Growth of the earthworm Eisenia foetida on microorganisms and cellulose

Growth of the earthworm Eisenia foetida on microorganisms and cellulose

0038-0717/84$3.00+ 0.00 Copyright 0 1984Pergamon Press Ltd Soil Biol. Eiochem.Vol. 16, No. 5, pp. 491495, 1984 Printed in Great Britain. All rights r...

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0038-0717/84$3.00+ 0.00 Copyright 0 1984Pergamon Press Ltd

Soil Biol. Eiochem.Vol. 16, No. 5, pp. 491495, 1984 Printed in Great Britain. All rights reserved

GROWTH

OF THE EARTHWORM EISENIA FOETIDA ON MICROORGANISMS AND CELLULOSE

FRANK M. FLACK and ROY HARTENSTEIN School of Biology, Chemistry and Ecology, State University College of Environmental and Forestry, Syracuse, NY 13210, U.S.A.

Science

(Accepted 4 January 1984) Summary-The growth of Eisenia foetida (Savigny) (200-300 mg live weight) was measured on mixtures of inorganic salts, vitamins, and cellulose upon a base of ashed loam or sand. High concentrations of inorganic salts, exceeding an ionic conductivity of about 8 mS cn-’ were detrimental, and lower concentrations failed to promote growth. Hatchlings (5 mg live wt) grew to 233 k 14 mg (SE) on 22 species of bacteria (100 mg dry wt) at 24 f l”C, in 8 weeks. There were no significant differences in growth on Gram-negative versus Gram-positive bacteria. Human pathogens were no less nutritive th& non-pathogenic spicies. There was no growth on 6 of 19 species of fungi. Only 31% weight (70 + 16 mg) was gained per unit weight of fungi as per unit weight of bacteria (1OOmg dry microbial biomass). Worms grew as well (274 f mg) on three species of protozoa as on 22 bacterial species. Growth on any microorganisms tested was less than on activated sludge. E. foetida doubled in weight on the fungus Coriolus hirsurus with the addition of sand or ashed loam. With either of these two forms of grit and microorganisms there was no growth. For growth, a combination of carbohydrate (cellulose), microorganisms and grit appeared necessary. Lyophilized microorganisms had to be washed free of ionic conductivity (and presumably organic residues) from their culture medium for earthworms to grow favorably.

INTRODUCTION Earthworms are important in turnover of soil (Satchell, 1967), contribute most to soil invertebrate biomass (Birch and Clark, 1953), contain the highest international units of cellulase and peroxidase (Hartenstein, 1982), and are believed to be capable of producing cellulases and chitinases, other than through intestinal microflora (Tracey, 1951; Parle, 1963). Nevertheless, little is known about interactions between earthworms, soil, microflora and humification. No qualitative differences were found between 50 species of bacterial isolates from the gut of Lumbricus terrestris and their surrounding soil (Bassalik, 1913). Nor were any of 343 isolates from 60 species of mainly Pheretima found to be indigenous to these earthworms (Khambata and Bhatt, 1957). Similar results were reported by Parle (1963). Even Marialigeti (1979), who identified 70% of 473 isolates from Eisenia kens as Vibrio spp, did not prove that the isolates were essential to survival or growth of the earthworm. Numerous investigators have reported increased numbers of microorganisms in either the gut or cast material of earthworms relative to the surrounding soil. Satchel1 (1967) reviewed this literature and concluded that it was unlikely that earthworms had an indigenous gut microflora. This statement probably holds true despite the results of Marialigeti (1979). Kozlovskaya (1969) stated that the increase in microbial activity in casts indicated an intensification of the mineralization process. Brown and Mitchell (198 l), and Hartenstein and Hartenstein (198 l), using direct counts and respirometric measurements respectively, supported this conclusion, especially in 491

view of what is now known about the humification process (Hartenstein, 1981). Domsch and Banse (1972) reported total destruction of fungi following their passage through earthworms, and Atlavinyte and Pociene (1973) reported a negative correlation between numbers of earthworms and algae in pots. Algae (and to a lesser extent fungi) were considered most important to the nutrition of several earthworm species. Dash et al. (1979) concluded that earthworm grazing on the soil microflora enhanced microfloral growth by preventing senescence. Neuhauser et al. (1980a) found significant weight increases of E. foetida in the presence versus absence of each of two bacterial, three fungal and two protozoan species. There were also significant differences between weight gained on live, as opposed to dead, Arthrobacter sp., Aspergills niger, Geophyllum trabeum and Tetrahymena pyriformis. Our objectives, using growth of Eisenia foetidu (Savigny) as the criterion, were to determine (a) whether E. foetida can grow on a non-microbial substrate, (b) whether microbes must be washed free of their culture medium before being suitable as food, (c) the ranking of bacterial, fungal or protozoa1 species as nutrients for E. foetida, (b) whether bacteria pathogenic to humans are less suitable as food to E. foetidu than non-pathogenic species, (e) whether Gram-negative bacteria are as good as Gram-positive bacteria as food, and (f) the minimum ingestible entities required for growth of E. foetida. METHODS AND MATERIALS Ail biochemicals were purchased from Sigma Chemical Co., St Louis, MO. Sand was ashed (SSO’C,

492

FRANKM. FLACKand ROYHARTENSIEIN

4 h), and washed in distilled water until the ionic conductivity of the used water was 50pScm-‘. Bacteria were obtained from Dr A. Phillips (Syracuse University), Dr T. Metcalf (University of New Hampshire), Miss Lenny Love (SUNY Upstate Medical Center) or purchased from Sigma Chemical Co. Fungi were obtained from the University’s collection. Protozoa were obtained as axenic species from Dr D. Beach (SUNY Upstate Medical Center). The microorganisms were cultured in 4 1 flasks aerated continually at room temperature, with 0.8% nutrient broth for bacteria, 1.5% malt extract broth for fungi, and the following medium for the protozoans: 50 ml 5% casein, 1 g K,HPO,, 5 g yeast autolysate and 950 ml distilled H,O. The protozoan culture medium was adjusted to pH 7.3, autoclaved 20 min at 120°C and supplemented with 25 ml 40% glucose sterilized separately. The cells were harvested by centrifugation and washed with distilled water until the used water read 50 PS cm-‘. The cells were then lyophilized to facilitate weighing and handling. To measure earthworm growth on inorganic salts and vitamins, 50 ml dry sand was placed into a 20 x 100 mm Petri dish and wetted to saturation with tapwater. A layer of moist cellulose (50 g of 3 parts H,O, 1 part cellulose), containing inorganic salts and vitamins, were placed upon the sand. The salts included NH,N03 or (NH&S04, KC1 or K-acetate, K,HPO,, MgCl, and CaCO,. The vitamins included biotin, folic acid, pyridoxine, riboflavin, thiamine, niacinamide, pantothenic acid, thioctic acid and paminobenzoic acid. A single gut-voided pre-clitellate earthworm (20&300mg live wt) was rinsed in distilled water, placed in the dish and rinsed and weighed every week for 4 weeks. To measure earthworm growth on microorganisms, approximately 12g ashed (SSOC, 4 h) loam was placed in a 15 x 60 mm Petri dish. Microorganisms were added in quantities given in Tables 14 after mixing with 5 g of one part cellulose to three parts distilled water. After placing the microbialcellulose mixture upon the ashed loam or sand, 1 non-voided hatchling (3-10mg) was rinsed and added. Specimens were weighed after 2,4,6 and 8 weeks at 24 f 1“C after rinsing and drying them on blotting paper. All values of results are expressed as X f SE. Earthworms failed to grow on cellulose with sand or ashed loam as substrate with any combination or concentration of salts and vitamins. Combinations of salts exceeding concentrations that would cause an

Table 1. Mean live weight (mg + SE) of E. fiefida after 8 weeks of feedine on cellulose, ashed loam and Sigma microorganisms, washed and unwashed 1 worm per-dish. N = 5* Species

Ration of microbial biomass 25 mg 50 mg 100mg

Arorobacfer uinelandii

Unwashed Washed

X(15) 74 (6)

48 (28) 152 (22)

90 (45) 245 (28)

76 (22) 182 (23)

Dead 267 (22)

32 (8) 99 (6)

70 (13) fl(18)

Escherichia coli

Unwashed Washed Saccharomyces cereuisiae

Unwashed Washed

16 (2) 21(b)

lP < 0.05 between washed and unwashed microbes at each ration level.

0

2

4 Tams

6

6

11

1weeks)

Fig. 1. Growth of E.foetida on 25 (O), 50 (A), and 100(m) mg lyophilized Serratia marcescens, cellulose and ashed loam, N = 5. The curve with hollow circles represents growth of E. foetida on activated sludge (Neuhauser et al., 1980b).

ionic conductivity in excess of about 8 mS cm-’ were lethal, and combinations with lower ionic contents caused losses in weight. Initial experiments with microorganisms revealed the need to wash them before lyophilizing. Failure to do this caused a significant reduction in growth, or death (Table 1). Growth on three amounts of Serratia marcescens (Fig. 1) did not follow a logistic curve at the lowest microbial amounts (25 mg) for the first 8 weeks, and decreased after this. Data from Neuhauser et al. (1980b) illustrate the fastest growth rate achieved by E. foetida in any substrate tested to date (Fig. 1). Growth on activated sludge compared with that on any microbe tested suggests a need for growth factors other than those in the present study. Growth rates of E.foetida on 22 species of bacteria (Table 2) indicate a range in live weight of about 150-400 mg after 8 weeks, with a mean weight of 233 + 14mg. On Gram-negative bacteria, the mean weight of the worms was 239 f 16 mg which did not differ significantly from the weight of the worms fed Gram-positive bacteria. After 8 weeks on a diet of the human pathogens Salmonella typhimurium, Shigella sonnei, Staphylococcus aureus and Strepto coccus faecalis the worms weighed 170 &-24 mg and on four other bacteria-Azotobacter vinelandii, Bacillus subtilis, Lactobacillus arabinosus and Serratia marcescens-189 f 38 mg. Growth rates on 19 species of fungi (Table 3) indicate there was no growth on 6 species. Among the remaining 13 species growth was least (2&30 mg) on Phanerochaete chrysosporium (Parent), Poria monticola, Sistotrema brinkmanii and Trichoderma koningii. Only 31% as much growth was produced by E. foetida (ignoring differences in initial weight) on 100 mg fungi as on 100 mg bacteria. The mean weight of E. fietida on the 19 fungal species was 70 f 16 mg. Growth rates for the three species of protozoa (Table 4) were about 20% faster than on bacteria,

Earthworm

493

nutrients

Table 2. Mean live weight (mg + SE) of Efoetidu after 8 weeks of feeding on cellulose, ashed loam and Iyophiiized bacteria. t wormperdish (N = 5 except where indicated)*

Bacteria

--

Gram stain affinity

Aerobucter aerogenes .&a&genes faecalis Atotobactcr vinelandii Bacillus cereus Bacillus sub&s Cfostridfum welchii Enterobacier sp. Escherichia cali Fiavobacrerium aquarile Klebs~ei~ap~e~on~e Lactobaciflus arabinosus Micrococcus

-

+ t “I-

+

luteus

+

Mycobacrerium smegmatis Noeardiu sp. Proteus vufgaris Proteus vulgaris Pseudomonas aeruginosa SulmonelJa typhimurium Serratia marcescens Shikclla sonnei Staphylococcus aureus Sfreptococcus faecalis _Yibrio comma

+ + -

+ +

XkSEof;ii’s

Ration of microbial biomass 100 mg 25 mg 50 mg 42 (8) z8(15) 74 (6)

1OI (13)

l53(38)

125 cwt 152 (22) 236 (IS)?

23.5 1Vf

18U11 @ (9)f so (3) 64 (9H 57(19H 14 (4) [email protected](I21 59 (9Q ss (23) 53 (18) 91(24)? 99 (5) 69(14) 9 f2It Il(llH 26(16)§ 42 (w Z(4) 54 (9H

K&(21)

69 (22)$ 262 (53) 165 (29) 99 @ff 173(38) 202(11) B(U 132 (3W 51 (w§ 75 (13H fz5(wt 93 (28H

245 (28) 298 (0% 173fi3) 26x (39)? 296 (37) 267 (22) 297 wt 200 (69) -is:, (42) 272 (67).I 394(14) 202 (76)? 200(22) 276 (27)t 350 (27) 149 (29)# 242 (29) Ip6 (60)$ I14 (35)t 224 (44)? z! (W

54f5)

l22(12)

233(14)

42(8)

tss(l?)f 130 (9) 187 (23) 176{19)? loa(17) @ (52)

*Underlined values significant at P < 0.05. TN=4. $N=3. $N= I.

Tabie 3. Mean live weight (mg f SE) of E. foe+& after 8 weeks of feeding on cellulose, ashed toam and lyophilized fungi. I wormper dish (N = 5 except where indicated)* ~-..~

-.

Fungi

Ration of microbial biomass 25 mg 50 mg 100 mg

~_

Agaricus bisporus Byssochlamys nivea Coriolus hirsutus Daeduiea quercina Geomyees asperuilatus Geotrichum candidum Monilia sp. Paecilomyces carneus Paecifomyces varioti Penicillium nigricans Penicillium viridicatum Phnnerochaete chrysosporium (Parent) Phaneruchaete ~hrysosporium (BKMC mutant) Phellillus pini Poria monricola Saecharomyces cerevisiae Sis&urema br~nkmun~i Trichoderma koningii rrichoderma longibrachiatum

26wt U (2) 70(14) 22 (6g 10(j) 8(l) 7 (21 14(4) Is (2) 9 (2)

14 (3) 20)

X&SE of x’s *Underscored tN=4. $N=3. $N=l.

$9 (81 19 (3) 162 (27) 60 (6) 9 (2) 17 (6) 7 (O)t 27 (5) x!(2) 18 (2)

I~(3

30 (14N 26(17) Is (4R

34 (8) 26 (2)t Z.(6) 501 S(l) lo (3)

If! (14x! 43 (8) 99 (6N 15(7) &(I) 27 (5)

18 (3)

39 (8)

61 (81 26 (W &!I (65) I&! (57) 12(3) 62 (26)$ 7(L) !?!(rw 66f5) I8(5)

79 (12H 23 (3) 47fll) 44Ul) 29 (3)t I7I(IS)F, 2f(7% 21. (2) 61(4)

values are significant at P < 0.05.

Table 4. Mean live weight (m8 k SE) of .E. foetida aRer 8 weeks of feeding on cellufose, ashed loam and ~yoph~z~ protozoa. I worm per dish (N = 5 except where indicated\*

Protozoa EugrerrO gracilis Ochromonas dmicu Tetrahymena pyrtyormis

X+SE

of x’s

~~““_

Ration of microbial biomass 25 mg 50 mg 100 mg 23 (6)

sit GW

334 (92ff

56(17)t

83(W

186Owt

33 (11) 37(10)

*Underscored values are significant at P c 0.05. TN=4.

82(12)

0~ (68)

sl(l)

274(45)

70(16)

FRANK M. FLACK and ROY HARTENSTEIN

494

but almost four times that on fungi. The mean weight of worms grown on the three protozoa was 274 f 45 mg. The minimum number of essential ingredients required for growth is given in Table 5. Worms did not grow on sand and microorganisms on ashed loam and microbes. By contrast growth or cellulose, microbes, and on sand or ashed loam, was similar and greatest. DISCUSSION The limits of growth of E. foetida on a wide range and combination of mineral salts in a substrate of cellulose upon grit were assessed. At tolerable ionic conductivities of less than about 8 mS cm-’ nitrogen had to be kept below the 1.2% concentration previously shown to be supportive of weight gain (Hartenstein, 1983). At higher concentrations of inorganic N the earthworms died. These observations, together with those of Neuhauser et al. (1980a), suggest that E. foetida requires its minerals and vitamins in the form of microbial biomass. Microorganisms were washed before testing them as food for E. foetida to avoid high ionic conductivity or putrefactive or fermentative metabolites (Neuhauser et al., 1980a), and to remove microbial metabolic wastes (Kaplan et al., 1980). Since growth on Gram-negative bacteria was similar to that on Gram-positive bacteria, E. foetida can apparently decompose bacterial cell walls with a wide variety of polysaccharides. Similarly, E. foetida appears able to destroy human pathogens as well as non-pathogenic microbes. Ranking microorganisms as nutrients places the bacteria on a par with the protozoa, and both of these superior to fungi as a group. Earthworms contain large amounts of N (Abe et al., 1979; Hartenstein et al., 1980), and although bacteria may provide more than twice as much N as fungi (Long, 1961), E. foetida grew three times as fast on a bacterial diet. A gain of more than 300 mg live weight on the fungus C. hirsutus compared with a higher weight than this

on only 2 of the 22 bacteria, suggests that factors other than N content alone were involved. Each of the three protozoa was inferior to the two best bacteria as nutrients for E. foetida. This suggests that factors other than presence or absence of protozoa can be invoked to account for the differences in development of E. foetida in the study reported by Miles (1963). Table 5. Live weight of E. foetida after 8 weeks on 100 mg lyophilized Coriolus hirsutus in relation to presence or absence of cellulose, ashed loam and sand. 1 worm per dish (N = 5 except where indicated)* Variable Cellulose alone Sand alone Ashed loam alone Cellulose and sand Cellulose and ashed loam

_ Weight X mg (SE) 134 (22) 13 (4)t Dead 249(20) 278(11)

*Lack of significance (P > 0.05) between variables is denoted by underscoring. tN=2.

Greater growth rates may have resulted with larger concentrations of these microbes, but use of 200 mg or more of lyophilized bacteria in previous work increased earthworm mortality. Assuming a proportion of 10% N in the bacteria, the final concentration in the ingesta tested was about 0.8% N on a dry weight basis. This value lies below the value of 1.2% N in mixtures of activated sludge and cellulose on which E. foetida grew as rapidly as on mixtures with higher levels of activated sludge (Hartenstein, 1983). Previously obtained data suggest that E. foetida requires microorganisms, in contrast to acellular soluble nutrients, for growth (Neuhauser et al., 1980a). This is confirmed in the present study, since washed microorganisms, in contrast to unwashed microbes, did not cause earthworm mortality, and allowed the earthworms to grow faster. The data of this study also suggest, but do not prove, that E. foetida requires cellulose in its diet. Nonetheless, in an unpublished study 14Cwas present in the tissues of E. foetida and Lumbricus terrestris Linnaeus after transferring these earthworms daily for 7 days from a 3-week retention period in 2.5: 1 “C-cellulose: H,O to fresh 2.5 : 1 cellulose: H,O, followed by gut voidance for 3 days as described by Hartenstein et al. (198 1) and rinsing in water until the used water was free of radioactivity. Also, earthworms are a rich source of cellulase (Hartenstein, 1982). Finally, the data suggest that grit is necessary if optimal growth is to be achieved. Sand and ashed loam were equally effective. Acknowledgement-This research was supported National Science Foundation.

by the

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Earthworm nutrients Hartenstein R. and Hartenstein F. (1981) Physicochemical changes effected in activated sludge by the earthworm Eiseniu foe&a.

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Khambata S. R. and Bhatt J. V. (1957) A contribution to the study of the intestinal microflora of Indian earthworms. Archives Mikrobiology 28, 69-80. Kozlovskaya L. S. (1969) Der Einfluss der Exkremente von Regenwurmen auf dieAktivierung der Mikrobiellen Prozesse in Torfboden. Pedob~o~ogia 9, 158-164.

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