Cultivation and preservation of methanogenic bacteria

Zentmlbl. Mikrobiol. 143 (1988), 149 - 155 VEB Gus t a v Fischer Verlag J e na [University of South Carolina, Department of Pharmacy, Columbia, USA; Institute of Biotechnology, Microbiology and Waste Trea tment, Technical University of Graz, Austria]

Cultivation and Preservation of Methanogenic Bacteria K. HUBER and R. M. LAFFERTY With 4 Figures

Summary Medium requirements and growth yields of Methanosarcina barkeri, Methanospirillum hungatei, M ethanococcus vanielii and Methanobacterium /ormicicum were evaluated. All strains grew well in completely defined media; only M. barkeri required additional vitamins. The maximum achievable biomass yields with H 2 /C0 2 (80: 20) as an energy source w ere 1.5 g dry weight/I. Ga s transfer is the limiting factor for growth as was determined in the case of the fermentation of M. vannielii. All strains can b e preserved in a viable state either a t - 20 °C or in liquid nitrogen provid ed that cells from the exponential phase of growth are used. The addition of 2 g cysteine/l to the media results in floculation of M. bryantii and M. /ormicicum.

Zusammenfassung FUr M ethanosarcina barkeri , M ethano8pirillum hunyatei, M ethanococcu8 vannielii und Methanobacterium /ormicicum wurden Medienzusammensetzung und Waehstumsrate ermittelt. AJle Stamme wuehsen in dem besehriebenen Medium gut, nur M. barkeri b enotigte zusatzlich Vitamine. D er maximal erreichbare Biomasseertrag betrug bei H 2 /C0 2 (80: 20) als EnergiequeJle 1,5 g Trockengewicht je I. Bei der Fermentation von M. vanidii stellte sich h eraus, daB der Gastransfer der limitier ende Faktor filr das Wachstum ist. Aile Stamme konnen bei - 20 °C oder in flussigem Stickstoff im lebensfahigen Zustand gehalten werden, so fern die Zellen d er exponentiellen Wachstums phase verwendet werden. Die Zugabe von 2 g Zystein/l zum Medium verursachte bei M. bryantii und M. /ormicicum eine Flockung.

Methanogenic bacteria have been classified as a new division of the procaryotes due to their special requirements for reducing growth conditions, their unique need of nickel ions and on the basis of different molecular components of the cells. The Gel AT ratios of their DNA has permitted their being classified together with the Halobacteria in the new kingdom Archaebacteria (BALCH et al. 1979, KANDLER and KONIG 1978, ZILLIG et al. 1982)_ Studies of carbon fixation in the case of these organisms revealed the presence of a new pattern of a reversed incomplete Krebs cycle beginning with the condensation of two one-carbon molecules to form acetic acid (WEIMER and ZEIKUS 1979; THAuER et al. 1982). Furthermore, the increasing importance of environmentally compatible waste disposal methods and the recognized necessity of obtaining energy from biomass have led to a greatly increased interest in this group of organisms during the last 10 to 15 years (SCHARER and Moo YOUNG 1979; HUNGATE 1950). In this report aspects pertaining to the handling and culturing of different species of methanogenic bacteria are described. Such cultures could be employed to inoculate methane generators in order to improve overall efficiency. 10

Zeotra\b\. Mikrobiol., Bd. 143

150

K. HUBER and R . M. LAFFERTY

Materials and Methods Organisms : M ethanosarcina barkeri, M ethano8pirillum hungatei, M ethanococcus vannielii , iWethanobacterium bryantii and Methanobacterium /ormicictt1n were obtained from the GernJa n Co llection of Microorganisms (DSM, Goettingen, FRG.) and cultured in d e fined media with v arying a mounts of K 2HP0 2, KH 2P0 4, NH. Cl, MgCI 2 , CaCI 2 , cysteine, N a 2 S, acetate, NaHC0 2 , and N aC!. The media were supplemented wi th trace elements using Wolfe's solu tion plus 1 X 10- 7 M Sc+ 4 (S e0 2 ), 5X10- 7 MMo+B (Na 2Mo0 4 x2 H 2 0) and 5X10- 7 M W+6 (Ha(P(WaOlO)4XH20 and even · tually with vitamins (WOLFE and H IGGINS 1979). The pH-value of the media for all strains was 7.2 with t h e exception of the medium for M. barkeri which was anjusted to 6.8 . A mixture of the gases H z a nd CO 2 (80 : 20) served as a n energy so urce. Subcultures were obtained b y inoculating fresh m edia every 1 - 2 weeks. Growth cur ves were obtained b y d r y weig ht d eterminations u s ing membrane filters . For cell co unts a h emocytometer was employed.

Analytical procedures Dry weight (DW) determinat ions were carried out using the membrane filter method (0,2/W! pore diamet er). Total cell nitrogen was measured by the Kj eldahl met hod and total phosphorus photometrically determined according to the standard pro cedures for water examination. A cetic acid was m eas ured gas chromatograp hica lly (Hewlett Packard Mode l 5840) using a glass column packed with "Chromosorb 110" (80-100 mes h; Supelco Ltd.). This materia l was first washed with acetone, supplemented with 150 mg metaphosphoric acid/5 g packing material and then fill ed into glass co lumns. H 2 , CO 2 , CH 4 a nd H 2 S were quantitat ively d et ermined using a "Poropak Q" stai nless steel column.

Medium preparation Distilled water was boiled for 10 minutes during which time it was deoxygenated u s ing oxygen· free N z. After co oling to ro om temperature, the r equ ired components of t h e medium were added; cy~t eine and Na 2S were added last to finally reduce the redox potential of the medium to - 300 mY. This procedure was controlled on the basis of the reduction of resorcin. The addition of a sma ll quantity of Na 2 S 2 0 3 further reduces the redox potential of the medi um if required. The medium wail then filled into either 100 ml or 1,000 ml flasks to 20 % of the v olume under an oxygen-free N 2 atmosphere. The flasks were then gassed with the H 2 /C0 2 mixture to a total pressure of 2 bar. Media prepared in this manner cou ld be stored for up to three weeks before use.

Fermantation L aborator y ferme ntations we l's carried out in a 10 I " Biostat V " (B. Braun, Melsungen, FRG. ) fermentor with the media a s d escribed. The a ddition of 0.2 ml polypropylene glycol (PPG) was u sed to prevent foaming . Trace e le ments s nch as CaCl" MgCl 2 a nd PPG were sterilized in t h e fermentor, wh3reag other c::.mp one:lts of the m e::l ium were st e rili zed using membrane filters. The fer mentor was gasse::l with the mixture of H2 and CO 2, To improve gas transfer from the gas to the liquid phase, a gas recycle system was installed with a circu la tion capacity of 50 ml/min. Gas volumes were determined wi th a conventional wet displacement gas meter. H 2 S was add ed to the gag mixture by circulating a fraction of the total gas stream through a saturated N a 2 S solution. The H 2 S concentl'ation w as held constant between 0.03 and 0.25% (v Iv) of the exhaust gas a, m eas ured gas chrom at ographically. The gas mixture was sterile filtered with membrane fi lters. Tracfls of oxygen were r em o ved by co ntinually passing the g as mixture through a pallad iulll' asbestos unit. Both t h e redox p otent ia l and the pH value o f the m e dia were cont inuousl y measured. The gas compos it ion was semi· co ntinuo usly moni t or"ed b y m ea ns of gas chromatographic analy.> is. Samples we re periodically tak en fram the ferm entor for t he a na lysis of bacteria l growth and medium co mponents.

Results Nutrient requirement s Organisms were grown with varying concentrations of the individual medium components. The concentration ranges which allow grO\vth are shown in Table 1.

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151

Table 1. Concentration ranges of media components for growth of methanogenic bacteria (gil)

1'0 4NH4Cl MgCl 2 • CaC!2 Cysteine Na2 S NaHC0 3 NaC! CH 3 COONa

0.5-10 0.5-10 0.5- 1 0.5- 2 0.1- 1 0.0-10 0.0- 3 1.0- 7

(M. vannielii 0.5-4)

None of the species of methanogenic bacteria examined required a complex medium with yeast extract or peptone for example. Only the growth of M. barkeri is vitamindependent as was established by subculturing with a 1 % inoculum in media without vitamins for at least 7 generations. M. barkeri resumes growth if vitamins were added after the fourth subculture. The addition of Ca ++ and Mg++ ions to the media resulted in the formation of precipitates which complicate the determinations of dry weight and nutrient requirements. The addition of cysteine (1 gil) gave positive effects. Higher concentrations of cysteine (2 gil) in the media causes flocculation of M. jormicicum and M. bryantii. Even higher concentrations of cysteine are toxic to most strains examined. With a concentration of 4 g cysteine/l only M. vannielii was still capable of growing. The best nutrient concentrations routinely used for seed cultures and for the fermentations are given in Table 2. The maximum dry weights of biomass which were achieved are given in Table 3. M. vanielii yielded up to 3 g dry weightil when the medium was supplemented with both yeast extract and peptone (2 gil). Table 2. Medium composition for growth of methanogenic bacteria (gil)

K 2 HP0 4 3 1 KH 2 P0 4 NH4 Cl 4 0.1 MgCI2 0.05 CaCl 2 1 CY3teine 0.5 Na 2S CH3 COONa 4 6 NaHC0 3 1 NaC! Wolfe's trace element solution 10 mIll Wolfe's vitamin solution (optional) 10 1111 The pH-value was adjusted to 7.2 except for M. sarcina. For this organism the optimal pH-value was 6.8. The gas atmosphere was mixture of H2 and CO 2 (80: 20).

Table 3. Maximum biomass yields attained with medium of Table 2 (gil)

M ethanobacterium formicicum Methanobacterium bryantii M6thanocOCCtlS vannielii Methanospirillum hungatei 10'

1.70

lAO 1.30 1.40

152

K.

HUBER

and R. M.

LAFFERTY

Maintenance of stock cultures If cultures are to be maintained in the exponential growth phase for more than 3 d, the gas mixture as described must be added to the flasks every 24 h, otherwise growth completely ceases and eventually the organisms either totally lyse (M. vannielii) or form spheroblasts (M. hungatei). When subculturing is performed once a week (in the case of M. barkeri every second week) with I % inoculum (v/v), viable cultures can be maintained at 30°C without additional gas dosage. All strains experimentally examined could be maintained in a frozen state at -20°C in 100 ml flasks if the culture used was in the phase of exponential growth. The most susceptible strain is M. vannielii with which lag phases of up to two weeks were routinely determined after thawing. After freezing in liquid nitrogen (-196°C) it is possible to obtain fully viable cultures from all strains both with and without the addition of 10 % dimethylsulfoxide (DMSO). However, freezing and thawing periods must be as short as possible to avoid cell damage.

Fermentation Fermentations were carried out at 30°C with M. vanielii in media as described in Table 2. An inoculum of 4 % (v/v) from a 72 h seed culture was used. The stirrer speed was varied from 675 to 924 rpm. No susceptibility to shear forces was observed with rapidly growing bacteria under these conditions. Figs. 1 to 4 show the results of a fermentation during which gas limitation is observed at a bacterial concentration of approximately I g DW/l. To a certain extent, an increase of stirrer speed can overcome this gas supply limitation. However, at biomass concentrations above 1.30 g DW/I, gas transfer capacity further limits exponential growth in the system used for these experiments.

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Cultivation a nd Preservation of Methanogenic B acteria

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Fig. 3. N utrient uptake by Methanococcus vannielii. Only the concentration of anommium ions decreases significantly. All other components of the medium are consumed in very small amounts. Ce ll carbon is mainly derived from the CO 2 of the gas. During growth the redo x potential dccrea~es. Growth cond itions as in Fig. 1. Fig. 4. Gas uptake by M ethanocOCCtt8 vannielii. The gas volume taken up by M . vannielii increases r apid ly during the first 50 h. When a certain cell concentration is reached, gas transfer beco mes limiting a nd growth is inhibited . The 1)H-valueg change ag exp ected. Growth co nditions as in Fig. 1.

Fig. 1. F erm entation of 2"lethanococcus vannielii. Exponen tial growth is limited after 45 h. An increase of s tirrer speed to 924 rpm. improved gas transfer which allowed a short period of expon ential growth. Temperature 30 °C Medium of Table 2, stirrer speed 675 or 924 rpm., continuous gassing with 80 % H2 and 20 % CO 2 (50 rnljmin), working volume 10 I. F ig. 2. Exponential growth of Methanococcus vannielii. The cell count indicates that exponential growth could only be maintained up to 40 h. Growth conditions as in F ig. 1.

154

K. HUBER and R. M. LAFFERTY

Discussion The culture of methanogenic bacteria has led to the recognition of important factI' pertaining to the biochemical pathways unique to this group of organisms (SMITH and HUNGATE 1958; BALCH and WOLFE 1976; SMITH 1965; BRYANT 1972). However, in order to exploit the full potential of these bacteria, limitations in connection with the ease of handling and the yields of the cultures have to be overcome. It could be shown that all strains examined can be cultured in completely defined media without serious complications other than those normally encountered when handling anaerobic bacteria. The redox potential, however, must be maintained below the critical value of -300 m V. This can be easily monitored on the basis of the reduction of resorcin as an indicator. All strains can be maintained viable in the form of frozen cultures either in liquid nitrogen or at -20°C. Media preparation is simple even at this low redox potential and the formation of precipitates can be prevented by the addition of 1 g cysteine/I. The formation of cellular flocks with a concentration of 2 g cysteine/I which was observed is a very interesting phenomenon since flocculation can be used for the separation of bacterial biomass. The fermentation of M. vannielii revealed that with H 2 /C0 2 gas mixtures as an energy source, one major limitation in obtaining higher cell yields is gas transfer. This could possibly be overcome by using fermentors at higher operating pressures within a practical range. The long residence times required in conventional anaerobic waste water treatment plants is mainly due to the low growth rates of the methanogenic bacteria (MAH and SMITH 1981; TAYLOR 1982). Although considerable technical progress in the field of methane fermentation has been made respect to new, more efficient fermentor types, small private biogas plants could operate much more efficiently if pure cultures of methanogenic bacteria were available at acceptable cost. These could be added directly to the bioreactor in the case of problems or to even decrease residence times (BOONE 1982; MATSUMOTO et aI. 1981). The biotechnological production of methane for the disposal of organic wastes other than those from agricultural operations has attained considerable significance in connection with many aspects of environmental protection. Thus, the use of "starter cultures" of methanogenic bacteria could offer new, practical possibilities for process improvement.

References BALCH, W. E., WOLFE, R. S.: New approach to the cultivation of methanogenic bacteria: 2-mercaptoethane sulfonic acid (HS-CoM)-dependent growth of Methanobacterium ruminantium in a pressurized atmosphere. Apo!' Env. Microbiol. 32 (1976), 781-79l. Fox, G. E., MAGRUM, L. J., WOESE, C. R., WOLFE, R. S.: Methanogens: Reevaluation of a unique biological group. Microbiol. Rev. 43 (1979), 260-296. BOONE, D. R.: Terminal reactions in the anaerobic digestion of animal waste. App. Env. Mikrobio!. 43 (1982), 57 - 64. BRYANT, M. P.: Commentary on the Hungate technique for culture of anaerobic bacteria. The American Journal of Clinical Nutriation. (1972), 1324-1328. HUNGATE, R. E.: The anerobic mesophilic cellulolytic bacteria. Bacterio!. Rev. 14 (1950), 1-49. KANDLER, 0., KONIG, H.: Chemical composition of peptido-glucanfree cell walls of methanogenic bacteria. Arch. Microbiol. lI8 (1978), 141-152. MAH, R. A., SMITH, M. R.: The methanogenic bacteria. In: The procaryotes. A handbook on habitats, isolation and identification of bacteria. (STARR, STOLP, TRUPER, BALOWS and SCHLEGEL, eds.) Vol. 1. Springer Verlag 1981. MATSUMOTO, J., NOIKE, T., ENDO, G.: Microbial growth and organic acid formation in acidogenesis phase of anaerobic digestion. Technology Reports of the Tahoku University 46 (1981),293- 311

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SCHARER, J. M., Moo YOUNG, M.: Methane generation by anaerobic digestion of cellulose coni' aining wastes. Adv. Biochemical Engineering II (1979), 85-101. SMITH, P. H., HrNGATE, R. E.: Isolation and characterization of Methanobacterium rllminantium n. sp ..J. Bacteria!. 75 (1958), 713 - 718. - Pure culture studies of methanogenic bacteria. Proc. Int. Waste Conference 20 (1965), 583. TAYLOR, G. T.: The methanogenic bacteria. Progress i n Industrial Micro biology 16 (1982), 231 - 330 THArER, R. K., BRANDIS-HEEl', A., DIEKERT, G., GILLES, H. H., GRAF, E. G., JAENCHEN, R., SCH<'iNHEIT, P.: Drei neue Nickelenzyme aus anaeroben Bakterien. Vortrag Ges. Deutscher Xaturforscher und Arzte. 19. Spt. 1982. \VEniER, P. J., ZEIKl:'S, J. G.: Acetate assimilation pathway of Methanosarcina barkeri. J. Bacteriol. 137 (1979), 332-339. \VOLl'E, R. S., HIGGINS, I. J.: Microbial biochemistry of methane - a study in contrasts. Int. Rev. Biochemistry 21 (1979), 268-300. ZILLIG, W., SCHNABEL, R., TI', J., STETTER, K. 0.: The phylogeny of archaebacteria, including novel anaerobic thermoacidophiles in the light of the RNA polymerase structure. Die Naturwissenschaften 69 (1982). Authors' addresses: Dr. K. HUBER, University of South Carolina, Department of Pharmacy, Columbia, South Carolina, USA; Prof. Dr. R. M. LAFFERTY, Institut fUr Biotechnologie, Mikrobiologie und Abfalltechnologie, Technische Universitiit Graz, SchlogelgaEse 9, A - EOI0 Craz, ALstria.