Ms. Mu 27
STERILIZATION
OF PEAT BY GAMMA
RADIATION
by F. E. PARKER* and J. M. VINCENT** School of Microbiology, University of New South Wales
KEY WORDS G a m m a r a d i a t i o n Legume inoculants Peat P e a t sterilization R a d i a t i o n resistant a m o e b a e R a d i a t i o n resistant a r t h r o b a c t e r R a d i a t i o n resistant micrococcus Radiation resistant mycobacteria Sterility testing
SUMMARY The effect of gamma-radiation on the survival of microorganisms has been quantified for the natural population of two types of peat. Data for several microbial types have been separately determined by regular plating and by indirect statistical probability estimates including, a wholly enclosed 'invertedbottle' technique for higher dose levels to exclude any possibility of post-treatment contamination. The most persistent microorganisms at intermediate dosage (2.5-3.5 Mrad) were commonly a micrococcus (which closely resembled Micrococcus radiodurans) arthrobacter-like rods, myxobacteria and amoeboid forms. The persistent organisms all survived because of high resistance to yirradiation, not because of high initial numbers. The most numerous true bacteria (including sporeformers), actinomycetes, filamentous fungi and yeasts were all readily destroyed. Although the safety margin with the commercially recommended dose of 5 Mrad is low for some of the more resistant organisms, no change is justified at this stage since the organisms most likely to survive such a dose do not seem to seriously affect the subsequent growth and survival of rhizobia. Moreover there would be some risk of radiation-induced peat toxicity if higher doses were applied and some post-irradiation contamination will be difficult to avoid in commercial production.
INTRODUCTION
Finely ground peat has long been the carrier of choice in legume inoculants. It is however an extremely complex and variable material in its physical, chemical and biological properties and not all sources are equally suitable for the multiplication and survival of rhizobia. In fact samples taken from nearby locations have proved quite variable. Elimination or reduction and simplification of the microbiological component by autoclaving or y-irradiation considerably improved peat as a medium for the growth and survival of rhizobia s. Present addresses: * Hawkesbury Agricultural College, Richmond, N.S.W. 2753, Australia. ** Department of Microbiology, University of Sydney, N.S.W. 2006, Australia.
285 Plant and Soil 61,285-293 (1981). 0032-079X/81/0612-0285501.35. 9 1981 Martinus Nijhoff/Dr W. Junk Publishers, The Hague. Printed in The Netherlands.
286 Gamma-radiation
F. E. PARKER AND J. M. VINCENT has several p r a c t i c a l a d v a n t a g e s o v e r h e a t s t e r i l i z a t i o n a n d
a d o s a g e o f 5 M r a d has, for several years, b e e n s t a n d a r d p r a c t i c e in A u s t r a l i a . T h e r e a r e h o w e v e r little p u b l i s h e d d a t a as to the details o f its sterilizing effect w h e n u s e d for this p u r p o s e . T h e p r e s e n t a c c o u n t a i m s to m a k e g o o d s o m e o f the deficiency b u t is r e s t r i c t e d to s t e r i l i z a t i o n p a r a m e t e r s for the m i c r o b i a l p o p u l a t i o n in g e n e r a l a n d for s o m e of the m o r e r e a d i l y d i s t i n g u i s h e d g r o u p s . M o r e d e t a i l e d s t u d y of m o r e r e s i s t a n t o r g a n i s m s e n c o u t e r e d in this w o r k will be t h e s u b j e c t of s e p a r a t e p u b l i c a t i o n .
EXPERIMENTAL
Peats used in present investigation Two different peats were used: (i) the neutral Badenoch (Mt. Gambier, South Australia) peat 5 (ii) an acid peat from Wingecarribee, (Southern Tablelands, New South Wales) which required neutralization with CaCO 3. All peat had been air dried (15-20% on a wet weight basis) and milled to pass a 200 mesh (approx. 0.1 mm) screen.
Irradiation Three 6~ sources were with mean dose rates of: (i) 15 krad/hour, University of New South Wales (UNSW): (ii) 80 krad/hour, Australian Atomic Energy Commission (AAEC): (iii) 458 kraal/hour (AAEC). The required total doses were obtained by withdrawing samples after the appropriate exposure times, calculated from the experimentally determined dose rate. A minimum of duplicate samples was irradiated at each dose. Irradiation was at room temperature. Dosimetry was based on ferrous ammonium sulphate, using the methods of Weiss 7.8 and Fregen 1. Doses were determined in the 21 fixed positions available in the smaller UNSW unit and in representative positions of the larger number avialable at AAEC. There was good agreement between replicates in the same position but positional effects had to be provided for in establishing treatment levels. Adjoining peat had no shielding effect.
Recovery of microorganisms Recovery conditions were rigorously tested to ensure maximal recovery. Samples from the UNSW unit were plated immediately; those from AAEC were held at 4~ and plated within 24h. Winogradsky's salt solution4: NaCI, 0.001%; MnSOa.4H20, 0.001%; MgSO,~.7HzO, 0.002%; FeSO.7H20, 0.001%; KzHPO4, 0.1%) was used as diluent. The peat was suspended in this solution with 20 minutes dispersion by means of a wrist shaker. This method was found to be the most suitable for routine use, particularly for definitive recovery of low numbers of survivors where strict asepsis was essential. None of seven dispersing agents gave any increase in viable count. Peat extract agar, used for the recovery of bacteria (including actinomycetes), was prepared by adding 0.02~ K~HPO4 and 2% agar to the supernatant from 200 g peat autoclaved in 1200 ml distilled water for 20 min at 121~ and adjusting pH to 7.0. Littman's oxgall agar 3, with peat extract base and containing 30 Isg/ml streptomycin, was the most
STERILIZATION OF PEAT BY GAMMA IRRADIATION
287
suitable of a wide range of media for the recovery of yeasts and other fungal propagules. This medium contained (as percentage in peat extract): peptone, 1.0; glucose, 1.0; oxgall, 1.5; crystal violet, 0.0001; streptomycin, 0.003; agar 2.0. For plate counts aliquots of appropriate dilutions were added to the dried surface of prepoured media by adding 0.2 ml and spreading with a sterile glass rod or by gently rotating 0.4 ml over the agar surface. These methods showed good agreement. Colonies were counted after 7, 14 and 28 days at 26~ Colony separation was sometimes difficult due to the presence of large amounts of peat (in the lower dilutions needed to determine recovery at higher doses), and to spreading myxobacterial and amoeboid growth. Counts were therefore checked against an indirect method (the 'most probable number 'a or the 'likely number'S). Where valid comparison was possible there was good agreement between direct plate counts and indirect methods; the latter was the only practical method where there were too few survivors for a reliable plate count.
Definitive demonstrations of irradiation survival The determination of absolute freedom from surviving microorganisms requires confidence as to successful exclusion of post-irradiation contamination. This is difficult to ensure if the irradiated material will not support microbial growth as is the case with peat irradiated 'dry' (15-20~o moisture) where the contents need to be manipulated after irradiation to provide conditions suitable for
Fig. I.
'Inverted bottles' used for the recovery of low numbers of radiation survivors from peat.
288
F. E. PARKER AND J. M. VINCENT
50~---L. ~ 9
05a..
9
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ffl
.~ oz ~5 ~
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3 1
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11.0
'
2:0"
'
3.0
DOSE (Mrad)
Fig. 2. Survival curves of micro-organisms irradiated in Mt. Gambler peat. Estimates based on duplicate samples plated on a minimum of duplicate PA spread plates for total bacteria, actinomycetes and bacterial spores (dilutions heated 80~ for 20 min.); by likely number method (10 x dilutions plated in duplicate on PA) for bacterial spores at 0.8 Mrad or greater, myxobacteria and amoebae (incubated 14 days at 26~ on duplicate spread plates of Littman's oxgall agar with P base and containing 30 ~tg per ml streptomycin for filamentous fungi and yeasts (incubated 5 days at 26~
microbial growth. Irradiating the peat moist and storing it unopend to allow multiplication of low numbers of survivors to readily detectable levels would have eliminated the problem of contamination, but the radiation resistance of microorganisms is affected by moisture content. A method of sterility testing using air-dry peat, without opening the container was therefore developed (Fig. 1). Two small Bijou bottles, screwed into a brass sleeve which carried a piece of filter paper between two O-rings were heat-sterilized. For the irradiation test 1 g peat was then placed in the upper bottle and 2
STERILIZATION OF PEAT BY GAMMA IRRADIATION
289
7, 6
o}_~4 ~3
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I'-
II
9
9
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5
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4 3 2
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0
\ 1!0
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'
210
~
3.0
DOSE (Mrad)
Fig. 3, Survival curves of micro-organisms irradiated in Wingecarribee peat. The treatmentsare the same as in Fig, 2,
ml peat'-extract broth in the lower. The filter paper retained the peat in the upper bottle but permitted broth to be added to the peat by inverting the unopened assembly after irradiation. The peat-broth mixture was then incubated for 8 weeks at 26~ suspended in 2 ml Winogradsky's salts solution and streaked on test agar medium. Plates were subsequently scored for presence or absence of growth and the types of growth characterised by microscopic examination of preparations from representative colonies and by subculturing, where appropriate, The number of survivors able to multiply in the medium-enriched peat was then calculated.
290
F. E. PARKER AND J. M. VINCENT
RESULTS
Results with the two peats showed substantial agreement in the relative sensitivity of the major groups of microorganisms (Figs. 2 and 3). As the radiation dose increased segments of the microbial population were selectively inactivated so that the survival curves of the mixed populations have to be interpreted as the resultant of composite curves, each for a different part of the population with its own sensitivity. Regression analysis of such survival curves provides therefore only a first approximation of the overall sensitivity of the population to y-irradiation. Bacterial spores, actinomycetes, filamentous fungi and yeasts were clearly the most susceptible (Dlo values*, calculated between 0 and 2.6 Mrad, in the order of 0.15-0.33). Total bacteria were intermediate (0.42-0.52); in both peats the most resistant were myxobacteria and amoebae (0.55-0.75), so that these became increasingly a significant part of the population as the irradiation dose was increased (Table 1). The dose required to eliminate the various groups from peat depends on initial numbers as well as their resistance. The peats were most readily cleared of detectable filamantous fungi (0.6-0.8 Mrad), yeasts (0.8-1.0), actinomycetes (1.12-1.2) and bacterial spores (1.0-1.2). Resistant bacteria, myxobacteria and amoebae (present in relatively low numbers in irradiated peat) survived 2.5 Mrad. Diverse colony types were obtained at highest doses permitting recovery of filamentous fungi and yeasts with both peats, and of actinomycetes in the case of the Wingecarribee peat. On the other hand the number of actinomycete colony
Table 1. Irradiation dose (Mrad) needed for tenfold reduction (Dlo) and dose required for nonrecovery (L.D.) D~o Organisms Filamentous fungi Actinomycetes Bacterial spores Yeasts Total bacteria Myxobacteria Amoeboid forms
LD.
Mt. Gambler
Wingecarribee
0.15 0.20 0.23 0.33 0.42 0.55 0.75
0.23 0.22 0.21 0.18 0.52 0.75 0.75
* Dlo value, irradiation dose needed for tenfold reduction.
Mt. Gambier 0.52 1.1 1.2 1.0 > 2.5 > 2.5 > 2.5
Wingecarribee 0.80 1.2 1.0 0.8 > 2.5 > 2.5 > 2.5
S T E R I L I Z A T I O N O F P E A T BY G A M M A I R R A D I A T I O N
291
T a b l e 2. T y p e s o f m i c r o o r g a n i s m s i s o l a t e d f r o m p e a t i r r a d i a t e d at 2.5-2.6 M r a d Type
Micrococcus b Arthrobacter-lik& Myxobacteria d Amoeboid e Others f
P e r c e n t of e a c h t y p e i s o l a t e d f r o m Mt. G a m b l e r peat a
Wingecarribee peat"
50 8 1 26 15
16 34 22 22 6
a Total isolates from Mt. Gambier peat: 157; from Wingecarribee: 32. b A representative isolate was subsequently found to be physiologically and taxonomically similar to Micrococcus radiodurans. c Close st udy of representative isolates indicated that this type most closely resembled Arthrobacter although in some respects it was like Nocardia or Mycobacterium. d Includes some which appeared to be Chondrococcus. e Includes some resembling myxomycetes. f Most of this group appeared to be post-irradiation contaminents.
types decreased wit.h increasing dose in the peat from Mount Gambier as did the types of spore-formers in both. At, and in excess of 2.5 Mrad the diversity of either bacterial colonies was markedly reduced and these were likely to be either a micrococcus, arthrobacterlike, myxobacteria or amoeboid (some resembling myxomycetes) (Table 2). In these samplings micrococci were the dominant bacterial form in the peat from Mt. Gambier; representation was more evenly distributed between micrococci, arthrobacter-like forms and myxobacteria in that from Wingecarribee. The inverted bottle technique generally recovered the same types of microorganisms as direct plating but gave considerably lower estimates of the most persistent types in 2.5 Mrad peat. This indicated that not all the survivors were able to multiply in the broth-enriched peat. This could be due to the microorganism's intrinsic properties, sub-lethal radiation damage, radiationinduced peat toxicity, competition and/or antagonism by other survivors or, in the case of myxobacteria and amoeboid forms, lack of a suitable food source. There could, of course, be interaction between such factors. It follows that the estimate obtained with 2.5 Mrad inverted bottles underestimated the number of radiation survivors in the strict sense. On the other hand it could provide a better estimate of these likely to be able to multiply in irradiated peat. D~o values calculated for the more resistant organisms between 2.5 and 3.5 Mrad, were: microcococcus, 0.88; arthrobacter-like, 0.70; myxobacteria 0.69; amoeboid forms, 0.57.
292
F. E. PARKER AND J. M. VINCENT
DISCUSSION
The sterilising dose for any material depends on the initial number and radiation resistance of the different types of microorganisms it contains. In a practical sense the degree of 'sterility' will be determined by acceptable risk and economic considerations. Unirradiated peat contains many more microorganisms (approx. 107-108 per g moist peat) than most other materials treated by irradiation. It is also likely to afford some protection against radiation damage to the exposed microorganism. Doses safe for routine processing must be established by direct experimentation because of possible 'tailing-out' effects but care has to be taken to avoid false positives (due to low level post-treatment contamination). Attention also has to be given to difficulties (statistical and technical) in demonstrating survivor levels of the order acceptable for practical purposes. In this investigation some of the problems associated with sterility testing were avoided by studying organisms which were consistently encountered at doses which restricted survival to the most persistent. These were then examined at additional dosage levels. In addition the inverted bottle technique, which removed all risk of post-treatment contamination, avoided false positives and related reasonably well to the practical evaluation ofT-irradiated peat as a carrier for inoculants. The general survival pattern of the peat microorganisms agreed with such data as are available for soils. Filamentous fungi and yeasts were most easily eliminated; actinomycets though quite as susceptible, required a larger dose to offset their higher initial count. The relative sensitivity of bacterial spores was rather unexpected but could reflect the recorded wide range of sensitivity encountered with such organisms. The high resistance level of myxobacteria and the amoeboid forms could be attributed to cyst or microcyst formation. The most resistant bacteria included micrococci (very similar to Micrococcus radiodurans, but with strains which differed in cell size and colony colour) and a form which resembled Arthrobacter. The most persistent organisms survived because of high radiation resistance, either intrinsic or induced by the environment, and not because of high initial numbers. Their resistance was extremely high and will be examined in more detail in separate publications. Data for higher levels of 7-radiation (~< 2.5 Mrad) permit a statement of probability of survival of the more resistant microorganisms at 5 Mrad (a commercially practical dose). On average one surviving bacterium of the micrococcus type could be expected in samples of about 50 g irradiated peat; 7.4 Mrad could be required to reduce the chance in this quantity to 10-3. The likelihood of
STERILIZATION OF PEAT BY GAMMA IRRADIATION
293
encountering other resistant forms (arthrobacterlike, myxobacteria and amoebae) in 100 g peat samples is much less ranging from 2 • 10- 2 to 5 • 10- 3. There are good economic, and possibly microbiological reasons (such as the risk of development of radiation-induced toxicity in the peat) for avoiding excessive irradiation if a milder treatment, short of that required for absolute sterility, suffices for the attainment and long term maintenance of high rhizobial numbers. The significance of any survivors in this regard is therefore important; some such data will be presented in a separate account. It does not seem likely on our data that actinomycetes found after long storage of commercial 5 Mrad yirradiated peat represent true survivors. Rather they seem to be due to the growth of post-treatment contaminents, either due to culture manipulation or 'pinhole' leaks in the polythene package. The findings of this investigation as well as storage data for commercial inoculants show quite convincingly that 5 Mrad y-irradiation is a practical way of securing near-sterility and a good quality carrier for rhizobial multiplication and survival. ACKNOWLEDGEMENTS The authors of this article appreciate the invitation to submit it in the issue of the journal which commemorates the long and distinguished career of Professor E. G. Mulder: both as a teacher, administrator and research worker in the field of agricultural microbiology. It has always been a pleasure to visit his department at Wageningen where so much has been achieved despite the difficulties experienced during and some time after the occupation of the Netherlands in World War If. The excellence of the institution has always been matched by the warmth of the hospitality extended to its visitors. Received 10 February 1981
REFERENCES 1 2 3 4 5 6 7 8
Fregene, A. O. 1967 Calibration of the ferrous sulphate dosimeter by ionometric and calorimetric methods for radiations of a wide range of energy. Rad. Res. 31,256~272. Halvorson, H. O. and Zeigler, N. R. 1933 Application of statistics to problems in bacteriology. I. A means of determining bacterial population by the dilution method. J. Bacteriol. 25, 101-121. Johnson, L. F., Curl, E. A,, Bond, J. H. and Fribourg, H. A. 1959 Methods for studying Soil Microflora-Plant Disease Relationships. Burgess Publishing Co. Minneapolis. Pochon, J. 1954 Manuel Technique d'Analyse Microbiologigues du Sol. Mason et Cie, Paris. Roughley, R. J. and Vincent, ~J. M. 1967 Growth and survival of Rhizobium spp. in peat culture. J. Appl. Bacteriol. 30, 362-376. Vincent, J. M. 1970 A manual for the practical study of the Root-nodule bacteria. IBP Handh. 15, Blackwell Scientific Publications, Oxford. Weiss, J. 1952 Chemical dosimetry using ferrous and ferric sulphates. Nucleonics 10, 28-31. Weiss, J. 1958 A survey of chemical dosimetric systems. Int. J. Appl. Rad. Isotopes 4, 89-95.