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Growth of bacteria at low oxygen concentrationsGerritse, Jan

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Chapter 4

Two-membered mixed cultures of methanogenic and aerobic

bacteria in 02-limited chemostats

Jan Gerritse and Jan C. GottschaJ

In: JournaJ of GeneraJ Microbiology (1993) 139:1853-1860

Chapter 4

Two-membered mixed cultures of methanogenic and aerobic bacteria in 02-limited chemostats

Jan Gerritse' and Jan C. Gottschal

Department of Microbiology, University of Groningen, Kerklaan 30, 9751 NN Haren, The Netherlands

Summary

Three different co-cultures composed of a methanogenic and a strictly aerobic bacterium were grown under 02-limitation in continuous cultures. The combinations used were (1) Methanobacterium fonnicicum with the aerobic heterotroph Comamonas testosteroni; (2) M. fonnicicum with a methanotrophic Methylocystis species; and (3) Methanosarcina barkeri with C. testosteroni. A1though true steady states were not obtained, growth and metabolic activity of the methanogenic and aerobic organisms occurred during 02-limited growth of these n:llxed cultures over extended periods of time. Co-cultures with C. testosteroni were considerably more stabIe than those with Methylocystis. Co-cultures with M. barkeri were less 02-sensitive than those with M. fonnicicum. C. testosteroni exhibited a higher 02-affmity than Methylocystis, resulting in a lower dissolved oxygen tension and a superior protection of the methanogenic bacteria against 02-poisoning than in n:llxed cultures with Methylocystis. The dissolved 02-concentrations in the n:llxed cultures were below the detection-limit of the 02-probes used (0.2 f1M). Calculations based on growth properties of pure cultures of C. testosteron i, M. barkeri and M. fonnicicum suggested that the dissolved 02-concentrations in the n:llxed cultures, as well as the 02-inhibition constants (apparent K; 02) of the methanogens were in the nano-molar range.

• Corresponding author: J . Gerritse

36

Introduction

Methanogenic bacteria are extremely sensitive to oxygen. They contain high concentrations of 02-labile compounds and require a relatively low redox potential for growth (Mah & Smith, 1981; Morris, 1976; Vogels et al., 1988). A1though methanogens may possess some enzymatic self­defence against 02-toxicity, growth in pure cultures is completely blocked in the presence of 02 (Jones et al., 1983; Kengen et al., 1991; Kiener et al., 1988; Kiener & Leisinger, 1983; Kirby, 1981; Patel et al. , 1984; Roberton & Wolfe, 1970; Smith & Hungate, 1958; Zehnder & Wuhrmann, 1977). Accordingly, the Iypical natural habitats of methanogens are environments conta=g relatively large anoxic areas, such as aq uatic sediments, flooded soils, landfills, stratified waters and the intestinal tract of animaIs. In addition, they may abide in anoxic micro-niches established in predominantly oxic environments in nutrient rich 'oases', such as detritus aggregates or faecal pellets (A1dredge & Cohen, 1987; Lloyd et al. , 1986; Oremland, 1988; Sieburth, 1987). In these habitats methanogenic bacteria are protected from 02-damage by the high respiratory activity of aerobic (micro-) organisrns.

Near oxic/anoxic interfaces, particularly in systems with very steep 02-gradients, methanogenic bacteria can live very c10sely to strictly aerobic bacteria. Metabolic coupling of anaerobes and aerobes may occur through exchange of metabolites (Wimpenny, 1981). Au illustrative example is the methane cyc1e in freshwater environments characterized by the production of methane by strictly anaerobic methanogenic bacteria within the anoxic zone of the sediment or the water column and the oxidation of methane by strictly aerobic methanotrophic bacteria located in a narrow zone at the oxic/anoxic interface where the diffusioD

profiles of methane and 02 overlap (Rudd & Taylor, 1980).

Several methanogenic bacteria Methanococcus maripaludis, Methanosarcina barkeri, Metanobacterium thermoautotrophicum, Methanobaclerium bryantii andMethanobrevibacler arboriphilus - though metabolically inactive, have been shown to survive oxic periods quite weU (J ones et al., 1983; Kiener et al., 1988; Kiener & Leisinger, 1983; Patel et al., 1983; Roberton & Wolfe, 1970; Zehnder & Wuhrmann, 1977). Hence, it can be anticipated th at in areas exposed to alternating oxic/anoxic conditions the microbial population can contain methanogens as weU as strict aerobes, though their activity is separated in time, depending on the (temporal) presence or absence of 02'

In spite of their apparent mutuaUy exclusive type of metabolism, there are some indications that methanogenic and aerobic bacteria may also share the same micro-habitat (growth without separation in time andlor in spa ce). This paradox may be explained by the fact that under 02-limitation aerobic respiring micro­organisms can maintain such very low dissolved 02-concentrations that complete inhibition of methanogens is avoided (Scott el al., 1985; Lloyd el al., 1989; Gerritse el al., 1990).

Co-existence of strictly anaerobic and aerobic bacteria is interesting because it facilitates interspecific microbial interactions including substrate competition, cross-feeding and co­operation during the degradation of organic matter. Defmed laboratory cultures have been used successfully in revealing information on the co-existence of and interactions between such metabolically diverse organisms (Chapman el al., 1992; Gerritse et al. , 1990; 1992; Gottschal & Szewzyk, 1985; Ohta el al., 1990; Van der Hoeven & Gottschal, 1989 and Wimpenny & AbdoUahi, 1991). However, the anaerobic bacteria used thusfar in mixed cultures with aerobes are Desulfovibrio, Streptococcus, Clostridium and Veillonella species, all of which are known to tolerate low concentrations of 02'

The aims of the present study were to investigate whether methanogenic bacteria, renowned for their very high sensitivity to 02' can also grow in defmed mixed cultures with strict aerobes. In addition, such cultures mayalso provide quantitative information on the factors responsible for the very narrow range of 02-concentrations which may allow coexistence of such bacteria. To this end, growth of the

Two-membered cultures ol' methanogens and aerobes

methanogens, Melhanobacterium fomlicicum and Methanosarcina barkeri, and the aerobes Comamonas testosteron i and Methylocystis sp. was studied both in pure cultures and in mixed cultures in the presence of growth-limiting concentrations of 02'

Methods

Organisms. Methanobacten"um formicicum strain Wolfe was isoJated and identified at the Department of Microbiology of the University of Nijmegen, The Netherlands, and kindly supplied by Dr. E. Raemakers-Franken. Mcchanosarcjna barken" DSM 800 Îs maintained În lhe slrain collection of Dur laboratol)'. The methanotrophic bacterium MethyJoCYSlis sp. strain T-l was obtained frorn Dr. K. Takeda who isolated th is organism from a soil which leaked methane gas (Takeda, 1988). Comamonas testosteronisp., was isolated in our laboratOly and described previously (Gerritse et al. 1990).

Media and growth ronditions. The medium used was a modified version of that described by Zehnder and Wuhnnann (1977). Basal medium contained (values in g 1"): KH2PO" 0.54; Na2HPO" 1.34; NH,CL, 0.3; NaCI, 0.3; CaCI2.2H20 . 0.04; MgCI2.6H20 , 0.04; Nitriloacetic acid (tri­sodium salt), 0.02; FeCI2.4H20, 0.01 ; KAI(SO')2' 0.0001 ; Na2Se0 3.5H20, 0.00003; Na 2 Wo,.H 20, 0.00003; ZnSO,.7H20 , 0.0001 ; MnCI2.4H20 , 0.00003; H3B03, 0.0003; CoCI2·6H20 , 0.0002; CuCl2.2H20. 0.00001; NiCI2.6H20. 0.00002; Na2Mo4 .2H20, 0.00003; resazurin, o.eX)]; yeast extract (BBL) 0.1; gelysatc peptone (BBL) 0.1; and I mi 1. ' of a filter·sterilized (0.2 j.Lm pore-size) vitamin sol ut ion according to Heijthuisen & Hanscn (1986).

For oxic batch cultures of Methylocystis and C testosteronithe basal medium was supplemented with 0.2 g r 1

Na2S04. Potassium·L·lactate (20 mM) was added as the carbon and energy sou ree for C testosterom~ whereas methanol (50-100 mM) or methane (50%, v/v in the gas­phase) were provided for growth of Methylocystis.

Methanogenic bacteria were routinely maintained in anoxic batch cultures with medium amended with NaHC03 (30 mM) and volatile organic acids (1.2 mM acetate, 0.4 mM propionate, 0.2 mM butyratc, 0.05 mM 2-methylbutyrate, 0.05 mM isobutyrate, 0.05 mM valerate and 0.05 mM isovalerate). Anoxic media were prepared under a N2I"C02 (80:20, v/v) atmosphere in culture bottles or hungate tubes sealed with butyl rubber stoppers and reduced with 10 mi 1-' of a (1.25%, w/v) Na2S.9H20/cysteine.HCI mixture. Sodium formate (100 mM) was supplied as the carbon and energy sou ree for M. [ormicicum from a separately autoclaved stock solution. For growth of M. barken' the NaCI concentration of the medium was raised to 0.6 g r' and methanol (100-150 mM) was added from an autoclaved (50%, v/v) stock solution.

Chemostat media weee the same as those used for batch cultures of methanogenic bacteria with the exception that the sulphide/cysteine solution was replaced by 10 mI ,-, of a thiosulphate/cysteine sol ut ion (pH 10) containing 5% (w/v) Na2S20 3.5H20 and 1.25% (w/v) cysteine.HCI. For chemostat cultures with M. formicicum peptone was omitted and the yeast extract concentration was increased from 0.1 te

1 g r'. Formic acid was pumped from a concentrated solution

37

Chapter 4

inta the culture vessel resulting in areseIVoir concentration (5,) of 430-460 mMo

Chemostat culture vessels had a liquid volume of 400-450 mi aod were equipped with sensors enabling continuous measurement and regulation of tempera tu re, redox potential and pH. Stabie measurement of dissolved 02 with Ingold sensors over extended periods of time (several weeks) was not possible as these electrocjes appeared to be poisoned in lhe reduced chemostat media. 02-concentrations in the gas phase weee measured with a Servomex 1100 oxygen analyzer. A description of the chemostat configuration was published previously (Gerritse et al., 1990).

Chemostat cultures of M. formicicum weee grown at 37"C aod pH 7.5, whereas for cultures with M. barken' the temperature was 35°C 3nd the pH 6.9. Tbc pH was maintained by automatic titration with 1 N NaOH or 1 N Hel. Tbe chemostats were stirred by means of a magnetic stirrer and the head-space of the culture vessel was gassed with N,IC02 gas (80:20, v/v), freed of 02-traces by passing over hot copper cu rls. Por 02-limited growth air was mixed with the N2"C02 Oow-gas resulting in mixtures containing 0 10 2% 02 (v/v) which were supplied both via the head-space as weil as via a submerged pipe into the culture Iiquid.

Cultures of M. fonnicicum had some lendency 10

adhere to the glass and electrodes present in the chemostat « 10% of the total biomass in the culture). In cultures with M. fonnicicum, wall-growth was removed befare co-culture experiments with aerobic bacteria were pcrformed. Wall­growth was not observed in cultures of Mbarken:

CeJ/-quantification in mixed cultures. The biomass of the populations of individual species in mixed chemostat cultures were estimated by combining cell-carbon measurements and microscopie cell-counts. Cells of M. barken' (large aggregated cocci), M. formicicum (Jong th in reds to fiJaments), ·C testosteroni (thick straight rods) and MethylDCyStis(small oval rods) could readily be distinguished morphologically. The ratio between cell-numbers of different species in mixed cuhures was determined on a Carl Zeiss G42-110-e phase-contrast microscape in samples taken from continuous cultures, flXed with 0.4% formaldehyde. Numbers of bacterial ce lis per unit volume were obtained with a Bürker-Türk counting chamber. Af ter appropnale dilution, 500-1500 cells were counted in at least duplicate samples. In aggregates of cells, individual cells were distinguished on the basis of a microscopically visible cell-walt. Subsequently, the amount of cell-carbon of the individual bacteria in the mixed cultures was calculated from the total cell-carbon concentration in the co-cultures and a cell-number/cell-carbon calibration detennined in pure cultures. Cell counts in batch cultures of M. formicicum, M. barkeá, C testosteron; and Merhylocysris yielded values of 6.90 x 10", 0.268 x 109,5.71 X

109 and 13.10 x 10" ce lis (mg cell-carbon)"' , respectively.

Respirometry. 02-consumption rates were determined at 37"C (Merhylocysris) or 35"C (C resrosrerom) in a Yellow Springs-type biological oxygen monitor equipped with a polarographic oxygen electrode connected 10 a Keithley model 485 picoamperometer for increased sensitivity al very low 02-concentrations (Gerritse et al., 1992). This method allowed detection of 02 at concentrations > 0.01 j.LM. 02-consumption of C testosteron; was analyzed with dilute resting cell suspensions in medium containing chloramphenicol and 20 mM of L-Iactate. To anoxic cell-suspensions of C testosteroni, 02 was added to a concentration of about 10 (J.M by injection

38

of air-saturated water. 02-utilization by Methylocystis was analyz.ed with resting cell suspensions saturated with an air/methane mixture (abaul 50%, v/v) or in air saturated suspensions containing 50 mM of methanol. Kinetic constants, apparent ~ 02 and Q02m

al(, values, were obtained with direct linear plots. It was assumed that air saturated suspensions contained 218 j.LM dissolved 02'

Determination of growth-kinetics. Maximum specific growth rates (j.Lmax values) were determined by following lhe change in optical density at 660 nm (ODssol in batch cultures, or in continuous cultures with the dilution (0) rate 20-50% above the j.Lmax (wash-out experiments). Half-saturation constants for growth (K,) were obtained from specific growth rales and residual formate or methanol concentratÎons (s) in steady state cultures of M. formicicum or M barkcá, respectively. h was assumed thaI growth of these baclena followed Monod.type of kinetics and the corresponding K, va lues were estimated from direct linear plots.

Analytical procedures. Quantification of gasses (CH 4 1

CO2 and 02)' tDtal organic carbon and cell-carbon was done as described previously (Gerritse er al., 1990). Formate concentrations were determined colorimetrically according 10 the method of Lang & Lang (1972). L-lactate concentrations were determined gas-chromatographically as described by Nanninga & Gottschal (1985). Methanol was measured on a Packard 427 gas-chromatograph quipped with a Porapack R column (SO/lOO mesh) kept at a temperature of 114°C. The temperature of injector and the (name-ionization) detector was 120"C.

Chemica Is. AH chemicals used were of analytical grade quality and obtained from commercial companies except L-lactic acid (F1uka) contained 90% L-Iactic acid in water.

Results

Growth and 0 zuptake kinetics of pure cultures of Comamonas testosteron i and Methylocystis sp.

Maximum specific consumption rates (002 max) and half-saturation constants (apparent ~,) for 02 were obtained respirometrically with cell­suspensions of Methylocystis and C. testosteron i, grown separately in oxic batch cultures.

The maximum specific growth rate (Il-max) of C. testosteron i in a batch culture at 35°C on 20 mM L-Iactate was 0.39 (± 0.02) h-1

. Rates of °2-

consumption by resting cell-suspensions of C. testosteroni revealed a 0 02 max of 22.4 Il-mol (mg cell-carbonr1 h-1 and an apparent ~ of 0.45 Il-M (Tabie 1). Cell-suspensions of C. testosteroni did not oxic:lize methane or methanol.

In a shake culture (150 r.p.m.) at 37"C with an air/methane mixture (50:50, v/v) in the head­space, Methylocystis grew with a Il-maxof 0.09 hol. When the carbon and energy source of an exponential-phase culture of Methylocystis was

Two-membered cultures of methanogens and aerobes

Tabel 1:

Kinetic parameters for oxygen consumption by resting eeJ/s of Comamonas testosteron; and Methylocystis sp. pre-grown on L-/aclate or methane, respeclively

Values afe means of at least three determinations ± standard deviation.

Species Substrate 0 02 ma>< Apparent K." 02 Q max~02

oxidized (!'-mol mg·' h· t ) (!,-M) (I~g.' h·)

C testosteroni L-lactate 22.4 '" 4.7 0.45 '" 0.14 49.8

Methylocystis Methane 7.4 '" 0.2 4.7 '" 0.75 1.6 Methanol 18.6 '" 0.3 1.9 '" 0.37 9.8

Table 2:

Growth parameters obtained from steady state cultures of Methanobacten"um {onnicicum and Methanosarcina barken

The dilution rate of the steady state cultures was 0.026 hol.

Species Substrate Qsubstrate OCH4 y subslrate 5subslrate !'-ma>< K, (mMin reseJVoir) (mmol g.' h·') (g mor') (mM) (h"') (mM)

M (onnicicum Formate 142.4 40.1 0.18 9.1 0.034 2.8 (460)

M. barken Methanol 10.6 6.94 2.44 0.3 0.042 0.18 (llS)

• Detennined from the rale of wash-out at a dilution rate of 0.05 hol.

switched from methane to methanol (100% air in head-space and addition of 60 mM methanol), exponential growth continued without a lag at a rate of 0.08 h-l . Methane grown ceils of Methylocysds oxidized methane as weil as methanoL The affinity for 02 was considerably higher with methanol than with methane as the substrate (Tab Ie 1).

Growth oj Methanobactenum jonnicicum and Methanosarcina barken in anoxic condnuous cultures

Some growth parameters of M. jormicicum and M. barken were determined in continuous cultures with formate or methanol, respectively, as the growth substrates.

Determination of fl-max through wash-out experiments using anoxic steady state cultures of M. jormicicum (D 0.026 h-I, pH 7.5, temperature 37'C) revealed relatively low values. Cell-yield values and the K,; for formate were also measured in this steady state culture (Tabie 2). In batch culture on 100 mM formate similar growth rate (fl-max = 0.028 h·l ) and yield data (0.15 g mo!"l) were obtained.

With M. barken, methanol-limited steady state cultures (D 0.026 hol, pH 6.9, temperature 35'C) were obtained (Tabie 2). At a dilution rate of 0.05 h·1 M. barken washed out at a rate which indicated a fl-maxof 0.042 h-I, very close to that found in batch culture (0.045 h·t).

The stoichiometries of methane production observed in the present study (Tabie 2) agreed weil with the known equations for the methanogenic conversion of formate and

39

Chapter 4

methanol reported previously (Vogels et al., 1988).

M. [onnicicum: 4HCOO' + 4H + -

CH. + 3CO, + ZH,O

M. barken 4CH30H - 3CH. + CO, + ZH,O

(I)

(z)

Washed cell-suspensions from chemostat cultures of M. fonnicicum or M. barken did not consurne measurable amounts of 0z « 0.04 !-LmOI (mg cell-carbon)"1 h-I) in the presence of either formale or methanol.

07limited mixed cultures with Methanobactenum fomûcicum

(1) Co-culture of M. fonnicicum and C. testosteroni. An anoxic formate-limited chemostat culture of M. fonnicicum (D = 0.026 h-I) was inoculated with about 150 mi of a L-Iactate-grown batch culture of C. testosteron i (Fig. lA). From t = 0 h-I the culture liquid was supplied with a flow of Nz/COz gas containing 0z 10 a concentration of 0.18% (v/v). This gas-mixture was supplied al a rate of about 2.5 lh-I (450 !-LM 0z h-It- 0z .was consu~.ed al a rate of 138 ±. 19 !-LM h- dunng this 1lli11al penod of 0z-luruted growth. Residual L-lactate remained > 17 mM and the redox-reading and dissolved 0z bel ow -175 mV and 0.2 !-LM,respectively.

A sharp decrease in the rate of methane formation occurred within the ftrst 5 h of the experiment, probably caused by dilution of the population of M. fonnicicum present in the chemostat (Fig. lC) . During the next 2.5 volume changes (65 hours), methane formation was restored partially but did not reach the initial anoxic value. Both M. fonnicicum and C. testosteron i washed-out, though at a lower rate Ihan the rate of dilution . Even a decrease of the dilulÎon rate (from 0.02610 0.020 h-I) at t=71 did nOl avoid a gradual decrease of methane formation.

After 0z-limited growth for 165 h (5.3 volume changes) the medium supply was stopped but the introduction of 0z into the culture was maintained at 450 !-LMh-I The populations of M. fonnicicum and C. testosteron i both increased at the expense of the residual formate and L-Iactate, respectively, present in the culture vessel, indicating simuItaneous growth of both organisms.

40

200 ~l --'-----'-'---' ,B)

_ 150

• t I ä 1 :

• ' 00 I i i I: ~

50

'Cl 1 .--. /" . .-."",

I I ""'. I

o ~---L----~--------------50 50 100 150 200

T;~lh)

Fig. 1: Time-course of a mixed continuous culture of Mcthanobacterium [onnÎcicum and C testosteroni. C testosteron; was inoculated into a farmate limited chemostat culture (D = 0.OZ6 h-', pH 7.5, temperature 37"C) of M. {ormicicum at t = O. Also al t = 0, the resulting co-cuhure containing 57 mg M formicicum eell-carbon 1" aod 32 mg C testosteron; cell-carbon r1 was exposed ta limiting amaunlS of 02 (0.18%, vlv, in the gas-flow). At t =71 , D was reduced from 0.OZ6 h·' to 0.Q20 h·' and at t = 165 the medium supply was stopped. Carbon and energy SQurces in the reservoir medium werc farmate (Sr=460 mM) faT M. formicicum and L-Iactate (5,=ZO mM) for C testosteroni. (A) Biomass of the population of M. formicicum (0) or C testosteron; (e ) , respeclively; ---, rate of wash-out in lhe absence of growth . (8) Residual formate (e ); ---, fannale accumulatien in the absence of consumptien. (C) Rate of methane format ion (e ).

(2) Co-culture of M. fonnicicum and Methylocystis sp. The aerobic methanotrophic bacterium Methylocystis sp. strain Tl was pre­grown on methane in a batch culture and inoculated into an anoxic formate-limited chemo-

1000 r---~---~--,..----' (A)

I ~o_o--o------q,.._

"--0 & 100

~ ~

i i

1 j

10 ............. _.-.-----. 1-----.

12~ ~==::::===::::==~===~ (BI

100

80

60

' 0

20

o L-=A~---==·~~·_---------_~ __ ~ __ __J

o 50 100 ISO 200

TIme (h)

Two-membered cultures of methanogens and aerobes

_~ 2 .0 l:~-(c-)--~-o-_-_-_-~~~~~_;~---' 100 ~

~ l. sl ,-, "'-0--( "'-0- 0 7S ~

f !i % U

Ö

1.0 /.""._ ............ - -. ! 0.5

{ E

50 ;I

8 o· Ö

25 !

o.o· ~:::==::::::=:=::;:I ===::" 0 .05.--

(0)

~~ 0.0 4

i O.OJ i ----j-I -:-::-::-L'---~ f- - : -l--- '--------------.1 i 0.02 ---- '1-

Q.Ol

f--' 0.00 L-__ ~ ___ ~ __ .c.... __ __'

o '0 100 "0 100

Time (hl

Fig. 2: Time-course of a mixed continuous culture of Methanosarcina barken and C tcstosterom: C testosteroni was inoculated iota a methanol limited chemostat culture (0 = 0.026 hO" pH 6.9, tempera tu re 35°C) of M. ·barken" at t =0. The co-culture containing 260 mg Mbarken' cell-carbon 1" and 17 mg C testosteroni cell-carbon 1" was subsequently exposed 10 limiting amounl'S of 02 (2%, vlv. in the gas.flow). Al t = 142, D was increased from 0.026 hO' 10 0.039 h-'. Carbon and energy sources available in the reselVoir medium were methanol (Sr = 115 mM) for M. barken and L·lactate CSr=21 mM) for C tcstosterom: CA) Biomass of the population of M. barken' (0) or C testosteroni (e ); ...• rate of wash.aut in the absence of growth. (B) Residual methanol (e ); .•. , methanol accumulation in the absence of consumption. (C) Rate of methane formation (0) and 02-consumption ce). (0) Specific growth rate of the population of M. barken ( .•.. ), C testosteroni (--) and the dilution rate (-) of the mixed culture.

stat (D=O.028 hol, S/orma(e=430 mM) of M. jormicicum. Although Methylocystis should be able to grow at the expense of methane produced by M. jormicicum, the poor affinity for 02 would preclude significant growth on methane (see above) at very low 02-concentrations. Therefore some methanol (initial concentration 57 mM) was added to the culture vessel to support 02-limited growth of tbis bacterium. Gradual introduction of air into the flow-1as resulted in an 02-supply rate of 400-500 ~Mh- and a rate of 0 2-consurnption of 30-50 ~M h -1 Nevertheless wash -out of the population of Methylocystis and methanol occurred at a rate eq ual to the dilution rate, indicating that tbis bacterium was not able to grow at the expense of methanol (or methane) oxidation under these severely 02-limited conditions.

Growth of M. jormicicum was also dramatically inhibited in this co-culture with Methylocystis. Under 02-limitation methane formation was reduced by more than 70% and

accumulation of formate and wash-out of the methanogen occurred at a rate nearly equal to the dilution rate. Af ter 3 volume changes the redox­reading had increased from about -200 to -80 mV. When strictly anoxic conditions were restored at that moment wash-out of Methylocystis continued, but growth of M. jormiciwm, formate consumption and methane production were resumed instantaneously.

0zlimited mixed cultures Dj Methanosarcina barken and C teslosteroni

In a preliminary experiment (not shown), a batch culture of C testosleroni was introduced into an anoxic methanollimited steady state culture of M. barken (D = 0.026 h-l, Srme'hanol = 115 mM, S,L-laC)a,e = 21 mM) . 02 (0.7%, v/v) was included in the N2/C02 gas and supplied at a rate of 260-280 ~M h-l Under these conditions the mixed culture consumed 02 at a rate of 11-13 ~Mh-l

41

Chapter 4

After 4.4 volume changes (170 hours) following inoculation, the density of C. testosteroni had dropped from 19 lO 4 mg of cell-carbon r1

However, the density of M. barkeri (248-251 mg cell-carbon I-I), the residual methanol concentration (0.3-0.4 mM) and the rate of methane production (1.89-1.95 fl-Mh- I ) had not changéd significantly within this period indicating that M. barkeri was capable of growth and methane formation under these 02-limited conditions, but that the density of C testosteroni dropped to unacceptably low levels.

The results of a very similar experiment though with a somewhat higher 02-supply (2%, v/v, at the same flow-ratel to stimulate growth of C testosteroni are shown in Fig. 2. The biomass of M. barkeri in tbis mixed culture remained about 10% lower than in the mono-culture. However, the time-course of the change in cell-carbon of M. barkeri (Fig. 2A) dearly showed that af ter the shift to 02-limited conditions the methanogen grew with a specific growth rate corresponding to the dilution rate of the culture (0.026 hol) (Fig. 2D). Yet, methanol consumption appeared slightly less than its supply, resulting in a slow increase of the residual methanol concentration (Fig. 2B).

When the dilution rate of the mixed culture was raised from 0.026 h-l to 0.039 b-l at t = 142 b-I the population ofthe methanogen washed­out gradually (Fig. 2A). Nevertheless the specific rates of growth (fl-) and methane production (QCH4), expressed per unit of biomass of M. barkeri, increased after tbis shift to a higher dilution rate.

1n these cultures, C testosteron i maintained a population density of about 10 mg r l at a dilution rate of 0.026 h- I and sbowed a drop to about 6 mg I-I at D = 0.039 h-I (Fig. 2A). Residual L-Iactate (not shown) remained at a saturating concentration for C testosteroni (> 17 mM) as a consequence of the Iimited supply of 02. In the mixed cultures of C testosteron i and M. barkeri the redox-reading always remained between -150 and -250 mV, and the dissolved 02-concentration remained below the detection-Iimit of the 02-probe « 0.2 fl-M).

Discussion

The results presented in this paper show that methanogenic bacteria can be grown in mixed cultures with aerobic bacteria in chemostats, under 02-limiting conditions. Similar results were

42

obtained earlier with co-cultures composed of strictly aerobic and anaerobic fermentative or sulphate-reducing bacteria (Chapman et al., 1992; Gerritse et al., 1990; 1992; Gottschal & Szewzyk, 1985; Ohta et al., 1990; Van der Hoeven & Gottschal, 1989 and Wim penny & Abdollabi, 1991).

Most of the growth parameters (K., fl-max and cel! yield) obtained in continuous cultures of Methanobacteriumformicicum andMethanosarcina barkeri with formate or methanol as the limiting substrates, respectively, were similar to those reported in other studies (Chua & Robinson, 1983; Schauer & Ferry, 1980; Schauer et al., 1982; Weimer & Zeikus, 1978).

Real steady-states of mixed cultures of the methanogenic and aerobic bacteria were not obtained. Without exception the introduction of small amounts of 02 led to a decrease in the rate of methane and biomass formation, together with a rise of the residual concentration of the carbon and energy source of the metbanogen. Nevertheless, metabolism and growth of M. formicicum as well as that of M. barkeri were evident in all the mixed cultures tested. The mixed culture of M. barkeri and C testosteron; came dosest to a true steady-state followed by M. formicicum plus C testosteron; and the combination of M. form;cicum plus Methylocystis doing less weil.

The observation that metabolism of M. formicicum was more inhibited in a mixed culture with Methylocystis than with C testosteron; is most Iikely explained by the considerably (5 to 30 times) higher apparent 0z-affinity of the latter organism. Particularly with methane as the substrate the O?-consumption capacity of Methylocystis was reÏatively low. This suggests that methane mono-oxygenase, catalysing the initial oxidative step in methane metabolism by methanotrophic bacteria (tbe oxidation of methane to metbanol) limits tbe rate of 02-utiIization by Methylocyst;s. Similar observations were reported for Methylos;nus trichosporium (Best & Higgins, 1981; Joergensen, 1985).

Because mixed cultures of C testosteron i and M. barkeri were relatively stabie and easy to grow, they were analyzed in more detail. Although M. barkeri could be maintained in a seemingly stabie 02-limited mixed culture with C testosteron; for more than 8 volume changes (> 8 days) , this did not represent truly stabie co­existence of the methanogen and the aerobe.

Apparently, the 02-affinity of C. testosteron; is slightly insufficient to establish a dissolved 02-concentration low enough to allow growth of the methanogen at a rate equal or above the dilution rate of the chemostat.

A major obstacle for the quantification of the inhibitory effect of 02 on the growth of M. fonnicicum and M. barkeri is that suppression of their growth-rate took place at 02- concentrations not detectable with the 02-probes in the cultures. The same problem was encountered by Scott et al. (1985) and Lloyd et al. (1989) in determining the effect of 02 on methanogenesis. In weil mixed samples of rurnen liquid, anoxic digesters or suspensions of M. barkeri, exposed to 02' methanogenesis was consistently inhibited before dissolved 02 became detectable even with a Photobacteriurn-probe or a membrane-inlet mass spectrometer (detection limits of 30 and 250 oM, respectively) .

Au estimate of the ~ 02, the 0z­concentration repressing the specific growth rate of the methanogeruc bacteria to V2f1max, may be obtained indirectly from our results of the mixed cultures with C. testosteroni. It is reasonable to assume that under 02-limiting conditions, with saturating concentrations of a carbon and energy source and other nutrients, the specific growth rate (f1)of a strictly aerobic bacterium depends on the dissolved 02-concentration ([02]) according to the Monod-equation (Owens & Legan, 1987).

f1 = f1 max • [Dj / (Ks + [Dj) (3)

By further assuming that the half-saturation constant (K,) for 02-limited growth of C. testosteron i is similar to the apparent K", for 02-consumption determined respirometrically, it follows that the dissolved 02-concentrations in the co-culture of M. barkeri and C. testosteroni can be assessed as follows.

(4)

In this equation f1represents the specific growth­rate of C. testosteron i in the mixed culture, f1maxis the maximurn specific growth-rate on L-lactate at saturating 02 and lactate concentrations (0.39 h· \ and K", is the half-saturation constant for 02-consumption obtained respirometrically (0.45 f1M) with C. testosteron; grown at 3SOC in a batch culture on L-lactate. This latter value is close to the apparent K", (0.35 f1M)determined previously for this bacterium grown in pure culture on

Two-membered cultures of methanogens and aerobes

L-lactate in an 02-limited chemostat culture (0 = 0.1 hol) at 30·C (Gerritse et al., 1992).

The dissolved 02-concentrations in the 02-limited co-cultures of C. testosteron i and M. barkeri thus calculated were in the range from 6 to 40 oM using the f1-valuesshown in Fig. 20. In these cultures the specific growth rate of M. barkeri varied from 0.022 to 0.032 h-l, which is within 50-75% of the anaerobically determined f1maxon methanol (0.042 hol) . Consequently, the apparent ~ 02 of M. barkeri is presurnably slightly higher than 40 oM (about 50-60 oM). Similar calculations for the mixed culture of C. testosteron; with M. formicicum suggest that this latter methanogen is less tolerant to 02 than M. barkeri with a ~ 02 of about 10-20 oM. This difference in 02-sensitivity could be based on the ability of M. barkeri to form cell-clusters which may help to reduce the 02-exposure of cells present in the interior of such cell-aggregates (Kiener & Leisinger, 1983). In our mixed culture experiments cell-aggregates of M. barkeri were composed of about 5-50 cells. Obviously, since these inhibition constants are estimates obtained in an indirect way they should be interpreted cautiously and regarded as maximum values.

lt is of interest to note that these experiments clearly indicate that the level of 02-tolerance of the methanogeruc bacteria used in this study is in the same order of magnitude as the dissolved 02-concentrations maintained under Oz-limitation by C. testosteron;. The fact that such metabolically diverse organisms as strict aerobes and methanogens can thrive simultaneously in the same habitat strongly suggests the possibility of a very direct coupling of the metabolic capacities of these bacteria. This not only influences our views on the flow of carbon substrates in natural low­oxygen environments but it mayalso prove particularly useful for the development of biological purification systems (Gerritse et al., 1992).

Acknowledgements

This investigation was supported by the Netherlands Integrated Soi! Research Project. We thank Dr. E. Raemakers-Franken and Or. K. Takeda for their kind gift of Methanobacterium fonniciwm and Methylocystis sp., respectively.

43

Chapter 4

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