat silicon rubber gaskets (1 mm thick) sealed the contacts between the glass cylinder, the grid with the membrane filter, and the steel bottom. One bore-hole in the base connected with an outlet outside was sufficient to collect the filtrate because the texture of the grid permitted a horizontal filtrate flow. The volume below the filter membrane amounted to about 3 ml. In the center of the upper compartment (360 ml working volume) a teflon-coated magnetic stirring bar suspended from the lid by an axis mounted on ball bearings was held just above the filter membrane to produce a cross-flow effect and prevent clogging of the filter membrane. It was driven by a magnetic stirrer driver at 600 rpm (Ikamag REO, equipped with an extra-strong magnet) positioned under the reactor. Furthermore, two baffles guaranteed thorough mixing, and additional bore-holes in the cover allowed the installation of fittings for sampling, feed supply and exhaust-gas outlet. Conductivity contacts triggered a peristaltic pump for filtrate removal and maintained the culture volume constant. The whole system. including all fittings and tube connections, was sterilized in an autoclave.
The system could be operated either as a chemostat or as a bioreactor with biomass retention. Any growth rate between p = D (no filtrate drawn off; operation as chemostat) and p + 0 (filtrate flow equivalent to the nutrient input; biomass completely retained and accumulated in the system) could be maintained. In order to permit the desired long-term operation without interruptions due to backflushing or changing the filter membrane, a filtrate flow far below the maximum filtration capacity of the system was used. Previous tests showed that maximum filtration rates of 250 to 500 ml hh’ for about 2 days with pure bacterial cultures (30-350 mg ll’ dry matter of Nitrosomorlus europaea. Nitrobacter bcinogrudsl\?i,
W. Tappe et al. / FEMS Microbiology
Pseudomonas jkorescens), and activated sludge (4000 mg l- ’ dry matter) were obtainable. A filtration rate of 50- 100 ml h-’ proved to be low enough to prevent the bacteria from clogging the membrane and permitted uninterrupted cultivation for up to several months. At these low filtration rates no increase in pressure was needed to obtain the filtrate. Therefore. all experiments with biomass retention were carried out at a hydraulic dilution rate of 0.1 h- ’ requiring maximum filtrate flows of 36 ml hh’. 2.2. I. Chemostatic operation Without the need for biomass retention the bioreactor could be operated as a simple chemostat. If no changes in operation mode were intended during the experiment the filter membrane was omitted and the filtrate outlet was closed. 2.2.2. Retention mode During operation in the retention mode the dilution rate of the feed and the growth rate of the bacteria were uncoupled. Depending on the ratio of the filtrate flow rate and the suspension removal rate any p between D and p + 0 could be selected. 2.2.3. Multi-stage operatiorz This simple device favours the performance of multi-stage experiments. Since every stage delivers a sterile filtrate, this can be used as feed for a culture in the following reactor. In this way, backfeeding and crossfeeding experiments could easily be carried out for the examination of mutual effects between different bacterial strains. 2.3. Bacterial
Ecology
I9
f 1996147-52
49
Nitrobacter winogradskyi (strain ‘Engel’, also obtained from H.-P. Koops) was grown on the same medium after oxidation of the NH, to NO; by Nitrosomonas. Pseudomonas j7uorescens (ATCC 174821 was grown on the same medium supplemented with 8.2 mM sodium acetate. The medium was supplied continuously at a rate of 36 ml h- ’ to give a dilution rate of 0.1 hh’, and the filtrate was removed at the same rate (complete biomass retention for the determination of maintenance energy demand) or with 9/ 10 of the input rate (partial biomass retention in the bioreactor cascade experiments). The bioreactors with the nitrifiers were aerated with 15 1 h-’ filtered air and the denitrifying culture was sparged with N, at the same rate. All experiments were carried out at 25 k 1°C. The nitrifying cultures were regularly checked for heterotrophic contaminations by incubating samples in tryptic soy broth (Merck) for two weeks at 30°C. 2.4. Analytical
methods
Ammonia, nitrite and nitrate were measured with a segmented-flow autoanalyzer (Skalar). Cell counts, cell volume distributions and mean cell volumes were determined with an electronic particle counter (Elzone PC 280; 18 pm orifice). In previous experiments, a linear correlation between biovolume. POC. and dry matter was found. Thus, only small samples (a maximum of 3 ml per day) needed to be taken for electronic particle counting to minimize disturbances in growth kinetics, and POC and dry matter were calculated from the measured biovolume. DOC was measured with a TOC-analyzer (Shimadzu TOC5000).
strains, media, and growth conditions
Nitrosomonas europaea (strain Nm 35, kindly supplied by H.-P. Koops. University of Hamburg) was grown in an inorganic medium containing (g 1-l): NH,Cl, 0.306: Na,HPO,. 12 H,O, 0.1; 0.05; CaCl, . 2H,O, 0.015; MgSO, . 7H20, NaHCO,, 1.13. trace elements ( pg 1-i 1: MnCl, . 4H,O, 99; ZnSO, 7H,O, 6.3; NiSO, .7H,O. 26.3; CoSO, .7H,0.2.8 1; CuSO, .5H,O, 4.99. Fe-EDTA, 1 ml of the stock solution (Titriplex III, 9.3 g 1-l and FeSO,, 6.95 g 1-l).
3. Results and conclusions One of the specific features of the retentostat as compared to a chemostat without biomass retention is that high cell densities at lowest growth rates can be attained. The apparatus is therefore appropriate for studying starvation phenomena and maintenance energy demand in continuous pure cultures of bacteria. Any low growth rate can be adjusted in spite of a constant dilution rate of the nutrients avoiding the
above mentioned problems in chemostats with homogeneity and low biomass concentrations at extremely low dilution rates. After preliminary tests had proven the reliability of the retentostat for extended axenic fermentations, an experiment with Nitrosomonas was started. A pure culture of N. europaea was continuously grown in a retentostat for 6 weeks with complete biomass retention. The low ammonia input concentration of 5.7 mM ensured energy-limited growth and excluded product inhibition (N0,/HN02) as found to occur in former chemostat experiments at higher input concentrations (30 mM NH,Cl). During the first 5 days after inoculation with 1.5 X 10’ cells ml-’ from a chemostatic preculture, growth was not limited by ammonia and the culture reached a p,,,,, of 0.05 hh’ while the mean cell volume increased from initially 0.32 pm’ up to 0.6 pm3. Subsequently, ammonia became growth-limiting and during the accumulation of biomass, the ratio of ammonia per cell and unit of time decreased continuously. The decrease in biomass-specific ammonia availability resulted in decreasing growth rates accompanied by a diminution in mean cell volume (Fig. 2). Finally, biomass concentration reached a stable maximum of 2.7 X 1O9 cells ml ’ equivalent to 180 mg 1~ ’ POC or 398 mg 1-l of dry matter. At this stage, energy generated from ammonia oxidation must have been used completely for meeting maintenance energy demand (1.43 pmol NH, -N mg drn-’ h-l). In order to verify that growth was limited by the spe-
10’0
Fig. 2. Cell concentration and mean cell volume of N. europarn during cultivation at a constant nutrient flow rate and complete biomass retention. The arrow indicates the shift-up in dilution rate from 0.1 hK’ to 0.2 h-‘.
.
0.000
/
0.20
0.25
/
0.30
I
I
0.35
0.40
mean
Fig. 3. Specific r*!xzea.
growth
cell
0.45
0.50
0.55
0
volume (pm31
rate and mean cell volume
of N. err-
cific ammonia availability, the input was shifted up in doubling the dilution rate from 0.1 hh’ to 0.2 hh ’ In this manner. the amount of ammonia provided per unit of time was doubled without changing the input concentration. An increase in cell concentration indicated that ammonia really was the growth limiting factor (Fig. 2). The specific substrate demand for maintenance determined for the non-growing bacteria was about 2-3 times lower than the values extrapolated from chemostat cultures of the same strain (C. Tomaschewski, thesis in preparation). This decrease in specific maintenance energy demand with decreasing growth rates has also been observed in other studies and is the subject of much debate [ 1, lo- 131. Pirt [l] accounted for a dormant population within a slowly or non-growing population giving rise to a lower m-value calculated for the whole population. Hence, especially for slowly grown bacteria, the homogeneity and activity of the whole population has to be analyzed. In order to test the homogeneity of Nitrosomonas at zero-growth in the retentostat we used the following procedure: The division rate obtained from the derivative of the increase in cell concentration with time correlated linearly with the mean cell volume (Fig. 3). Therefore, the mean cell volume could serve as an indicator for the actual growth rate and the cell volume distribution should reflect the homogeneity of the population. The narrow log-normal distribution of non-growing Nitrosomonas cells with a mean cell volume of 0.25 pm” already indicated a homogeneous population since faster growing subpopula-
W. Tappe et al. / FEMS Microbiology
the existence of a dormant fraction without substrate consumption. Amongst many further potential applications of the retentostat for studies related to microbial ecology one additional example shall be briefly presented: The complex biological processes of nitrification and denitrification were simulated with pure bacterial cultures by means of a bioreactor cascade (Fig. 5). Three retentostats were operated in a series. In the first one, Nitrosomonas europaea was grown on a medium containing ammonia (5.7 mM) and acetate (8.2 mM). The sterile filtrate of this culture was fed to the second retentostat and served as the substrate for a pure culture of Nitrobacter winogradskyi. Subsequently, the filtrate of the second stage was pumped into the third retentostat, where nitrate was denitrified by Pseudomonas sp. under anoxic conditions using acetate as a source of carbon and energy. Under steady state conditions, approximately 90% of the ammonia input was oxidized to NO, in the first stage by Nitrosomonas. Nitrobacter on its part oxidized nearly 95% of the NO, to NO,. Acetate uptake (measured as DOC) was not detectable in these two stages although Nitrobacter should be able to grow mixotrophically [ 141. In the third stage the concentrations of NH,, NO, and
-1 0.08
0.44
0.26
cell volume
0.62
02
(urn’)
Fig. 4. Cell volume distribution of N. ewopaea after incubation with 0.7 mM NH,Cl.
before and 12 h
tions would have led to an uneven distribution in cell volume classes. The reactivity of the non-growing cells was tested by incubating diluted samples (10’ cells ml-’ ) with 0.7 mM NH,Cl for 12 h to permit a growth response. From the almost perfect symmetrical shift of the cell volume from 0.25 to 0.31 pm3 after substrate addition (Fig. 41, we conclude that all cells were in an active state. Therefore, the lower m-values determined for the non-growing bacteria compared to the higher maintenance requirements of the chemostat cultures could not be explained with air in
51
Ecology 19 (1996147-52
air m
Nz in
gas out surplus biomass t
II [
ammonia + aceticacid
1
Fig. 5. Configuration of the bioreactor outflow of the different stages.
Nitrobacter
cascade and the concentrations
of ammonia,
Pseudomonas
-
nitrite and nitrate in the nutrient reservoir and the filtrate
52
W. Tuppe et al. / FEMS Microhiolog~
NO; ranged below the detection level (35pM) of the segmented flow analysis. Since acetate as the carbon source was given in excess, denitrification was limited by the NO; and NO, input from the second reactor. The ammonia not oxidized by Nitrosomonas and Nitrobacter was obviously assimilated by Pseudomonas during anoxic growth in the last reactor. These examples indicate the value of the retentostat for the experimental investigation of microbial interactions. In general, the same presuppositions are valid for the retentostat as for chemostats: The correct interpretation of the measurements depends on the system’s homogeneity. Therefore, the microorganisms should grow in suspension without flocculation and adhesion to the internal surfaces of the reactor walls and installations. However, if adhesion occurs, the error in calculations will be relatively smaller when based on the higher cell densities attainable in the retentostat as compared to the much lower densities in chemostatic cultures growing at the same growth rate.
References [I] Pirt. S.J. (1987) The energetics of microbes at low growth rateb: Maintenance energies and dormant organisms. .I. Ferment. Technol. 65. 173-177. [2] Poindexter, J.S. (1981) Oligotrophy. Fast and famine existence. Adv. Microb. Ecol. 5. 63-89. [3] Van Elsas, J.D. and van Overbeek, L.S. (1993) Bacterial responses to soil stimuli. In: Starvation in Bacteria (Kjelle-
Ecolog,v I9 (I 9%) 47-52
berg. S.. Ed.), pp. 55-79. Plenum Prehs. New York and London. 141Herbert. D.. Elsworth, R. and Telling. R.C. (1956) The continuous culture of bacteria. A theoretical and experimental sudy. J. Gen. Microbial. I-l, 601-631. [51 Hamford, G.S. and Humphrey. A.E. (1966) The effect of equipment scale and degree of mixing on continuous fermentation yield at low dilution rates. Biotech. Bioeng. 8. 85-96. [61 Bulthuix, B.A.. Koningstein, GM.. Stouthamer. A.H. and Van Veraeveld. H.W. (1989) A comparison between aerobic growth of Brrcillus /ichrr~ifwmi.s in continuous culture and partial-recycling fermentor. with contributions to the discussion on maintenance energy demand. Arch. Microbial. 152. 199-507. [71 Chesbro. W., Evans. W. and Eifert, R. (1979) Very slow growth of E. L.o/~.J. Bacterial. 139. 635-638. [Xl Matin, A.. Auger. E.A., Blum. P.H. and Schultz. J.E. (1989) Genetic basis of starvation survival in nondifferentiating bacteria. Ann. Rev. Microbial. 42. 293-3 16. [9] Beyeler. W., Rogers, P.L. and Fiechter. A. (1983) A simple technique for the direct determination of maintenance energy coefficient: an example with Z?./vonro~rc~.\ rnubi[is. Appl. Microbial. Biotechnol. 19. 277-280. [IO] Van Verseveld, H.W.. Chesbro. W.R.. Braster. M. and Stouthamer, A.H. (1983) Eubacteria have 3 growth mode?, keyed to nutrient tlow. Arch. Microbial. 1.17. 176- 184. [I I] Stouthamer, A.H. and Bettenhausen. C. (1973) Utilization of energy for growth and maintenance in continuous and batch cultures of microorganisms. Biochim. Biophys. Acta 301. 53-70. [I21 Pin, S.J. (1982) Maintenance energy: a general model for energylimited and energy-sufficient growth. Arch. Microbiol. 133. 300-301. [ 131Neijssel. O.M. and Tempest. D.W. (1976) The role of energy-spilling reactions in the growth of Klrhirlltr trero~rrws NCTC 318 in aerobic chemostat culture. Arch. Microbial. I IO.305-31 I. [ 141 Bock, E., Koopa, H.P. and Harms. H. (1986) Cell biology of nitrifying bacteria. In: Nitrification (Presser, J.I.. Ed.). 17-38. Special publications of the Society for General Microbiology. Vol. 30. IRL Press.