1924) and Vibrio amyloceua (Gray, 1939). Neither the Cellulomonas species nor. cellulose is only one constituent.

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1 CELLULOSE DECOMPOSITION BY ;AEROBIC MESOPHILIC BACTERIA FROM SOIL I. ISOLATION AND DESCRIPTION OF ORGANISMS' W. H. FULLER AND A. G. NORMAN2 Iowa Agricultural Experiment Station, Ames Received for publication April 12, 1943 A large amount of cellulose finds its way into the soil as the chief component of crop residues, and of natural vegetation. Under normal conditions of temperature and moisture this cellulose disappears almost completely and quite rapidly. There must exist in ordinary soils a vigorous aerobic mesophilic'population e,apable of utilizing cellulose. It has been claimed that many' fungi are particularly active in this respect, and although a heavy development of fungi,may frequently be observed shortly after the addition of cellulosic materials, they do not usually appear to remain domit for long. There seems to be no good reason for underestimating the importance of the aerobic bacteria in this process. Descriptions have been published of a considerable number of aerobic celluloseorganisms, but the information about many of them is scanty. In most cases little biochemical work was carried out, and the cultures were not maintained. To a considerable extent attention has been centered on certain organisms such as Cytophaga hutchinoonii that appeared to be specific in the utilization of cellulose. Almost all the aerobic mesophilic cellulose bacteria so far described have been placed in four genera though their classification is far from satisfactory. In the fifth edition of Bergey (1939), twenty-seven species of the genus Celulomonas are listed, four species of the genus Celvibrio, three species of a highly-questionable genus, CeUfalcicula, and, in an appendix to the Spirochaetales, five species of the genus Cytophaga. In addition there are a few other cellulose organisms in sundry other genera. This listing is hardly representative of the forms found in soil. Short curved vibrios are common. Out of 17 new species described by Kalnins (1930), 12 were curved rods of the genus Vibrio, 4 belonged to the genus Bacterium and one to the genus Bacillu8. Other vibrios have been described, such as Vibrio agarliquefaceno(microspira agarliquefaciens, Gray and Chalmers, 1924) and Vibrio amyloceua (Gray, 1939). Neither the Cellulomonas species nor the Vibrio species are in any sense specialized; all use a wide range of carbon and nitrogen sources though not necessarily vigorously. Unspecialized soil cytophagas have also been found (Fuller and Norman, 1943). It seems likely, therefore, that the aerobic cellulose organisms in soil are predominantly versatile organisms that individually may not be very vigorous on cellulose but which are likely to be able to maintain themselves in a heterogenous soil population that is normally utilizing not pure cellulose alone but a complex substrate of which cellulose is only one constituent. I Journal paper J-1108 of the Iowa Agricultural Experiment Station, Ames, Project Research Associate, and Research Professor of Soils, respectively. 273

2 274 W. H. FULLER AND A. G. NORMAN The purpose of the work described in these papers was to isolate from soil representative aerobic cellulose bacteria, and to determine their activity quantitatively, not only on filter paper (the standard form of cellulose commonly used as the criterion of cellulose decomposition), but also on cellulose preparations from plant materials, in the absence and presence of other cell-wall constituents. ISOLATION OF ORGANISMS Isolations were made from top soil sample of four soils: Clarion silt loam (Prairie), Ames fine sandy loam (Planosol, forest-derived), Fayette silt loam (Gray-Brown podzolic) all from Iowa, and Palouse silt loam (Chernozem) from Washington. The organisms described below were selected for detailed study because of their initial vigor in attacking cellulose and the apparent stability of this property over a period of several months. Other forms were obtained which have not yet been fully examined. No consistent procedure was followed in isolation and purification. Cellulose dextrin agar described by Fuller and Norman (1942) was found to be extremely helpful. Some of the isolations were made by plating soil suspensions directly on this medium. Others were taken from plain agar plates covered by filter paper moistened with Dubos' solution (1928), transferred to cellulose dextrin agar for purification, tested for cellulose-decomposing ability on filter paper strips partially immersed in Dubos solution, and carried either on cellulose, or, in a few cases, on starch. Purification of some of the forms was a matter of difficulty and was only accomplished by repeated dilution and plating on cellulose dextrin agar, the colonies being picked at an early stage. The fact that most colonies of cellulose-decomposers on this medium are surrounded by a halo was of considerable assistance. DESCRIPTION OF ORGANISMS The following appear to be new species: Pseudomonas ephemerocyanea n. sp. Ephemerocyanea is from the Greek ephemeros and cyaneos meaning "shortlived blue." Morphology. Vegetative cells: Straight to slightly bent rods with rounded ends, x microns, arranged singly. Spores: Absent. Motility: Present, one to three polar flagella. Staining: Gram negative. Cultural characteristics. Gelatin stab: Growth and liquefaction. Starch agar slant: Heavy, gelatinous, tan to light brown growth. The color deepens to brown in old cultures. Litmus milk: No visible growth. Indole: Negative. Nitrite formation: Nitrate is reduced to nitrite. Diastase: Positive. Carbohydrates: Glucose, lactose, maltose, galactose, arabinose, and to a lesser extent, xylose, are rapidly attacked. Polysaccharides: Cellulose, cellulosan, water-insoluble and water-soluble cellulose dextrins and pectin are readily utilized. Gum arabic and calcium gluconate are only slowly utilized. Filter paper strips: Strips of filter paper in mineral nutrient medium may be disintegrated at the

3 CELLULOSE DECOMPOSITION BY BACTERIA FROM SOIL 275 surface of the solution in 24 hours; however, this usually takes 36 hours. Transfers made from thirty-day-old starch agar slants fail to cause fragmentation of the cellulose strips until 4 or more days. The disintegrated region at the liquid-air interface is soft and pulpy and turns light brown after an initial short period during which it may be violet or blue in color. The cellulose below the surface is only slowly decomposed, becoming light brown and pulpy in about 10 to 15 days. The solution becomes turbid as the cellulose is attacked. Nitrogen: Peptone, yeast, nitrate, and ammonia are used. Oxygen: Highly aerobic. Temperature: 22-35oC. Habitat: Soil. Colony characteristics. Starch: Pin-point colonies appear on starch agar medium in 3 days. In 5 days, surface colonies, 1 to 2 mm. in diameter, have a chalkwhite color that soon takes on a tan cast. The raised colony has a glistening luster, is smooth, and has an edge that is smooth and entire. Subsurface colonies are small and angular. After 20 days the surface colonies are 3 to 4 mm. in diameter. Water-insoluble dextrin: Colonies on water-insoluble dextrin agar appear in 4 or 5 days, and are pin-point in size. Immediately upon the appearance of a colony a distinct enzymatic zone extending 2 to 1j mm. away from the edge of the colony may be seen. The zone grows as the colony grows. In 7 days the chalk-white colony, that has a convex elevation, is smooth edged and about 1 mm. or less in diameter. After 20 days the colony still looks much the same, but generally grows to a size of about 2 to 3 mm. Pseudomonas lasia n. sp. Lasia is from the Greek lasios, woolly or shaggy. Morphology. Vegetative cells: Short, slender rods with rounded ends, x microns. Generally singly; sometimes in chains. Spores: Absent. Motility: Present. Singly polar flagellum. Staining: Gram negative. Cultural characteristics. Gelatin stab: Thin growth occurs but no liquefaction. Starch agar slant: White growth turning to pale yellow in old cultures. Litmus milk: Reduction at the bottom of the tube but no peptization, curd, or change in reaction. Indole: Negative. Nitrate: Nitrate is reduced to nitrite. Diastase: Positive. Carbohydrates: Glucose, xylose, maltose, and starch are readily utilized. Arabinose and galactose and gum arabic are feebly attacked. No acid is formed. Polysaccharides: Cellulose, cellulosan, water-insoluble and watersoluble cellulose dextrins, hemicellulose, and pectin are readily attacked. Acid is not formed. Filter paper strips: A very decided clouding of the solution, is noticed in about 6 days and at the end of 7 days the filter paper becomes limp and rubber-like. Slight shaking causes partial disintegration of the paper at the surface of the liquid. Pigment usually is not produced in 7-day-old cultures. The paper strip swells, becomes light cream to pale yellow in color, and very flexible in about 12 days. Slight shaking causes the cellulose to disintegrate into a pulpy, shredded mass. Nitrogen: Peptone, yeast, nitrate and ammonia are used. Oxygen: Aerobic. Temperature: C. Habitat: Soil. Colony characteristics. Starch: Colonies growing on starch agar appear in 3 to 4 days, are 1-2 mm. in size, and convex in elevation. They are ivory to pale

4 276 W. H. FULLER AND A. G. NORMAN yellow in color, have an entire and smooth edge, and are round in shape with a flat surface. Subsurface colonies are slightly irregularly round and look like small woolly balls because of their loose surface. As the colonies become older, they change to a cream color. Water-insoluble dextrin: Chalk-white subsurface colonies about 1 mm. in diameter surrounded by cleared zones appear in 4-5 days. In 10 days the colonies become light cream to pale yellow in color and are about 2 mm. in diameter. The colonies do not spread on the surface, but prefer to grow down into the medium forming irregularly round shapes of loose woolly appearance. Colonies near the surface are convex. Pseudomonas erythra n. sp. Erythra is from the Greek erythros meaning "reddish." Morphology. Vegetative cell: Short rods with rounded ends, x microns, usually arranged singly. Spores: Absent. Motility: Present. Single polar flagellum. Capaiation: Present. Staining: Gram negative. Cultural characteristics. Gelatin 8tab: No growth. Litmus milk: No growth. Indole: Negative. Nitrate: Negative. Diastase: Negative. Carbohydrates: No growth. Polysaccharides: Cellulose and water-insoluble cellulose dextrins are used, the latter to a lesser degree than the former. Nitrogen: Yeast and nitrate are used. Oxygen: Highly aerobic. Temperature: 22-35C. Habitat: Soil. Colony characteristics. Starch: Starch does not support growth. Waterinsoluble dextrin: Cellulose dextrin supports growth only feebly. The colonies that appear after 8 to 10 days of incubation all grow beneath the surface of the medium. They are irregular or angular in shape, usually less than 1 mm. in size, and are surrounded by a clear zone 2 to 5 mm. in diameter. The colonies are buff or red-brown in color. No matter how heavily the medium is seeded, only a few colonies appear. Filter paper strips: Cellulose shows signs of being attacked in 4 to 5 days by the appearance of red-brown spots on the paper above the surface of the liquid and the solution becomes cloudy. The brown spotted areas enlarge, become viscous on the surface and the paper changes to a tough membrane with a reddish hue. After ten days incubation, the area involved extends 2-5 mm. upward from the level of the liquid. As decomposition progresses, the filter paper becomes pale brown, thin, tough and flexible. The cellulose does not break apart with moderate shaking but may be wound up in a slimy string. Achromobacter picrum n. sp. Picrum is from the Grelek, pikros meaning "sour", "bitter." Morphology. Vegetative cells: Short, straight rods with rounded ends, x microns, arranged singly. Spores: Absent. Motility: Absent. Staining: Gram negative. Cultural characteristics. Gelatin stab: Growth and liquefaction. Starch agar slant: White growth. Litmus milk: No growth. Indole: Negative. Nitrate: Nitrate is reduced to nitrite. Diastase: Positive. Carbohydrates: Glucose and starch are vigorously decomposed producing acid and no gas. Lactose, maltose,

5 CELLULOSE DECOMPOSITION BY BACTERIA FROM SOIL 277 galactose, arabinose and xylose are more slowly utilized. Poly8accharides: Cellulose, cellulosan, water-insoluble and water-soluble cellulose dextrins, hemicellulose, and pectin are utilized producing acid. The volatile acid is acetic and non-volatile acid is.lactic. Filter paper strips: The greatest attack on filter paper is at the air-liquid interface, where, after a period of about 7 days, it becomes pulpy and falls apart on gently shaking. Cellulose below the k&rfac6 of the liq.- uid swells and loses some of its original rigidity. -Pigment iq.-ot noticeable. Nitrogen: Peptone, yeast, nitrate, and ammonia are-,usd. :Oxygen: Aerobic. Temperature: C. Habitat: Soil. Colony characteristics. Starch: Colonies that appear on starch agar in 3 or 4 days are smooth, glistening, and have an entire edge. A "rough" strain with somewhat irregular margin has. also been isolated.. In 5 or 6 days the colonies measure 2-3 mm. in diameter and may turn a pale yellow color. Subsurface colonies are disc-shaped. Water-insoluble dextrinr: Very small colonies appear on water-insoluble cellulose dextrin in 5 or 6 days. In 9 days the colonies appear chalk-white, convex, and round with an entire edge. Submerged colonies are disc-shaped. Very distinct halos extend 2-5 mm. from the edge of the colonies. The latter usually measure 2 mm. in diameter. BaciUus aporrhoeus n. sp. Aporrhoeus is from the Greek, aporrhoe, meaning flowing outwaids. Morphology. Vegetative ceus: Slightly curved rods with rounded ends, x miarons, arranged singly. Spores: Present. Ellipsoid endospores are x microns, terminal to sub-terminal. Sporanpa generally swollen terminally. Motility: Present, by peritrichous fagella. Staining: Gram positive. Cultural characteristics. Gelatin stab: No growth or liquefaction. Starch agar slant: Heavy, slimy, white to transparent growth Litmus milk: No visible growth. Indole: Negative. Nitrate: Nitrates are reduced to nitrites. Diastase: Positive. Carbohydrates: Glucose, maltose, galactose, arabinose, xylose are utilized. Lactose is not attacked. Polysaccharides: Cellulose, cellulosan, water-insoluble and water-soluble cellulose dextrins, hemicellulose, and pectin are utilized. Filter paper 8trips: Cellulose below -the surface of the liquid becomes very limp, pulpy, and slightly swollen after about 7 days of incubation. The paper strips arebroken in two only after slight shaking. The most extensive attack occurs at the surface of the liquid, where a pale yellowing is sometimes noticeable. The solution is never more than slightly turbid. Nitrogen: Peptone, yeast, nitrate, and ammonia are used. Oxygen: Aerobic. Temperature: Optimum C. Habitat: Soil. Colony characteristics. Starch: Colonies appear in 3 or 4 days. They are irregularly raised, gray-white in color, and semi-transparent. Growth is viscid and very gummy in consistency. Colonies vary from 2 to 6 mm. in diameter. Colonies often extend themselves by moving about on the surface of the agar medium and piling up, giving a windrow effect. A portion of the colony may move in a thin line away from the main body at the rate' of 3 mm. in 2 hours.

6 278 W. H. FULLER AND A. G. NORMAN Not all portions of the same colony or neighboring colonies move as vigorously as this, however. The movement may be such that a hooked or whorled effect is produced at the margin. Movement seems to occur more frequently on glucose agar than on starch. Not all the colonies move. Water-insoluble dextrin: Small, pin-point colones are surrounded by a pronounced enzymatic zone and appear in 5 or 6 days. Ten-day-old colonies appear 1 to 2 mm. in diameter, convex in elevation, cloudy-white in color, and often irregular in shape. Growth is slow, but the enzymatic zone is pronounced, generally extending 2 to 3 mm. from the edge of the colony. Colony movement is infrequent. DISCUSSION OF ORGANISMS The taxonomy of the cellulose-decomposing bacteria has been confused by the policy of creating for these organisms special genera within the families to which they belong on morphological grounds. A physiological property has no place in a genus description unless the characteristic in question is obligate or so outstanding as to outweigh most other considerations. Almost all of the cellulose bacteria are versatile organisms, capable of utilizing other polysaccharides and carbohydrates to various degrees. In most cases the cellulose-decomposing ability does not outweigh the other characteristics. This property, therefore, is best relegated to the key, where it may well be conveniently used for separating species within the genus as morphologically described. The only exception to this might be the case of specific cellulose organisms. A few species which develop poorly on substrates other than cellulose are known but until their physiology has been more fully studied, it is probably unwise even to set these apart from oth-er organisms closely related morphologically. In view of the considerations above, the five new species described in this paper have been assigned to existing genera on morphological grounds. Four out of the five are versatile organisms; one has only been cultured on cellulose and cellulose-dextrin. Three have been placed in the genus P8eudomonas. Some curved cells were usually present in cultures of each of these three species, but the proportion of curved cells did not justify inclusion in the genus Vibrio. Pigment production occurred to some extent in each of the three cultures; P. ephemerocyanea, as the name implies, produces an intense blue to blue violet color which is quite shortlived. When developing on filter paper strips, the colotation is somewhat localized to the area of attack at and immediately above the surface of the liquid. In aerated cellulose suspensions, the whole medium becomes blue. The pigment is not confined to the fibers and appears to be water-soluble. It is transitory, however, and after only a few hours fades slowly to a tan or light brown color. The ephemeral blue pigment has not been observed to be produced from any substrate other than cellulose but colonies on other carbohydrates on plates become light brown with age. A reddish pigment, also water-soluble, is produced by P. erythra on cellulose. The color is not intense and is most readily seen in filter paper strip cultures. By capillary action the pigment is carried up the filter paper until the whole portion exposed has a reddish hue. The area of

CELLULOSE DECOMPOSITION BY BACTERIA FROM SOIL 279 most vigorous attack may be pale brown. Colonies on cellulose dextrin agar are buff to red brown, but the color is limited to the colony itself.

7 CELLULOSE DECOMPOSITION BY BACTERIA FROM SOIL 279 most vigorous attack may be pale brown. Colonies on cellulose dextrin agar are buff to red brown, but the color is limited to the colony itself. Pigment production is less evident in the third species of this genus, P. lasia. On filter paper strips the area attacked slowly becomes pale yellowish and colonies on cellulose dextrin and other substrates also assume this color as they age. It can hardly be claimed, however, that there is clear evidence of the formation of a watersoluble pigment in this case..% I' GI I \OILE C(OLONIE OFB13ACJILLI APRIOiliEJU- Pseudomonas erythra, unlike the othei twxo species of this genus here described, seems limited to the utilization of cellulose. Onily feeble growth is supported by cellulose dextrin, and no growth is obtained on starch or simple sugars. This organism is considerably less vigorous in its development on filter paper than the specialized cytophagas, several strains of which were isolated and studied in parallel with these new cultures. Two strains of Achromobacter picrum were isolated, differing only slightly in colony appearance. In its fully aerobic character, this organism differs from most species of Achromobacter, the great majority of which are facultative. This organism is unusual in producing acids from cellulose aerobically. Acids are similarly given from many other carbohydrate substrates.

8 280 W. H. FULLER AND A. G. NORMAN Aerobic spore-forming rods are rarely seen in soil unless some recent addition of plant materials has occurred. Even so, very few bacilli have been found to have the property of utilizing cellulose. One species, Bacillus latvianus, was described by Kalnins (1930), and two others, Bacillus (Cellulobacillus) myxogenes and Bacillus (Cellulobacillus) mucosus were studied in detail by Simola (1931). Bacillus palymyxa, B. macerans and B. amylolyticus have been reported to have feeble cellulose-decomposing powers though they are not ordinarily regarded as cellulose bacteria. Although its growth on filter paper is not as vigorous as some organisms, the cellulose-decomposing ability of B. aporrhoeus, here described, is unquestionable. It has in addition the extremely interesting and unusual property of giving motile or "amoeboid" colonies (fig. 1). Movement is frequently rapid but does not seem to occur on all media. Three other organisms of this genus, B. circulans, B. sphaericus, and B. alvei have been observed to behave similarly, but none of these utilizes cellulose. Inasmuch as colony motility is so distinctive a property this was made use of in arriving at the species name. SUMMARY Five new species of aerobic cellulose-decomposing bacteria are described. Three are species of Pseudomonas (P. ephemerocyanea, P. lasia, and P. erythra), one of Achromobacter (A. picrum), and one of Bacillus (B. aporrhoeus). With the exception of P. erythra all are versatile organisms capable of growing well on many carbohydrates. A. picrutm alone produces acid from cellulose and sugars. B. aporrhoeus gives motile colonies on starch and glucose-agar. ACKINOWLEDGMENT We are much indebted to Dr. R. E. Buchanan for assistance in the nomenclature of these organisms. REFERENCES BERGEY, D. H., BREED, R. S., MURRAY, E. G. D., AND HITCHENS, A. P Bergey's manual of determinative bacteriology. 5th ed., Baltimore. DUBOS, R. J The decomposition of cellulose by aerobic bacteria. J. Bact., 15, FULLER, W. H., AND NORMAN, A. G A cellulose-dextrin medium for identifying cellulose organisms in soil. Soil Science Society of America, Proceedings, 7, 243-6, FULLER, W. H., AND NORMAN, A. G Observations on some soil cytophagas. J. Bact., 45, GRAY, P. H. H Vibrio amylocella, n. sp., a soil organism that decomposes cellulose and produces glucose from starch. Can. J. Research, C. 17, GRAY, P. H. H., AND CHALMERS, C. H On the stimulating action of certain organic compounds on cellulose decomposition by means of a new aerobic micro-organism that attacks both cellulose and agar. Ann. Applied Biol., 11, KALNINS, A. Aerobic soil bacteria that decompose cellulose. Acta Univ. Latviensis Lauksaim-niecibas Fakultat (Ser. 1), 11, , SIMOLA, P. E. Uber den abbau der cellulose durch mikroorganismen. I. Zur morphologie und physiologie der aeroben sporenbildenen cellulose-bakterien. Ann. Acad. Sci. Fennicae (A), 34, No. 1,

ING MOBILE COLONIES ON THE SURFACE

A NEW SPECIES OF THE GENUS BACILLUS EXHIBIT- ING MOBILE COLONIES ON THE SURFACE OF NUTRIENT AGAR Department of Botany and Bacteriology, The Univer8ity of Texa8 Received for publication, July 23, 1934 During