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A small-scale, three-vessel, continuous culture system for quantitative studies of plant fibre degradation by anaerobic bacteria
Journal of Microbiological Methods 12 (1990) 173-182
173
Elsevier MIMET 00399
A small-scale, three-vessel, continuous culture system for quantitative studies of plant fibre degradation by anaerobic bacteria Albrecht Kistner and Jan H. Kornelius Laboratory for Molecular and Cell Biology, South African Council for Scientific and Industrial Research, Pretoria, South Africa (Received 3 January 1990; revision received 29 July 1990; accepted 13 August 1990)
Summary A continuous culture system is described which consists of three all-glass water-jacketted culture vessels of = 250 ml capacity, linked by electro-pneumatic valves and medium lines of low hold-up volume to a common, spherical medium reservoir. The contents of the reservoir and the culture vessels are agitated by efficient vibratory stirrers to keep the insoluble substrate in homogeneous suspension, and are blanketed against contact with atmospheric 0 2 by slow purging with a stream of Oz-free gas mixture containing 5 °70 H 2, 30% CO 2 and 65°70 N 2. Medium is dosed to the culture vessels by periodic activation of the pneumatic valves, and corresponding volumes of culture are displaced into sterile harvest vessels via lateral exit ports with the aid o f a slow flow of anaerobic gas through the culture vessels. Both pH auxostat (as defined by Martin and Hempfling [1]) and chemostat operation are possible. Performance specifications of the system are given and the usefulness of the system for investigations of the kinetics of growth on, and utilization of, solid substrates is illustrated by results obtained with one strain each of Ruminococcusflavefaciens and Fibrobacter (Bacteroides) succinogenes, grown on ball-milled filter paper cellulose.
Key words: Anaerobic bacterium; Continuous culture; Plant fibre degradation; Rumen bacterium; Solid suhstrate
Introduction
Fibre-degrading microorganisms play a crucial role in the ecosystem of the rumen in making available a large proportion of the energy and C locked up in insoluble cell wall carbohydrates of the plant material ingested by the host animal [2]. Other members of the microbial community of the rumen, themselves incapable of degrading cellulose and hemicelluloses, benefit from these activities in obtaining a share of the soluble sugars released, and the volatile fatty acids which are the end products of Correspondence to: A. Kistner, Animal and Dairy Science Research Institute, Private Bag X2, 1675 Irene, South Africa.
0167-7012/90/$ 3.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
174 carbohydrate metabolism by the complex microbial population of the rumen are the m a j o r energy source to the ruminant. A knowledge of the kinetics of microbial growth on, and utilization of, intact plant cell walls or their components is therefore, clearly, of importance to the animal nutritionist. However, as a result of the technical difficulties involved, little information on this aspect is to be found in the literature [3]. To obtain kinetic parameters from batch incubations of the insoluble substrate with either pure cultures or mixed microbial populations requires analyses on samples collected at a number of time intervals. If the incubations are started with dilute inocula, the changes in substrate concentration.sampling interval -~ are initially very small, become progressively larger as the microbial population proliferates, and then decrease again as the more readily digestible structures are depleted or the availability of sites for further enzyme binding becomes rate limiting. This results in a variable magnitude of analytical error. Furthermore, when the solid substrate is physically or chemically heterogeneous and it is desired to determine the rates at which the different fractions are solubilized in batch incubations, this becomes exceedingly laborious. In principle, continuous culture techniques offer advantages in this respect. Provided that steadystate conditions have become established, extents of solubilization of solid substrates in toto, or of their constituents can be calculated from concentration differences of these between in-flowing medium and the cultures. Similarly, the product of these concentration differences and dilution rate gives the volumetric rate of solubilization of the components or the solid substrate as an entity. Far fewer analyses are required than in the case of batch incubation experiments. Where detailed information of the solubilization of individual components, e.g., the constituent sugars of hemicelluloses, is desired, a reduction in the number of samples requiring analysis becomes important. Moreover, by running continuous cultures at different dilution rates, the effect of mean residence time on the rate and extent of solubilization of the substrate can be determined and it becomes possible to establish whether different constituents are removed at different rates or in a definite sequence. However, fermentors of conventional design are not readily adaptable to steady-state continuous cultivation of microorganisms on solid substrates inasmuch as it is difficult to ensure an even inflow of substrate into the culture, as illustrated by the work of Gray [4]. Sedimentation of the substrate in the medium lines and trapping of solids in pumps or other dosing devices are the greatest stumbling blocks. Earlier, we gave a brief description of a continuous culture system, operating in p H auxostat (as defined by Martin and Hempfling [1]) mode, designed primarily for determining specific growth rates of strictly anaerobic fibre-digesting bacterial species from the rumen on homogeneously dispersed cellulose [5]. The present paper describes the system and later extensions to it in greater detail, as well as its performance in chemostat mode and an example of its application to the study of the kinetics of growth, on pure cellulose, of one strain of each of two bacterial species which play a major role in fibre digestion in the rumen.
175 Materials and Methods
Apparatus The layout of the culture apparatus is shown in Fig. 1. A spherical reaction vessel of 10-1 nominal capacity (Code FR10LF; Jobling Laboratory Division, Stone, Staffordshire, UK) was used as medium reservoir because, together with the chosen agitation system, it promoted a rapid recirculating flow pattern within the entire volume of medium and thereby kept substrate particles in uniform suspension. The flanged lid of the vessel was modified by the addition of a Friedrichs-type condensor for removal of excess water vapour from the effluent gas, a screw-threaded port with polypropylene cap for periodic topping-up with sterile medium and a central flanged port to which was clamped the flexible membrane which provided a hermetical seal
TO pH CONTROl
'MPRESSED £NT
Fig. 1. Layoutof the culture apparatus, with only one culture vessel shown: 1, medium reservoi.r;2, stop valve, normally in open position; 3, vibratory stirrers; 4, dosing valve; 5, three-waysolenoid valve in compressed N 2 line; 6, culture vessel; 7, harvest vessel.
176 between the lid and the oscillating (maximum amplitude 2.5 mm) stirrer shaft. A nontoxic grade of silicone rubber sealant was used to seal the lid to the reservoir. The bottom of the reservoir was provided with an outlet port consisting of the tapered portion of a redundant 37-mm bore chromatography column with stainless steel fittings. Four short bends of 3.2-mm bore stainless steel tubing were welded to the end plate and terminated in stop valves of the same material (Code SS42S4; Whitey, Highland Heights, Ohio, USA). One of the valves carried a female Luer fitting sealed by a male Luer plug and was used for periodic aseptic withdrawal, by means of syringes, of samples of medium for p H measurements and the determination of the initial concentration of substrate. A vibratory stirrer (Vibro-mixer El; Chemap, M/~nnedorf, Switzerland) was mounted centrally above the medium reservoir with the hollow shaft extending to within 25 m m of the bottom of the reservoir. A 65-mm stainless steel stirrer disc with conical perforations tapering upwards was attached to the lower end of the shaft and a gas sterilizing filter to a short side arm 25 m m below the upper blind end. An O2-free gas mixture, containing 5% H2, 3007o CO 2 and 65°7o N2, at a controlled pressure of 3.5 kPa entered through the filter, escaped just below the vibrating stirrer disc and was well dispersed through the circulating medium. To obtain a constant pressure of medium at the outlet ports, independent of the changing liquid head, the reservoir was operated as a Mariotte flask [6]. A very low flow of gas ( ~ 300 ml. h - l ) was allowed to escape from a fine capillary attached to the exit gas filter downstream of the condensor to prevent a gradual build-up of partial pressure of O2 which could diffuse in through the elastomer seal around the vibrating shaft. The medium lines beyond the remaining three stop valves were kept as short and uniform of bore as possible to minimize sedimentation and trapping of substrate. In the original version of the apparatus 1.5-mm bore stainless steel lines were used and the dead volume between reservoir and culture vessels was < 1 ml [5]. However, when coarser cell wall preparations were used, clogging problems were experienced and the bore of the connecting lines was increased to 3.2 mm. Dosing valves were placed in the media lines immediately downstream of the stop valves. These consisted of short lengths of thin-walled silicone rubber tubing bridging a gap of = 25 m m in the medium lines and encased in glass or metal cylinders with a short side arm. The cylinders were pressurized with N 2 to ~ 155 kPa via three-way solenoid valves (Code MOFH-3-M5; Festo, Esslingen, FRG), to flatten the silicone tubing, thereby shutting off the medium supply. When the solenoid valves were energized, the compressed N 2 supply was interrupted, the pressure was vented and medium flowed to the culture vessels. The solenoid valves received trains of pulses either from simple p H controllers (pH meter 302, Digital Data Systems, Randburg, South Africa) for p H auxostat control [5], or from a programmable sequencer ('National' Sequencer PL20; Matsushita Electric Works, Osaka, Japan) for chemostat operation. In the latter case the duration of the pulses was adjustable in steps of 0.1 s and the intervals between pulses in steps of 1 s, with the three valves being programmable independently. Appropriate settings for a desired value of dilution rate were determined experimentally. The custom-built, pneumatically operated pinch valves were readily sterilizable, did not trap substrate particles and functioned reliably over many months without need for maintenance. The design of the culture vessels is also shown in Fig. 1. The lids had a c o m m o n
177
inlet for medium and anaerobic gas, as well as ports for the stirrer shafts, combination pH electrodes and Luer-lok cannulae for inoculation and sampling by syringe. As sterile medium was added, equal volumes of culture were displaced through side ports of the vessels and PTFE or polypropylene tubing to harvest vessels. Vibro-mixers with 45-mm stirrer discs attached to the ends of solid shafts were used to mix the contents of the vessels. Temperature of the cultures was controlled by circulating water at 39 °C from a constant temperature bath through the jackets surrounding the lower parts of the vessels. The whole apparatus was accomodated within a standard instrumentation rack. All electronic instrumentation was placed on the upper level. A laminate-covered 9-mm A1 platform at a lower level supported the medium reservoir and the three culture vessels, while the load cells from which the harvest vessels were suspended were attached to its under surface. The circulating thermostat was placed on a bottom platform (Fig. 2).
Fig. 2. Physicalarrangementof the componentsof the system; 1, data logger;2, pH controllers; 3, variable transformers for controlling amplitude of vibratory stirrers; 4, medium reservoir; 5, vibratory stirrers; 6, culture vessels; 7, harvest vessels.
178
Measurement of dilution rates Provision was made for suspending the harvest vessels from miniature load cells (Code SM-25; Interface, Scottsdale, USA) which communicated with a three-channel data logger (Systec, Pretoria, South Africa). This permitted automatic recording of the mass increase of the harvest vessels against time. At intervals the data were transferred to a microcomputer for plotting of the curves and calculation of the linear regression parameters. Mean dilution rates were calculated from the slopes of the curves and the density of the medium.
Organisms Ruminococcus flavefaciens FD1 was kindly donated by M.P. Bryant in ~ 1960. Fibrobacter (Bacteroides) succinogenes 24-M8 came from the culture collection of this laboratory.
Medium Medium No. 10 of Caldwell and Bryant [7] was modified by substitution of 0.1% ball-milled W h a t m a n No. 1 filter paper, washed free of soluble degradation products, for the mixture of carbohydrates. The stock mixture of volatile fatty acids (VFA) was adjusted to p H 7.5 and the concentration of NazCO 3 lowered to 18.7 mM to give a p H of ~ 6.8 when the medium was equilibrated with a gas phase of 5% H 2, 30°70 C O 2 and balance N 2 at 39°C. The medium was prepared in 10-1 volumes, with the bulk (8350 ml), containing cellulose, minerals, peptone, yeast extract, haemin and redox indicator, being heat-sterilized in a 15-1 aspirator bottle equipped for magnetic stirring, sparging with anaerobic gas mixture and aseptic transfer of medium to the nutrient reservoir. After sterilization the aspirator bottle was placed on a raised platform with built-in magnetic stirrer and immediately purged with the anaerobic gas mixture. To minimize volatilization of VFA and precipitation of phosphates, the stock solutions of VFA and NazCO 3 were combined and sterilized in a separate 1-1 aspirator bottle equipped similarly to the larger bottle. After the temperature had dropped to < 50 °C, this solution was also purged with the CO2-containing gas mixture to convert carbonate to bicarbonate, before draining, under a positive pressure of the gas, into the larger aspirator bottle. When yeast extract was replaced by a defined B vitamin stock solution [4], this was added as a filter-sterilized solution at this stage. Finally, heatsterilized stock solutions of cysteine and sodium sulphide were added and the medium drained by gravity into the medium reservoir via a port in the lid.
Estimation of cellulose Triplicate 10-ml volumes of medium or culture were placed in preweighed 15-ml tubes and centrifuged at 1240 × g for 20 min. The supernatants were discarded and the pellets resuspended in 3-ml volumes of 0.05o7o lysozyme in 3 mM Tris buffer (adjusted to p H 8) which was I m M with respect to EDTA. After 30 min incubation at 37 °C the suspension was recentrifuged and the supernatant discarded. The pellet was resuspended in 1 ml of a 2°7o cetyltrimethylammonium bromide solution in 0.5 M H2SO 4 and heated in a boiling waterbath for 30 min. After further centrifugation, the supernatant was discarded and the pellet washed twice with 3-ml volumes of distilled water and the tubes dried at 105 °C to constant weight.
179
Results
Growth of cultures On several occasions the modified Medium No. 10 of Caldwell and Bryant [7] supported little or no growth, especially ofF. succinogenes 24-M8, when prepared in 10-1 volumes, whereas small volumes prepared for batch cultures from the same stock solutions presented no problems. Circumstantial evidence pointed to heat inactivation, during the relatively long heat exposure period required to sterilize larger volumes of medium, of some essential growth factor in which older batches of yeast extract were near-deficient. Switching to fresh batches eliminated the problem but, in the interest of reproducibility of growth responses, yeast extract was eventually replaced by a stock solution of B vitamins [4]. Thereafter, consistently good growth of the strains of R. flavefaciens and F. succinogenes was obtained, with complete colonization of all exposed substrate surfaces at all but the highest dilution rates.
Constancy of flow rate Fig. 3 shows the increase in mass of a harvest vessel as a function of elapsed time for a constant setting of pulse duration and interval of the programmable sequencer. Each point represents the mean of three readings collected by the data logger from the same load cell at the same time after initiation of data collection on consecutive days. The error bars, inasmuch as they are not obscured by the symbols, represent stan10o0 Y-intercept : 4.03 Slope : 40.03 Corr. coeff. : 0.99996
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Time (h) Fig. 3. Constancy of medium addition to, and culture outflow from, a continuous culture fermentor as measured by the increase in mass of the harvest vessel as a function of time, for a constant setting of pulse duration and interval of the programmable sequencer. Each point represents the mean of three readings collected by the data logger from the same load cell at the same time after initiation of data collection on consecutive days. The error bars represent standard deviations.
180 dard deviations. The slope of the regression line indicates a gravimetric flow rate, over the 3-day period, of 40.03 g . h ~ from which a mean volumetric flow rate of 40.11 ml. h - ~was calculated. During this time, > 4.7 1 of medium was drawn from the reservoir by the three culture vessels, but the drop in head of liquid had no detectable effect on flow rates.
Substrate flow to culture vessels It was reported earlier that the coefficient of variation in cellulose concentration between 12 5-ml samples collected at equal volume intervals while draining the whole contents of the medium reservoir was 3.1% [5]. However it was still possible that sedimentation or trapping of substrate in the medium line or imperfect mixing in the culture vessels could cause fluctuations in substrate concentration in the vessels and thus interfere with attainment of steady-state conditions in the cultures. To test for longer-term constancy of substrate concentration, the contents of the reservoir were passed through a single, sterile culture vessel over a period of almost 5.5 days at a mean flow rate of 84.8 m l . h -1. With a working volume of the vessel of 250 ml, this corresponded to a dilution rate of 0.34. h -1 and an exponential turnover rate of > 98% in 12 h. 11 samples of ~ 35 ml each were withdrawn with aseptic precautions at 12-h intervals and the cellulose content determined gravimetrically in three 10-ml volumes of each sample. The coefficient of variation in cellulose concentration between the 11 samples was 3.38%. This was considered acceptable for the calculation of volumetric rates of substrate solubilization.
Solubilization of cellulose Fig. 4 shows the effect of dilution rate on the extent and volumetric rate of solubilization of filter paper cellulose by R. flavefaciens FD1 and E succinogenes. The extent of digestion by R. flavefaciens was close to 100% at D = 0.04. h-1 which corresponds to a mean residence time of particles within the system of 25 h. With increasing dilution rate, the extent of solubilization decreased, dropping to --20°70 at D=0.51 .h -~. The volumetric rate of solubilization initially increased with dilution rate, reached a 100 t
~
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Fig. 4. E f f e c t o f d i l u t i o n rate o n t h e extent ( [] t3 ) a n d v o l u m e t r i c r a t e ( o o ) of solubilization o f b a l l - m i l l e d W h a t m a n No. 1 filter p a p e r b y c o n t i n u o u s c u l t u r e s o f R. flavefaciens F D I (left) a n d E succinogenes 2 4 - M 8 (right).
181 maximum of 200 mg. (1 h)- 1 at D = 0.32. h - l and then decreased fairly sharply with further increase in dilution rate. Phase contrast microscopy of wet mounts of fermentor contents showed that around D = 0 . 3 2 . h -l every cellulose particle was densely colonized by cocci, mainly in chains, while there were relatively few unattached bacterial cells. With decreasing dilution rate, the rate of solubilization appeared to be limited by competition for available substrate surface, an increasingly larger proportion of the cocci being unattached. On the other hand, at D values > 0 . 3 2 . h -1 there appeared to be an oversupply of cellulose and most particles were only lightly colonized by bacteria. The maximal rate of solubilization of cellulose by E succinogenes was < 6007o of that found in the case of R. flavefaciens FD1, and even at a dilution rate of only -~ 0.02.h -I, somewhat < 80% of the cellulose supplied was digested. Furthermore, the decline in the extent of digestion with increasing dilution rate was steeper than in the case of FD1. It is possible that the semidefined medium used was not optimal for this relfitively recent isolate of E succinogenes, the species generally being regarded as being particularly well adapted to the digestion of highly ordered forms of cellulose. Discussion The solid-substrate continuous culture system described here has given reliable service over several years. Very few contamination problems were experienced, even though the contents of the medium reservoir were replenished many times during a run from the aspirator bottle in which the medium was prepared and sterilized. A gradual buildup of substrate particles on the inner surfaces of the reservoir over a period of weeks, which was more pronounced in the case of cryo-milled maize cell walls than in the case of ball-milled filter paper cellulose, necessitated its periodic dismantling and cleaning. The tendency of substrate particles to adhere to the surface could be reduced, but not entirely eliminated, by treatment of the glass with a siliconizing agent, e.g., 2°70 (v/v) dimethyldichlorosilane in 1,1,1,trichloroethane. The vibratory stirrer, although recommended for a maximum volume of 5 1, produced excellent mixing of > 11 1 medium in the spherical reservoir. However, a flaw in the design of the system was the removal of the mounting rods of the stirrers for the culture vessels and their replacement by brass dovetail slides which fitted into corresponding recesses in a common, solid metal bracket above the culture vessels. This arrangement facilitated placement of the stirrers after sterilization of the culture vessels and ensured proper alignment of the stirrer shafts within the vessels, but at the same time caused interference between the stirrers, notwithstanding the fact that vibration-absorbing rubber pads were inserted between the stirrer bodies and the dovetail slides. The effect of this interference was that good mixing in the fermentor vessels could only be ensured by running all three stirrers at full amplitude, which caused excessive noise, or by using not more than two fermentors simultaneously. The pneumatically operated pinch valves gave very satisfactory service and it was not unusual for the silicone rubber tubing to last for > 1 yr. Together with the programmable sequencer they provided a very cost-effective medium dosing system, the performance of which compared well to that of good commercial dosing pumps (Fig. 3). The downward slope of the short medium line and its smooth, uniform bore minimized sedimentation and trapping of substrate particles and ensured an even flow of substrate
182 to the fermentors, which is a prerequisite for a t t a i n m e n t o f steady-state c o n d i t i o n s . P a v l o s t a t h i s et al. [3] achieved the s a m e g o a l with c h e m o s t a t cultures o f R u m i n o c o c c u s albus by r a p i d l y circulating m e d i u m from a n d to the reservoir t h r o u g h a m e d i u m line o f 3 - m m b o r e with a peristaltic p u m p . W h e n a p i n c h - t y p e two-way s o l e n o i d valve in this line was a c t u a t e d via a PC, m e d i u m was diverted t h r o u g h a s h o r t side a r m to the culture vessel. This a p p e a r s to be an elegant s o l u t i o n to the p r o b l e m o f substrate s e d i m e n t a t i o n , b u t p r e s u m a b l y requires s o m e w h a t m o r e m a i n t e n a n c e t h a n the system d e s c r i b e d here. O u t f l o w f r o m the culture vessels o f Pavlostathis et al. was also controlled by peristaltic p u m p s , a n d as the result o f rising o u t f l o w lines, solid substrate a n d b i o m a s s settled b a c k into the culture vessels when the o u t f l o w p u m p s were stationary, resulting in slightly different retention times for liquid a n d s u s p e n d e d solids. O u r system was n o r m a l l y used in c h e m o s t a t m o d e as this allowed o p e r a t i o n at a wide range o f d i l u t i o n rates o r m e a n residence times. U n d e r these c o n d i t i o n s the ferm e n t o r s were n o t fitted with p H electrodes b e c a u s e s u l p h i d e in the m e d i u m t e n d e d to p o i s o n the reference electrodes a n d limit their o p e r a t i n g lives. W i t h the low concent r a t i o n s o f solid substrate used, the b u f f e r i n g c a p a c i t y o f the m e d i u m was a d e q u a t e to prevent m a j o r p H changes. O n l y when it was desired to o p e r a t e the c o n t i n u o u s cultures close to their w a s h - o u t points, a p o o r l y b u f f e r e d m e d i u m a n d p H a u x o s t a t control were used as d e s c r i b e d earlier [5]. T h e e x p e r i m e n t s o f which the results are shown in Fig. 4 were a starting p o i n t for studies o f the kinetics o f s o l u b i l i z a t i o n o f p l a n t cell wall c a r b o h y d r a t e s by growing m o n o c u l t u r e s o f fibre-digesting r u m e n bacteria. The c o n t i n u o u s culture system d e s c r i b e d is proving e q u a l l y useful for s t u d y i n g the degrad a t i o n o f intact p l a n t cell walls by d e f i n e d cocultures o f cellulolytic a n d n o n c e l l u l o l y t i c species. This will be r e p o r t e d elsewhere.
Acknowledgements T h e a u t h o r s wish to t h a n k G r e t a Thew, f o r m e r l y o f this laboratory, for expert technical assistance a n d the D i r e c t o r o f the Veterinary Research Institute, O n d e r s t e p o o r t , for the use o f facilities which have m a d e this w o r k possible.
References 1 Martin, G.A. and Hempfhng, W. P. (1976)A method for the regulation of microbial population density during continuous culture at high growth rates. Arch. Microbiol. 107, 41-47. 2 Hungate, R.E. (1966) The Rumen and Its Microbes. Academic Press, New York. 3 Pavlostathis, S. G., Miller, T. L. and Wolin, M. J. (1988) Fermentation of insoluble cellulose by continuous cultures of Ruminococcus albus. Appl. Environ. Microbiol. 54, 2655-2659. 4 Gray, W. M. (1978) Microbialinteractionsin defined continuous culture systems effecting anaerobic cellulose degradation. Ph.D. thesis, Clemson University. 5 Kistner, A., Kornelius, J.H. and Miller, G.S. (1983) Kinetic measurements on bacterial cultures growing on fibres. S. Afr. J. Anita. Sci. 13, 217-220. 6 Ricica, J. (1966) Technique of continuous laboratory cultivations. In: Theoretical and Methodological Basis of Continuous Culture of Microorganisms (Malek, I. and Fencl, Z., eds.), pp. 155 - 313, Academic Press, New York. 7 Caldwell, D. R. and Bryant, M. R (1966)Medium without rumen fluid for nonselective enumeration and isolation of rumen bacteria. Appl. Microbiol. 14, 794-801.
173
Elsevier MIMET 00399
A small-scale, three-vessel, continuous culture system for quantitative studies of plant fibre degradation by anaerobic bacteria Albrecht Kistner and Jan H. Kornelius Laboratory for Molecular and Cell Biology, South African Council for Scientific and Industrial Research, Pretoria, South Africa (Received 3 January 1990; revision received 29 July 1990; accepted 13 August 1990)
Summary A continuous culture system is described which consists of three all-glass water-jacketted culture vessels of = 250 ml capacity, linked by electro-pneumatic valves and medium lines of low hold-up volume to a common, spherical medium reservoir. The contents of the reservoir and the culture vessels are agitated by efficient vibratory stirrers to keep the insoluble substrate in homogeneous suspension, and are blanketed against contact with atmospheric 0 2 by slow purging with a stream of Oz-free gas mixture containing 5 °70 H 2, 30% CO 2 and 65°70 N 2. Medium is dosed to the culture vessels by periodic activation of the pneumatic valves, and corresponding volumes of culture are displaced into sterile harvest vessels via lateral exit ports with the aid o f a slow flow of anaerobic gas through the culture vessels. Both pH auxostat (as defined by Martin and Hempfling [1]) and chemostat operation are possible. Performance specifications of the system are given and the usefulness of the system for investigations of the kinetics of growth on, and utilization of, solid substrates is illustrated by results obtained with one strain each of Ruminococcusflavefaciens and Fibrobacter (Bacteroides) succinogenes, grown on ball-milled filter paper cellulose.
Key words: Anaerobic bacterium; Continuous culture; Plant fibre degradation; Rumen bacterium; Solid suhstrate
Introduction
Fibre-degrading microorganisms play a crucial role in the ecosystem of the rumen in making available a large proportion of the energy and C locked up in insoluble cell wall carbohydrates of the plant material ingested by the host animal [2]. Other members of the microbial community of the rumen, themselves incapable of degrading cellulose and hemicelluloses, benefit from these activities in obtaining a share of the soluble sugars released, and the volatile fatty acids which are the end products of Correspondence to: A. Kistner, Animal and Dairy Science Research Institute, Private Bag X2, 1675 Irene, South Africa.
0167-7012/90/$ 3.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
174 carbohydrate metabolism by the complex microbial population of the rumen are the m a j o r energy source to the ruminant. A knowledge of the kinetics of microbial growth on, and utilization of, intact plant cell walls or their components is therefore, clearly, of importance to the animal nutritionist. However, as a result of the technical difficulties involved, little information on this aspect is to be found in the literature [3]. To obtain kinetic parameters from batch incubations of the insoluble substrate with either pure cultures or mixed microbial populations requires analyses on samples collected at a number of time intervals. If the incubations are started with dilute inocula, the changes in substrate concentration.sampling interval -~ are initially very small, become progressively larger as the microbial population proliferates, and then decrease again as the more readily digestible structures are depleted or the availability of sites for further enzyme binding becomes rate limiting. This results in a variable magnitude of analytical error. Furthermore, when the solid substrate is physically or chemically heterogeneous and it is desired to determine the rates at which the different fractions are solubilized in batch incubations, this becomes exceedingly laborious. In principle, continuous culture techniques offer advantages in this respect. Provided that steadystate conditions have become established, extents of solubilization of solid substrates in toto, or of their constituents can be calculated from concentration differences of these between in-flowing medium and the cultures. Similarly, the product of these concentration differences and dilution rate gives the volumetric rate of solubilization of the components or the solid substrate as an entity. Far fewer analyses are required than in the case of batch incubation experiments. Where detailed information of the solubilization of individual components, e.g., the constituent sugars of hemicelluloses, is desired, a reduction in the number of samples requiring analysis becomes important. Moreover, by running continuous cultures at different dilution rates, the effect of mean residence time on the rate and extent of solubilization of the substrate can be determined and it becomes possible to establish whether different constituents are removed at different rates or in a definite sequence. However, fermentors of conventional design are not readily adaptable to steady-state continuous cultivation of microorganisms on solid substrates inasmuch as it is difficult to ensure an even inflow of substrate into the culture, as illustrated by the work of Gray [4]. Sedimentation of the substrate in the medium lines and trapping of solids in pumps or other dosing devices are the greatest stumbling blocks. Earlier, we gave a brief description of a continuous culture system, operating in p H auxostat (as defined by Martin and Hempfling [1]) mode, designed primarily for determining specific growth rates of strictly anaerobic fibre-digesting bacterial species from the rumen on homogeneously dispersed cellulose [5]. The present paper describes the system and later extensions to it in greater detail, as well as its performance in chemostat mode and an example of its application to the study of the kinetics of growth, on pure cellulose, of one strain of each of two bacterial species which play a major role in fibre digestion in the rumen.
175 Materials and Methods
Apparatus The layout of the culture apparatus is shown in Fig. 1. A spherical reaction vessel of 10-1 nominal capacity (Code FR10LF; Jobling Laboratory Division, Stone, Staffordshire, UK) was used as medium reservoir because, together with the chosen agitation system, it promoted a rapid recirculating flow pattern within the entire volume of medium and thereby kept substrate particles in uniform suspension. The flanged lid of the vessel was modified by the addition of a Friedrichs-type condensor for removal of excess water vapour from the effluent gas, a screw-threaded port with polypropylene cap for periodic topping-up with sterile medium and a central flanged port to which was clamped the flexible membrane which provided a hermetical seal
TO pH CONTROl
'MPRESSED £NT
Fig. 1. Layoutof the culture apparatus, with only one culture vessel shown: 1, medium reservoi.r;2, stop valve, normally in open position; 3, vibratory stirrers; 4, dosing valve; 5, three-waysolenoid valve in compressed N 2 line; 6, culture vessel; 7, harvest vessel.
176 between the lid and the oscillating (maximum amplitude 2.5 mm) stirrer shaft. A nontoxic grade of silicone rubber sealant was used to seal the lid to the reservoir. The bottom of the reservoir was provided with an outlet port consisting of the tapered portion of a redundant 37-mm bore chromatography column with stainless steel fittings. Four short bends of 3.2-mm bore stainless steel tubing were welded to the end plate and terminated in stop valves of the same material (Code SS42S4; Whitey, Highland Heights, Ohio, USA). One of the valves carried a female Luer fitting sealed by a male Luer plug and was used for periodic aseptic withdrawal, by means of syringes, of samples of medium for p H measurements and the determination of the initial concentration of substrate. A vibratory stirrer (Vibro-mixer El; Chemap, M/~nnedorf, Switzerland) was mounted centrally above the medium reservoir with the hollow shaft extending to within 25 m m of the bottom of the reservoir. A 65-mm stainless steel stirrer disc with conical perforations tapering upwards was attached to the lower end of the shaft and a gas sterilizing filter to a short side arm 25 m m below the upper blind end. An O2-free gas mixture, containing 5% H2, 3007o CO 2 and 65°7o N2, at a controlled pressure of 3.5 kPa entered through the filter, escaped just below the vibrating stirrer disc and was well dispersed through the circulating medium. To obtain a constant pressure of medium at the outlet ports, independent of the changing liquid head, the reservoir was operated as a Mariotte flask [6]. A very low flow of gas ( ~ 300 ml. h - l ) was allowed to escape from a fine capillary attached to the exit gas filter downstream of the condensor to prevent a gradual build-up of partial pressure of O2 which could diffuse in through the elastomer seal around the vibrating shaft. The medium lines beyond the remaining three stop valves were kept as short and uniform of bore as possible to minimize sedimentation and trapping of substrate. In the original version of the apparatus 1.5-mm bore stainless steel lines were used and the dead volume between reservoir and culture vessels was < 1 ml [5]. However, when coarser cell wall preparations were used, clogging problems were experienced and the bore of the connecting lines was increased to 3.2 mm. Dosing valves were placed in the media lines immediately downstream of the stop valves. These consisted of short lengths of thin-walled silicone rubber tubing bridging a gap of = 25 m m in the medium lines and encased in glass or metal cylinders with a short side arm. The cylinders were pressurized with N 2 to ~ 155 kPa via three-way solenoid valves (Code MOFH-3-M5; Festo, Esslingen, FRG), to flatten the silicone tubing, thereby shutting off the medium supply. When the solenoid valves were energized, the compressed N 2 supply was interrupted, the pressure was vented and medium flowed to the culture vessels. The solenoid valves received trains of pulses either from simple p H controllers (pH meter 302, Digital Data Systems, Randburg, South Africa) for p H auxostat control [5], or from a programmable sequencer ('National' Sequencer PL20; Matsushita Electric Works, Osaka, Japan) for chemostat operation. In the latter case the duration of the pulses was adjustable in steps of 0.1 s and the intervals between pulses in steps of 1 s, with the three valves being programmable independently. Appropriate settings for a desired value of dilution rate were determined experimentally. The custom-built, pneumatically operated pinch valves were readily sterilizable, did not trap substrate particles and functioned reliably over many months without need for maintenance. The design of the culture vessels is also shown in Fig. 1. The lids had a c o m m o n
177
inlet for medium and anaerobic gas, as well as ports for the stirrer shafts, combination pH electrodes and Luer-lok cannulae for inoculation and sampling by syringe. As sterile medium was added, equal volumes of culture were displaced through side ports of the vessels and PTFE or polypropylene tubing to harvest vessels. Vibro-mixers with 45-mm stirrer discs attached to the ends of solid shafts were used to mix the contents of the vessels. Temperature of the cultures was controlled by circulating water at 39 °C from a constant temperature bath through the jackets surrounding the lower parts of the vessels. The whole apparatus was accomodated within a standard instrumentation rack. All electronic instrumentation was placed on the upper level. A laminate-covered 9-mm A1 platform at a lower level supported the medium reservoir and the three culture vessels, while the load cells from which the harvest vessels were suspended were attached to its under surface. The circulating thermostat was placed on a bottom platform (Fig. 2).
Fig. 2. Physicalarrangementof the componentsof the system; 1, data logger;2, pH controllers; 3, variable transformers for controlling amplitude of vibratory stirrers; 4, medium reservoir; 5, vibratory stirrers; 6, culture vessels; 7, harvest vessels.
178
Measurement of dilution rates Provision was made for suspending the harvest vessels from miniature load cells (Code SM-25; Interface, Scottsdale, USA) which communicated with a three-channel data logger (Systec, Pretoria, South Africa). This permitted automatic recording of the mass increase of the harvest vessels against time. At intervals the data were transferred to a microcomputer for plotting of the curves and calculation of the linear regression parameters. Mean dilution rates were calculated from the slopes of the curves and the density of the medium.
Organisms Ruminococcus flavefaciens FD1 was kindly donated by M.P. Bryant in ~ 1960. Fibrobacter (Bacteroides) succinogenes 24-M8 came from the culture collection of this laboratory.
Medium Medium No. 10 of Caldwell and Bryant [7] was modified by substitution of 0.1% ball-milled W h a t m a n No. 1 filter paper, washed free of soluble degradation products, for the mixture of carbohydrates. The stock mixture of volatile fatty acids (VFA) was adjusted to p H 7.5 and the concentration of NazCO 3 lowered to 18.7 mM to give a p H of ~ 6.8 when the medium was equilibrated with a gas phase of 5% H 2, 30°70 C O 2 and balance N 2 at 39°C. The medium was prepared in 10-1 volumes, with the bulk (8350 ml), containing cellulose, minerals, peptone, yeast extract, haemin and redox indicator, being heat-sterilized in a 15-1 aspirator bottle equipped for magnetic stirring, sparging with anaerobic gas mixture and aseptic transfer of medium to the nutrient reservoir. After sterilization the aspirator bottle was placed on a raised platform with built-in magnetic stirrer and immediately purged with the anaerobic gas mixture. To minimize volatilization of VFA and precipitation of phosphates, the stock solutions of VFA and NazCO 3 were combined and sterilized in a separate 1-1 aspirator bottle equipped similarly to the larger bottle. After the temperature had dropped to < 50 °C, this solution was also purged with the CO2-containing gas mixture to convert carbonate to bicarbonate, before draining, under a positive pressure of the gas, into the larger aspirator bottle. When yeast extract was replaced by a defined B vitamin stock solution [4], this was added as a filter-sterilized solution at this stage. Finally, heatsterilized stock solutions of cysteine and sodium sulphide were added and the medium drained by gravity into the medium reservoir via a port in the lid.
Estimation of cellulose Triplicate 10-ml volumes of medium or culture were placed in preweighed 15-ml tubes and centrifuged at 1240 × g for 20 min. The supernatants were discarded and the pellets resuspended in 3-ml volumes of 0.05o7o lysozyme in 3 mM Tris buffer (adjusted to p H 8) which was I m M with respect to EDTA. After 30 min incubation at 37 °C the suspension was recentrifuged and the supernatant discarded. The pellet was resuspended in 1 ml of a 2°7o cetyltrimethylammonium bromide solution in 0.5 M H2SO 4 and heated in a boiling waterbath for 30 min. After further centrifugation, the supernatant was discarded and the pellet washed twice with 3-ml volumes of distilled water and the tubes dried at 105 °C to constant weight.
179
Results
Growth of cultures On several occasions the modified Medium No. 10 of Caldwell and Bryant [7] supported little or no growth, especially ofF. succinogenes 24-M8, when prepared in 10-1 volumes, whereas small volumes prepared for batch cultures from the same stock solutions presented no problems. Circumstantial evidence pointed to heat inactivation, during the relatively long heat exposure period required to sterilize larger volumes of medium, of some essential growth factor in which older batches of yeast extract were near-deficient. Switching to fresh batches eliminated the problem but, in the interest of reproducibility of growth responses, yeast extract was eventually replaced by a stock solution of B vitamins [4]. Thereafter, consistently good growth of the strains of R. flavefaciens and F. succinogenes was obtained, with complete colonization of all exposed substrate surfaces at all but the highest dilution rates.
Constancy of flow rate Fig. 3 shows the increase in mass of a harvest vessel as a function of elapsed time for a constant setting of pulse duration and interval of the programmable sequencer. Each point represents the mean of three readings collected by the data logger from the same load cell at the same time after initiation of data collection on consecutive days. The error bars, inasmuch as they are not obscured by the symbols, represent stan10o0 Y-intercept : 4.03 Slope : 40.03 Corr. coeff. : 0.99996
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Time (h) Fig. 3. Constancy of medium addition to, and culture outflow from, a continuous culture fermentor as measured by the increase in mass of the harvest vessel as a function of time, for a constant setting of pulse duration and interval of the programmable sequencer. Each point represents the mean of three readings collected by the data logger from the same load cell at the same time after initiation of data collection on consecutive days. The error bars represent standard deviations.
180 dard deviations. The slope of the regression line indicates a gravimetric flow rate, over the 3-day period, of 40.03 g . h ~ from which a mean volumetric flow rate of 40.11 ml. h - ~was calculated. During this time, > 4.7 1 of medium was drawn from the reservoir by the three culture vessels, but the drop in head of liquid had no detectable effect on flow rates.
Substrate flow to culture vessels It was reported earlier that the coefficient of variation in cellulose concentration between 12 5-ml samples collected at equal volume intervals while draining the whole contents of the medium reservoir was 3.1% [5]. However it was still possible that sedimentation or trapping of substrate in the medium line or imperfect mixing in the culture vessels could cause fluctuations in substrate concentration in the vessels and thus interfere with attainment of steady-state conditions in the cultures. To test for longer-term constancy of substrate concentration, the contents of the reservoir were passed through a single, sterile culture vessel over a period of almost 5.5 days at a mean flow rate of 84.8 m l . h -1. With a working volume of the vessel of 250 ml, this corresponded to a dilution rate of 0.34. h -1 and an exponential turnover rate of > 98% in 12 h. 11 samples of ~ 35 ml each were withdrawn with aseptic precautions at 12-h intervals and the cellulose content determined gravimetrically in three 10-ml volumes of each sample. The coefficient of variation in cellulose concentration between the 11 samples was 3.38%. This was considered acceptable for the calculation of volumetric rates of substrate solubilization.
Solubilization of cellulose Fig. 4 shows the effect of dilution rate on the extent and volumetric rate of solubilization of filter paper cellulose by R. flavefaciens FD1 and E succinogenes. The extent of digestion by R. flavefaciens was close to 100% at D = 0.04. h-1 which corresponds to a mean residence time of particles within the system of 25 h. With increasing dilution rate, the extent of solubilization decreased, dropping to --20°70 at D=0.51 .h -~. The volumetric rate of solubilization initially increased with dilution rate, reached a 100 t
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Fig. 4. E f f e c t o f d i l u t i o n rate o n t h e extent ( [] t3 ) a n d v o l u m e t r i c r a t e ( o o ) of solubilization o f b a l l - m i l l e d W h a t m a n No. 1 filter p a p e r b y c o n t i n u o u s c u l t u r e s o f R. flavefaciens F D I (left) a n d E succinogenes 2 4 - M 8 (right).
181 maximum of 200 mg. (1 h)- 1 at D = 0.32. h - l and then decreased fairly sharply with further increase in dilution rate. Phase contrast microscopy of wet mounts of fermentor contents showed that around D = 0 . 3 2 . h -l every cellulose particle was densely colonized by cocci, mainly in chains, while there were relatively few unattached bacterial cells. With decreasing dilution rate, the rate of solubilization appeared to be limited by competition for available substrate surface, an increasingly larger proportion of the cocci being unattached. On the other hand, at D values > 0 . 3 2 . h -1 there appeared to be an oversupply of cellulose and most particles were only lightly colonized by bacteria. The maximal rate of solubilization of cellulose by E succinogenes was < 6007o of that found in the case of R. flavefaciens FD1, and even at a dilution rate of only -~ 0.02.h -I, somewhat < 80% of the cellulose supplied was digested. Furthermore, the decline in the extent of digestion with increasing dilution rate was steeper than in the case of FD1. It is possible that the semidefined medium used was not optimal for this relfitively recent isolate of E succinogenes, the species generally being regarded as being particularly well adapted to the digestion of highly ordered forms of cellulose. Discussion The solid-substrate continuous culture system described here has given reliable service over several years. Very few contamination problems were experienced, even though the contents of the medium reservoir were replenished many times during a run from the aspirator bottle in which the medium was prepared and sterilized. A gradual buildup of substrate particles on the inner surfaces of the reservoir over a period of weeks, which was more pronounced in the case of cryo-milled maize cell walls than in the case of ball-milled filter paper cellulose, necessitated its periodic dismantling and cleaning. The tendency of substrate particles to adhere to the surface could be reduced, but not entirely eliminated, by treatment of the glass with a siliconizing agent, e.g., 2°70 (v/v) dimethyldichlorosilane in 1,1,1,trichloroethane. The vibratory stirrer, although recommended for a maximum volume of 5 1, produced excellent mixing of > 11 1 medium in the spherical reservoir. However, a flaw in the design of the system was the removal of the mounting rods of the stirrers for the culture vessels and their replacement by brass dovetail slides which fitted into corresponding recesses in a common, solid metal bracket above the culture vessels. This arrangement facilitated placement of the stirrers after sterilization of the culture vessels and ensured proper alignment of the stirrer shafts within the vessels, but at the same time caused interference between the stirrers, notwithstanding the fact that vibration-absorbing rubber pads were inserted between the stirrer bodies and the dovetail slides. The effect of this interference was that good mixing in the fermentor vessels could only be ensured by running all three stirrers at full amplitude, which caused excessive noise, or by using not more than two fermentors simultaneously. The pneumatically operated pinch valves gave very satisfactory service and it was not unusual for the silicone rubber tubing to last for > 1 yr. Together with the programmable sequencer they provided a very cost-effective medium dosing system, the performance of which compared well to that of good commercial dosing pumps (Fig. 3). The downward slope of the short medium line and its smooth, uniform bore minimized sedimentation and trapping of substrate particles and ensured an even flow of substrate
182 to the fermentors, which is a prerequisite for a t t a i n m e n t o f steady-state c o n d i t i o n s . P a v l o s t a t h i s et al. [3] achieved the s a m e g o a l with c h e m o s t a t cultures o f R u m i n o c o c c u s albus by r a p i d l y circulating m e d i u m from a n d to the reservoir t h r o u g h a m e d i u m line o f 3 - m m b o r e with a peristaltic p u m p . W h e n a p i n c h - t y p e two-way s o l e n o i d valve in this line was a c t u a t e d via a PC, m e d i u m was diverted t h r o u g h a s h o r t side a r m to the culture vessel. This a p p e a r s to be an elegant s o l u t i o n to the p r o b l e m o f substrate s e d i m e n t a t i o n , b u t p r e s u m a b l y requires s o m e w h a t m o r e m a i n t e n a n c e t h a n the system d e s c r i b e d here. O u t f l o w f r o m the culture vessels o f Pavlostathis et al. was also controlled by peristaltic p u m p s , a n d as the result o f rising o u t f l o w lines, solid substrate a n d b i o m a s s settled b a c k into the culture vessels when the o u t f l o w p u m p s were stationary, resulting in slightly different retention times for liquid a n d s u s p e n d e d solids. O u r system was n o r m a l l y used in c h e m o s t a t m o d e as this allowed o p e r a t i o n at a wide range o f d i l u t i o n rates o r m e a n residence times. U n d e r these c o n d i t i o n s the ferm e n t o r s were n o t fitted with p H electrodes b e c a u s e s u l p h i d e in the m e d i u m t e n d e d to p o i s o n the reference electrodes a n d limit their o p e r a t i n g lives. W i t h the low concent r a t i o n s o f solid substrate used, the b u f f e r i n g c a p a c i t y o f the m e d i u m was a d e q u a t e to prevent m a j o r p H changes. O n l y when it was desired to o p e r a t e the c o n t i n u o u s cultures close to their w a s h - o u t points, a p o o r l y b u f f e r e d m e d i u m a n d p H a u x o s t a t control were used as d e s c r i b e d earlier [5]. T h e e x p e r i m e n t s o f which the results are shown in Fig. 4 were a starting p o i n t for studies o f the kinetics o f s o l u b i l i z a t i o n o f p l a n t cell wall c a r b o h y d r a t e s by growing m o n o c u l t u r e s o f fibre-digesting r u m e n bacteria. The c o n t i n u o u s culture system d e s c r i b e d is proving e q u a l l y useful for s t u d y i n g the degrad a t i o n o f intact p l a n t cell walls by d e f i n e d cocultures o f cellulolytic a n d n o n c e l l u l o l y t i c species. This will be r e p o r t e d elsewhere.
Acknowledgements T h e a u t h o r s wish to t h a n k G r e t a Thew, f o r m e r l y o f this laboratory, for expert technical assistance a n d the D i r e c t o r o f the Veterinary Research Institute, O n d e r s t e p o o r t , for the use o f facilities which have m a d e this w o r k possible.
References 1 Martin, G.A. and Hempfhng, W. P. (1976)A method for the regulation of microbial population density during continuous culture at high growth rates. Arch. Microbiol. 107, 41-47. 2 Hungate, R.E. (1966) The Rumen and Its Microbes. Academic Press, New York. 3 Pavlostathis, S. G., Miller, T. L. and Wolin, M. J. (1988) Fermentation of insoluble cellulose by continuous cultures of Ruminococcus albus. Appl. Environ. Microbiol. 54, 2655-2659. 4 Gray, W. M. (1978) Microbialinteractionsin defined continuous culture systems effecting anaerobic cellulose degradation. Ph.D. thesis, Clemson University. 5 Kistner, A., Kornelius, J.H. and Miller, G.S. (1983) Kinetic measurements on bacterial cultures growing on fibres. S. Afr. J. Anita. Sci. 13, 217-220. 6 Ricica, J. (1966) Technique of continuous laboratory cultivations. In: Theoretical and Methodological Basis of Continuous Culture of Microorganisms (Malek, I. and Fencl, Z., eds.), pp. 155 - 313, Academic Press, New York. 7 Caldwell, D. R. and Bryant, M. R (1966)Medium without rumen fluid for nonselective enumeration and isolation of rumen bacteria. Appl. Microbiol. 14, 794-801.