Allen Paul W. Industrial Fermentations

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THE MICROBIOLOGY OF THE SOIL 415

gen, and the gradual liberation of phosphorus, potassium, calcium,

magnesium, sulphur and iron from minerals normally abundant in

average soils.

Nitrogen is accumulated in the soil by two methods, non-symbiotic

nitrogen fixation and symbiotic fixation. The former may be of

greater importance than we at present believe but the latter is of

great importance in soil systems of permanent soil fertility. Liebig in

1840,

advanoed the theory that plants obtain their nitrogen by the

assimilation of the NH

of the air: but this view was found to be in-

correct by Laws and Gilbert of Rothamstead. They showed that

elementary nitrogen cannot be assimilated by plants and that there

is not enough ammonia in the air to go very far in fulfilling the nitrogen

needs of plant. Berthelot showed that only plants grown in unsterilized

soils are able to make nitrogen gains.

In 1893, Winogradsky demonstrated that certain anaerobic butyric

acid bacteria as Bacillus chlostridium pasteurianum are able to form

nitrogenous compounds' from the elementary nitrogen of the air.

In 1901, Beyerinckr showed that there is a large group of aerobic

bacteria which are able to fix atmospheric nitrogen. He called this

group of bacteria azotobacter. It has been enlarged by Lipman,

Lohnis, and Westennann to contain the following members:

Azotobacter Vinelandii... ]

Azotobacter Beyerinoki...

Lipman

Azotobacter Woodstownii. J

Azotobaoter Vitreum Lohnis and Wefltennann

Other organisms which have been reported as oapable of fixing a

small amount of atmospheric nitrogen are Radiobacter, Bacillus radi-

cicola, B. pyocyaneus, B. pneumonia, B. lactis viscorum, B. prodi-

giosus, B. ruminatus, B. simplex, and B. mesentericus.

It is reported that the salts of iron and that approximately neutral

reaction are important factors in the development of the azotobacter

group. Some have estimated that members of the azotobacter group

may fix from 15 to 60 lbs. of nitrogen per acre annually.

The existence of alga in soil is supposed to be of benefit to azo-

tobacter and some believe that azotobncter and algse often live in a

sort of a symbiotic fashion. The algae are said to produce mannite

sugars' from cellulose material thereby furnishing azotobacter with a

proper carbohydrate.

The amount of nitrogen fixed by the activity of azotobacter

generally is not great as it is estimated that azotobacter requires

100 lbs. of carbohydrate for each pound of nitrogen fixed.

Gainey says that soils more acid than pH6 do not usually contain

azotobacter. He says, "A definite and very close correlation was

established between the absolute reaction of the soil and the presence

416 INDUSTRIAL FERMENTATIONS

or absence of azotobacter in the soil." He further says that since a

very limiting H ion concentration was found for pure cultures of azo-

tobaoter in laboratory media, it is believed that the very close correla-

tion existing between the reaction and the presence of azotobacter

indicated that the absolute reaction is of paramount importance if

not the actual limiting factor in controlling the presence of this group

of organisms' in soils.

Symbiotic Nitrogen Fixation.

In ancient Roman times acoording to Columella, fanners con-

sidered that the growing of beans, alfalfa, lupines and vetches improved

the soil and acted as manure. However more definite knowledge was

not obtained until 1888 when Hellriegel and Wilfarth established

the fact that certain plants and bacteria work together in the fixing

of atmospheric nitrogen in the soil. Legumes were found to fix nitro-

gen by a symbiotic activity between the plant root and a bacillus

which was called B. radicicola, the symbiosis resulting in tubercle

formation on the plant roots. These tubercles had first been observed

by Malpighi in 1687, who erroneously thought they were pathological

processes. Marshall Ward studied these root tubercles and discovered

that they were due to outside infection of the root, but it was left for

Hellriegel and Wilfarth to interpret their true importance as atmos-

pheric nitrogen fixers.

C. V. Piper in explaining how muoh nitrogen legumes may add to

the soil by the practice of green manuring says:

"Attempts have been made to determine the quantities of nitrogen

fixe4 by the action of the nodule bacteria, but no method has yet

been found that will give more than an approximately correct answer.

Analyses have been made of the quantities of nitrogen in various

leguminous crops, both tops and roots, but the total nitrogen thus

obtained includes some that was

taken directly from the soil. De-

terminations have been made of the total nitrogen in soils before and

after growing a leguminous crop, but the result represents merely the

net difference between gain and loss resulting from the interplay of a

number of factors, and even the net gain cannot be safely credited to

the legume alone. Lastly, oertain legumes have been grown with and

without inoculation and the difference in the quantities of nitrogen in

the crops determined.

"The Michigan Agricultural Experiment Station grew soy beans

inoculating the soil on one field and leaving another uninoculated.

Samples taken and analyzed showed that about two-thirds of the

total nitrogen was taken from the soil and one-third from the air. The

Illinois Agricultural Experiment Station grew alfalfa, both in pots

and in field plats, some inoculated, others not. On the inoculated

plate the alfalfa in the crops for a season contained nearly 160 pounds

more nitrogen per acre than the crop from the uninoculated plats.

"At the Central Experimental Farm at Ottawa, experiments were

THE MICROBIOLOGY OF THE SOIL 417

;onduoted with red clover by growing this crop year after year on

specially prepared plats of sandy soil with a known nitrogen content.

£ll the clover grown was turned back to the soil during nine years,

^t the end of this time the soil contained 472 pounds of nitrogen per

icre more than at the beginning. As a result of a similar experiment

carried on by the New Jersey Agricultural Experiment Station, the

3onclusion seems warranted that in this case the legumes took from

:he air and made available to the grain crops at least 64 pounds of

litrogen per acre each year.

"The Rhode Island Agricultural Experiment Station found that

after growing legumes in pots for five years, removing the above-

ground portions of the summer cropB but turning under the hairy

vetch grown in winter, there was a gain at the rate of a ton of nitrogen

oer acre, seven-tenths of which was removed in the crops. Many more

axamples might be given.

"On the other hand, workers in Kansas and in Wisconsin ooncluded

that alfalfa took more nitrogen from the soil than from the air and

that when the hay was removed there was a loss of nitrogen. In

general, however, it has been accepted as an approximation to the

truth that legumes get about two-thirds of their nitrogen from the

air and one-third from the soil. If these orops are turned under,

the soil will be richer in nitrogen; if they are removed, there will be

in general little or no change in the nitrogen content of the soil so far

as this is influenced by the fixation of nitrogen in the nodules and by

the removal of ^the crop."

The inoculation of soils with B. radicicola may be accomplished

in several different ways but the inooulation of the seed by some

method before planting is perhaps most efficient. Commercial prepa-

rations for broadcasting over the field to be inoculated have been

used considerably but are not considered very efficient. The transfer

of inoculated soil from one field to another is quite successful but

undesirable soil infection often results from the practice of this

method.

radicicola was

at first cultivated in the laboratory on a medium

made of hard wood ashes, saccharose or maltose and agar devised by

Harrison. The organism will remain alive for several years on media.

radicicola enters the plant through the root hairs or by way of

a bruise. The bacteria produced in the nodule are said to be absorbed

bodily by the plant tissue. With them the nitrogen whioh they have

fixed is also assimilated by the plant. When the nodule becomes

inactive it softens and disappears. As to how the plant utilizes the

nitrogen fixed by the bacteria there are three theories:

(a) Winogradsky advanced the theory that the free (N) and

nascent (H) form ammonia within the organism. (Reduction Theory.)

(b) Gerlack and Vogel believe the nitrogen is not reduced but is

oxidized. (Oxidation Theory.)

pounds. (Direct Union Theory.)

418

INDUSTRIAL FERMENTATIONS

It was.reported at N. J. Agr. Exp, Station that 200 lbs. of nitrogen

per acre was., fixed by B, radicicola in one year.

The conservation of the fertility of the soil is principally the con-

servation of nitrogen. Whatever the form in which nitrogen exists in

soil it is moving in the nitrogen cycle. Organic nitrogen is mainly

in the form of protein or protein derivatives of animal or vegetable

products. Nitrogen in protein exists in five different forms as:

1 amino nitrogen

2 diaminp nitrogen

3 ammonia nitrogen

4 guanidin nitrogen

5 pigment forms

Whatever the form of the organic nitrogen product it is attacked

in normal soil by bacteria.

Some of the organisms which split protein to ammonia are B. sub-

tilis,

B. mesenterious, B. mycoides, B. fluorescens liquefaciens, B.

fluorescens putidus, Saroena lutea, B. arboreacens, B. proteus vulgatus,

pyocyaneue, B. panthimiB. All the above organisms are strong

aerobee with the exception of B. proteus and B. fluorescens putidus.

In the breaking down of protein in soil the arnmonifiers carry the

simplicationof the protein molecule to a point where the main de-

rivatives are hydioxy aeids and ammonia. Other products are formed

in a lesser degree.

mycoides according to Kruse oonverte 46% of the nitrogen in

protein into ammonia while B. pyocyaneus converts 70% of the nitro-

THD NITROGEN CTCLK PBOM PEOTEN TO PBOTEIN (A POSSIBLE CYCLE). KIIUBE.

Trypsiiiand similar enzymes (alkaline proteinase)

| Pepsin and similar enaymes (acid proteinaae)

Complex Protein Molecule

Amino

acid

Proteoses

Ami-no

acid

Peptones

Amino

acid

Polypeptlds

Amino

acid

Peptida

Amino

acid

Amino

acids

Erepsin and similar enzymes

•HTDHOLTTIO CHAN0E8

THE MICROBIOLOGY OF THE SOIL 419

gen in protein into ammonia. No foul gases are formed in the aerobic

decomposition of protein.

The secret of the conservation of the nitrogen iii the soil is in the

annual nitrogen return, the prevention of the denitrification and of the

leeching away of the nitrates which are readily soluble in water. It

is found that the greater the amount of humus' in the soil the less the

nitrate loss due to leaching. It appears that the humus material holds

the nitrates by absorption or some kind of linking.

The amino acids anaerobically break up into hydroxy acids and

.CHNH

.COOH = CeH

.CH

COOH + NH

according to Kruse.

phenyl alanin phenyl propionio acid

The amino acids aerobically break up into simple products and

CHuNOa + 150 = 6CO

+ NH, + 5H

O according to Kruse.

leuoine

is absorbed by soil moisture as NH

H NH*0H.

Much CO

produced at the same time is absorbed as C0

-j- H

O =

HjCO^NH^OH + H

= 2H

0 + (NH

)

CaC0

Nitrosomonaa oxidizes (NHJ

+ 60 = 2HNO

+ H

Nitrosomonas is the only organism which can cause this re-

action.

The strong acidity produced by nitrous acid must be neutralized if

nitrification is to proceed as:

(PO

)

+ 2HN0

= CaACPO*), + Ca(NO

)

(PO<)

+ 4HN0

= CaH*(P0

)a + 2Ca(N0

)

The oxidation of nitric to nitrate is caused by nitrobacter as:

Ca(NO

)

+ 2O = Ca(NO

)

The calcium nitrate is utilized by plants and by them made into

proteins again.

Denitrification.

In some very rich soils as those used for truck farming and garden-

ing, there is considerable loss of nitrogen due to the activities of B.

denitrificans. There are also twenty-eight different soil bacteria which

are able to reduce nitrate to nitrite. Other forms reduce only nitrites.

The biological reactions taking place in extremely rich soils may be

grouped as follows:

Reduction of N0

to N0

or NO

Reduction of NO

to TtfH

Reduction of N0

to N

Liberation of N from organic compounds.

420 INDUSTRIAL FERMENTATIONS

Carbohydrates as starch, sugars and other residues of crops may

furnish the carbon for these reductions.

Denitrification in ordinary soils is not extensive and causes very

little loss of nitrogen but in heavily manured and wnter logged soils

it may be an important faotor.

Phosphorus.

The slow but gradual liberation of phosphorus, potassium, sulphur

and other essential mineral elements of plant growth is quite satisfac-

tory in average soils. While no phosphorus fermenting organism has

ever been found still Stoklaaa found that P

in phosphate was made

soluble by bacteria as follows:

Unmodulated 3.8%

B.myoaides 23 %

megatherium 21 %

B.coli 20 %

meeentericus 15 %

B.butyrioua 15 %

B.proteus 14 %

B.fluorescens 9.2%

It was found that acetic acid and formic acid are better solvents

of phosphates than carbonic acid. Lipman thinks that sulphur makes

phosphorus in phosphates available.

Potassium.

Some of the sources of potassium in soils are

Orthoclase

Feldspar (muscovite)

Mica (biotite)

Peroxines

Labradorite

There is evidence that potassium is made available to plants by

the action of bacteria.

Soluble potassium may be made available to plants by the action

of biological end products upon .the zeolrtjic compounds of the soil.

Maclntire has shown, however, that the economic additions of calcic

and inorganic materials functioj/ to protect native soil potassium and

give a repressive rather than a liberative chemical effect.

^ According to Kruse, urea is an important constituent of manured

soilB.

Urea C0(NH

)

the chief nitrogenous constituent of urine and

the principal end point of tissue metamorphosis is attacked by the urea

organisms and converted into ammonia and CO

as:

C0(NH

)

+ H

O = CO

+ 2NH

(Hydrolysis (enzyme)

(urease)

THE MICROBIOLOGY OF THE SOIL 421

The baoteria concerned are mostly micrococci (M. ureae)

+ 2NH

-f H

O = (NHJaCOs or

+ 2H

O = (NH

)

+ 14.3 Calories.

Lentils and broad beans have much urease and perhaps this may

act in the soil when the Heeds decay.

Bassalik found that some of the intestinal bacteria of earthworms

are able to make the potassium of orthoclase available to plants.

Many of the products of bacterial action in soils bring potassium

of minerals into solution as acetic acid.

Sulphur.

There are several members of the non-filamentous

sulphur baoteria

which are important in liberating sulphur. Winogradsky found that

these bacteria consume hydrogen rather than produce it. They oxidize

hydrogen sulphide in their cells BB:

S + O

= 2H

O -f- S

They store free sulphur in their cells and later oxidize it to HaSO*,

whioh is changed by contaot with calcium compounds as CaH

(CO

and finally exists in the spil as sulphates'. These bacteria are very

important in nature as they furnish plants with available forms of

sulphur. Some of these organisms producing bacterio-purpurin are

Spirillum rubrum, Spirillum volutans, chromatium Okenii, etc.

INDEX

Acetaldehyde, 12

Acetic Acid, 104

Acetic Acid Fermentation, 296

Acetone, 106

Activated Sludge, 116

Alcohol from Cellulose, 17

Alcohol Manufacture, 17

Alcoholic Fermentation, 11

Alcoholic Yeasts, 11

Analysis of Apple Juice, 298

Anthrax, 41

Appert, 243

Bacillus Typhosufl, 71

Barms, 134

Bating, 44

Beet Sugar, 263

Blue-Stain, 83

Bone Black Filters, 183

Botulism, 256

Bread, 128

Bread Constituents, 151

Bread Improvers, 143

Bread-Making Losses, 146

Buffalo Skins, 40

Cane Sugar, 263

Carbolic Acid, 72

Cellobiose, 18

Cellulose, 19

Centrifugahzation, 263

Chemical Retting, 51

Chromogenic Breads, 150

Citric Acid, 105

Cocoa, 335

Coefficient of Disinfection, 71

Coffee, 335

Cold Storage, 210

Composition of Vinegar, 292

Concentration, 230

Confirmed Test, 341

Contact Filters, 120

Corn, 27

Corn Oil, 188

Corn Products, 168

Creosote, 88

Damaged Corn, 194

Decay in Eggs, 347

Denatured Alcohol, 15

Denitrification, 319

Depilation, 43

Dew Retting, 49

Dill Pickles, 232"

Disinfectants, 66

Disinfection of Hides, 44

Distillery, 29

Drinking Water, 337

Dry Heat, 67

Drying, 218

Dry Salt Pork, 276

Egg Preservatives, 354

Eggs,

347

Enaymea of Yeast, 14

Ethyl Alcohol, 13

Flax 53

Food Preservatives, 205

Formaldehyde, 72

Fuel Gasea, 26

Fusel Oil, 13

Gasoline, 66

Grape Sugar, 58

Green Molt, 30

Glycerin, 110

Glycerol, 12

Glucose, 181

Grit Chambers, 115

Hemp, 47

Imhoff TJink, 116

Incineration, 66

Indigo, 92

Indigo Fermentation, 91

Inoculation. 57

Industrial Alcohol, 12

Invertase, 14

Kippered Salmon, 289

Lactic Add, 104

Leather, 39

Legume Cultures, 84

Malt, 144

Malt Vinegar, 305

Maple Sugar, 357

Maple Syrup, 357

Marine Products, 278

423