HUMUSORIGIN, CHEMICALCOMPOSITION,ANDIMPORTANCEINNATUREHUMUSORIGIN, CHEMICAL COMPOSITION,AND IMPORTANCE IN NATUREBySELMANA. WAKSMANProfessor of Soil Microbiolofl1/, RutgersUniversity, and Microbiologist,New JerseyAgricultural Experiment Station"Humus istheproduct of livingmatter, and the 80urce of it."-A. Thaer.BALTIMORETHEWILLIAMS&WILKINSCOMPANY1986COPYRIGHT, 1936THEWILLIAMS&WILKINSCOMPANY- ' Y s 7 t f ~Made intheUnited Statesof AmericaPublished January, 1936COMPOSEDANDPRINTEDATTHBlWAVERLYPRESS, INC.FORTHEWILLIAMS&WILKINSCOMPANYBALTIMORE, MD., U. S. A.PREFACEAlthough the importanceof humus in agriculturewasrecognized bytheearlyGreekandRomanphilosophers, it is onlyduringthelastcentury and a half that attempts have been made to disclose the natureof this groupof organic complexes, their formation and decomposition,andtheir role inplant nutrition. Although a number of problemsconcernedwiththestudyofhumusstillawait solution, sufficient evi-dence has accumulated to warrant an attempt to summarize theavailable information ina monographicmanner.Several reviews have beenpublisheddealing with certainaspectsof the origin, chemical nature, properties, and functions of humus.It isof particular importance to note here Wollny'sbook"DieZerset-zungder organischenStoffe"publishedin1897, Baumann'spaper onthe "History of the humic acids" in 1910, Oden's monograph on "Humicacids," several recent handbook summariesofchemical or agriculturalaspectsof humus, aswell asthenumerousbooksoncoal, peat, forestsoils, andcomposts. None of these, however, fully covered thesub-jectofhumusasa whole. Theapproachtotheproblem hasusuallybeenmade either froma purelychemical, physico-chemical, agricul-tural, or technological point of view. Wollnyis about theonlyonewhohas madeanall-embracingstudyof humus as anatural body, ofits formation, andtransformation. Unfortunately, soil microbiologywasstill in itsinfancy at thattime, andlittlewasknownconcerningtheroleof microorganisms in the formationof humus.Noother phaseofchemistry hasbeensomuchconfusedas that ofhumus, as a result of whichit frequently becomes necessarytolayconsiderableemphasis uponthe proper definition of theterms used.Vague generalizations, as "decay" and "putrefaction," havebeenusedfor specific processes of decomposition; the great complexity of the num-eroushumicacids, all ofwhichdesignated not definitechemical com-pounds but mere preparations, and the unjustified comparisons betweenthe natural humus compounds, formed in soils, composts, or bogs, withartificial preparations produced in the laboratory by the action of strongmineral acidsoncarbohydrates, servednot toadvancethesubjectofhumus, but rather toconfuseit.vvi PREFACEStudiesof the origin and chemi,cal natureof humus have been carriedout at the NewJersey Agricultural Experiment Station, during more thanfifteen years. Inthis work, the author has been assisted bysometwenty-fiveresearchworkers, most of whomhaveobtainedtheirad-vanced degrees from Rutgers University, using some phase of the studyofhumusasthethesissubject. To them, grateful acknowledgmentshouldfirst of all be made. Theauthor is particularlyindebtedtoR. L.Starkey, of the New Jersey Agricultural Experiment Station,forreading and criticizing the manuscript as a whole; he is also gratefulfor criticismandassistancerenderedbyC. F. Marbut, oftheUnitedStates Department of Agriculture, Washington, D. C.; by H. Jenny,Columbia, Missouri;byR. Bradfield, Columbus, Ohio;byW. Fuchs,of RutgersUniversity; and by C. E. Renn, whoassistedinpreparingthefigures.New Brunswick, N. J.CONTENTSINTRODUCTORYTher6leof humus in theorganiccycle in nature. .. . . . . . . . . . . . . . . . . . . . . . . . .. ixPART A. HISTORICAL DEVELOPMENT OF OUR KNOWLEDGE OF THECHEMICAL NATURE OF HUMUS, ITS FORMATION, ANDITSROLEINPLANTNUTRITIONC H A P ~ E R INature and characteristics of humus. Typesof humus... . . . . . . . . . . . . . . . . . 3CHAPTER IITher6Ieof humus in plant nutrition. .. . . . . . . .. . . . . . . . . . . . . . . . . . . . . .. . . .. . 10CHAPTER IIIThe changing conceptionsconcerning thechemical natureof "humus"and"humic acids"..... . ..... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 21CHAPTERIV"Humification"oforganic matterinsoilsandin composts, and methods ofhumusdetermination. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 63PARTB. ORIGINANDNATUREOFHUMUSCHAPTERVOriginof humus. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 91CHAPTERVIIsolation of definiteorganicchemical compounds fromhumus.. . . . . . . . . . . .. 129CHAPTERVIIChemical natureof humus asawhole. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 153CHAPTERVIIIHumus- formationincomposts, animal manures, andgreenmanures.. . . . .. 191CHAPTER IXHumus in forest and heathsoils. . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 212CHAPTERXHumusinmineral soils (field, grassland, garden, andorchard). . . . . . . . . . .. 242CHAPTERXIHumus inpeat and in coal. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 259viiviii CONTENTSCHAPTER XIIOrganic matterformationsinwater systems. ~ 288CHAPTER XIIIPhysical and physicochemical properties of humus. . . . . . . . . . . . . . . . . . . . . . . .. 305PARTC. DECOMPOSITIONOFHUMUS, ITSFUNCTIONSANDAPPLI-CATIONSCHAPTERXIVDecompositionofhumus innature....................................... 331CHAPTERXV.r Presenceinhumusofsubstancesinjuriousandbeneficial tothegrowthofhigher plants 351CHAPTERXVIUtilization of various forms of humus for agricultural and industrialpurposes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 361CHAPTERXVIISoil humus and the scienceofpedology 371OUTLOOKHumus as anorganic system 397ApPENDIXMethods of analysisof humusandof certainhumus constituents. . . . . . . . .. 399BIBLIOGRAPHY. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 413INDEX 481INTRODUCTORYTHE ROLE OF HUMUS IN THE ORGANICCYCLE IN NATUREWith the exception of certain limited groups of organisms, known asautotrophic bacteria, only the green or chlorophyll-bearing plants,amongall theliving forms, arecapableof synthesizingorganic matterfromsimple elements and mineral compounds; these nutrient elementsarederivedpartlyfromtheatmosphereandpartlyfromthesoil, the'sea, or other substrates where plants grow. The plant kingdom capableof bringingabout this processis representedonthisplanetbyformswhich range in size frommicroscopic organisms to giant trees: theformerrequirenofixed habitat, andcomprise. thevarious diatoms andotheralgaeinhabitingseas, rivers, andlakes, their life beingonlyoflimitedduration; thelatterlivefor manycenturiesinfixedhabitats.Thecommonabilityof theseforms tosynthesizeorganicmatteris ahighly importantcharacteristic.Theanimal kingdomuses theplants as themajor source of food.Theanimals, however, donot utilizetheplantsubstanceasa whole.Apart ofthelatter, frequentlya major part, iseithernot consumedat all or is left inan undigested or partially digested form. Thisresidual material is subjected to decomposition bymicroorganisms,including bacteria, fungi, and protozoa inhabiting the soil, the sea, andthe compost, andrepresentedthere bynumerous types possessingagreat varietyofactivities.The animals, as well, sooner or later die, and their bodies, in additionto the products of their metabolism, also become subjected to microbialAB a result of these microbiological processes, theelements which have originally been consumed by the plants for organicsynthesisarereturnedtocirculation, thuscompleting thecycleof theelementsintheprocess of life.However, theplantandanimal residues donot becomecompletelymineralized. Acertain part ofthese residuesismoreorlessresistanttomicrobial decomposition and remains for a periodof time inanundecomposed or in a somewhat modified state, and may even accumu-lateunder certainconditions. Thisresistant material is darkbrownto black in color and possesses certain characteristic physical and chem-ixx INTRODUCTORYCarbon of atmosphere .Carbon content of hydrosphere .Carbon contentof anthracitecoal. .Carbon content ofbituminouscoal. .Carbon content of browncoals .Carbon content of allformsof peat .content of all soils, to adepth of 30 em .C,arbon contentof all living matter .ical properties; it is usually called HUMUS. Asaresult of the forma-tion andaccumulationofthishumus, apartoftheelementsessentialfor organic life, especiallycarbon, nitrogen, phosphorus, sulfur, andpotash, becomelocked upandremovedfromcirculation. Inviewofthefact that themost important of theseelements; namely, carbon,combined nitrogen, and available phosphorus, are present innaturein only limited concentrations, their transformation into an unavailablestate, in the formof humus, tends toserveasacheck upon plant life.On the other hand, since humus can undergo slow decomposition undercertain favorableconditions, ittendstosupplya slow butcontinuousstream of theelements essential for new plant synthesis.Humusthusservesas a reserveanda stabilizerfor organiclifeonthisplanet.Theactualconcentration oforganic matter in the formof humus inthesoil andin thesea farexceeds thatpresentinall theliving formsof plants and animals. It is sufficient to call attention to the fact thatthehumus content of thesoil is considerablygreater thanthetotalamountof organic matter present in all thecrops harvested inagivenyear fromall thefields, orchards, and gardens, or that available in theformofreservefoodstuffs. Thelargequantitiesofhumuspresentinpeat andinbrownandhardcoal, whichfar exceedthe supplies oforganicmatterinourforests, represent organicaccumulationsduringmanythousands of years. The amount of organic matter foundinseas, rivers, and lakes, whether in true solution, in colloidal suspension,or inthebottom material, alsoexceedsmanytimes (593) theorganicmatter content of all plant andanimal life in thosewaters.Thecarboncontent of thevariousforms of humusonthis planet,aswell asthatpresentinall livingformsandin theatmosphere, hasrecently (181, 1206a, 645) beencalculatedas follows:,',metric tons tl 0 600,000,000..J}OO, . N

422,000,000,000 I2, 732,000,000,000 1,499,000,000,0001,123,000,000,000 _400,000,000, 0, 40 ...,0 '700,000,000,000Lundegardh(656) calculated thattheamountofCO2consumedan-nually by the green plants is about 59 billion tons;the total supplyof

i- \b0""HUMUS INORGANIC CYCLE INNATURExi l; .'.I ,...,o.K ILJ( CO2intheatmosphere wouldthus last onlyabout thirty-five years r(989).Anunderstandingof theoriginandchemical natureof humus, itsformationandaccumulation, andits transformationanddecomposi-tionrepresentsa highlyimportant phaseofourknowledgeof organiclife. Themicroorganisms influence the cycle of humus innatureinmore than one way: 1. They bring about its formation from plant andanimal residues. 2. They continuously transform humus, under favor-ableconditions, andfinallydecomposeit completelyor "mineralize"it. 3. Their own cell substance contributes directlyas a source ofhumus. Theroleofmicroorganisms in thecycleoforganic matter inthesoil, as well as innatureingeneral, is, therefore, indispensable.Without them, thecontinuedexistenceof lifeuponthisearth, as weknow it, would havedisappearedlongago, since mostof theavailableelements essential for plant growthwouldhavebeenstoredaway inthe form of inanimate plant and animal residues.Thisbookisanattempttotellthestoryofhumus, itsoriginfromplant and animal residues, itschemicalcomposition, its physical prop-erties, its importance in nature, especially in soil processes and inplant growth, andfinallyits decomposition. Humus is treatedhereas a natural body, universally innature, whereverplantoranimal residuesareundergoingdecomposition. Without denyingtheroleof purelychemical reactionsin theformationandtransformationofhumus, especiallythoseofoxidationandreduction, hydrolysisandpolymerization, it should be recognized that the primary agents in theformationandtransformationof humus arethe microorganisms; byoverlookingtheir functions, theearlier chemists failedtounderstandthe origin and significance of humus, in spite of the many.years of effort,fromthe beginnings of organic chemistry until recent times. Thesoil, compost, peat bog, and ocean are living systems, where the organicare continuously modified by numerousprocesses about bymicroorganisms whichliveinthese, systemsandreproduce,utilize energyandgive off heat, decompose organic 'substances, andconstruct newcomplexes. Humus andliving microorganisms arecon-nected by numerous,invisibleties, which must be appreciated in orderto understand the origin and nature of humus.PARTAHISTORICAL DEVELOPMENT OF OUR KNOWLEDGE OF THECHEMICALNATUREOFHUMUS, ITSFORMATION, ANDAITSROLEINPLANTNUTRITIONCHAPTERINATUREANDCHARACTERISTICSOFHUMUS. TYPESOFHUMUS...pinguishumusdulciqueuliginelaeta,Quique frequens herbiset fertilis uberecampus ...-GEORGICS OF VIRGIL.The study of the origin, chemical nature, and transformation ofhumus has beenlimitedchieflytotherole that humus playsinsoilprocesses andplant nutrition. Theimportanceof humus inthesoilis manifold: it serves as asource of nutrients for plant growth; it modi-fies thephysical andchemical natureof the soil invarious ways; itregulates anddetermines thenatureof themicrobial populationanditsactivities, bysupplyingsourcesofenergyandvariousorganicandinorganicnutrientsessential fortheirgrowth, andbymakingthesoila more favorable substratefor their development. Humus gives tofertilesoilssomeof their mostimportantphysical andchemicalprop-erties. Humus characterizes the soil, since differences inthe origin,abundance, andchemical natureof humusresult intheformationofdistinct soil types.Humusmaybelookeduponas astorehouseof important chemicalelementsessential for plantgrowth, especiallyofcarbon andnitrogen,and to a less extent of phosphorus, calcium, iron, manganese, andothers. The utilization of some of these elements held in the inorganicfractionofthesoil is alsoinfluencedbyhumus, throughits chemicalinteraction with the inorganiccomplexes. One should consider furtherthecolloidal effects of humus on the soil; itsbuffering properties whichmodify thesoil reaction; itscombining power withbases;itsinfluenceupon the oxidation-reductionpotential of the soil; its adsorption ofcertaintoxicmaterialsinjurioustoplantgrowth; itsability tosupplycertain c a t a l y ~ i c agentsandsmall quantities of certainrareelementsessential for plant growth; itsinfluenceuponsoil structure, uponthemoisture-holdingcapacityof thesoil, anduponsoil temperature; aswell as numerous other reactionswhich are of direct or indirect impor-tancetoplantgrowth. Onewill, therefore, not exaggeratein stating34 DEVELOPMENT OF KNOWLEDGE OF HUMUSthat a knowledgeofsoil humusismost essential for a properunder-standing of theorigin and natureof the soil aswell asof theprocessesthat control plantgrowth.Thechangingconceptionsof humus. Theterm"humus"datesbacktothetimeoftheRomans, when it wasfrequentlyusedtodesignatethesoil asawhole. I;t was later applied to the organic matter of soilsand composts or to different fractionsof this organic matter, aswell asto complexes formed by the action of chemical reagents upon avarietyof organic substances. FromTheophrastus (373-328 B.C.) to Wal-lerius(1709-1785),the conception of"oleum unctuosum,"which stoodfor"thesoil or fatnessof theland,"dominatedtheideasof the natu-ralists. The great botanist Linnaeus (1707-1778) classified soils ina manner similar tohis classificationof plants. Amongthevarioussoil types thus recognized are found: Humus daedalea (gardensoil),Humus ruralis(field soil), Humuslatum(mucksoil), Humus damascena(claysoil), Humuschistosa(redclay soil), etc. Wallerius (1245) firstdefined "humus," in 1761, in terms of decomposed organic matter.However, the prevailing ideas concerning the chemical nature of humusandthemechanismofitsformationwereveryvague. Most often itwasconsideredasa complex, formedinsoils, inbogs, or incomposts,from plant residues, by aspecial process of "humification."De Saussure, in his famous work "Recherches chimiques sur lavegetation" (942), devotedconsiderableattentiontohumus (terreau).Hefoundthat itisnot ahomogeneoussubstance, but that itconsistsof various complexes (extractives, oils, salts) which can be readilyremoved. Humus was foundcapable of absorbing oxygen,which com-bined with the carbon of the soil to give CO2 About that time humuswas showntocontaina substancewhichwas solubleinalkalies andprecipitated on acidification of the alkaline solution. Thaer (1140)differentiated between "acid humus" or peat, formed with limitedadmissionof oxygen, and "mildhumus," formed inthe presence ofsufficient oxygen. Liebig(638) spoke of humus as "a brown substance,easilysolubleinalkalies, but onlyslightlysolubleinwater, andpro-duced duringthe decomposition of vegetable matters bythe actionofacidsoralkalies."Theterm"humus" cameintogeneral useat a timewhenorganicchemistrywasstill initsinfancyandwhenall organicandinorganiccompoundswereconsideredtobesubstancesverysimpleinchemicalcomposition. Unfortunately,itwasnotalwaysusedtodesignatethesameorganicsubstancesorpreparations. SomeappliedthistermtoNATURE AND CHARACTERISTICS OF HUMUS 5thetotalorganic matterof thesoil, whileothersdesignated by it thatpart of the organicmatter which is readily oxidized by certain reagents,such as hydrogen peroxide or potassiumpermanganate (568, 345).Frequently"humus"meant onlythat fractionof theorganicmatterwhich is soluble in dilute alkali solutions(361, 1014); in many instancesthecomplexformedonprecipitationof thealkalineextract byacidswas so designated (801, 79). More recently certain investigators(371, 1077) havereferredtothatpart oftheorganicmatterwhichisnot acteduponbytheacetyl bromidereagent as"humus." Theuseof thetermtodesignatesoil organicmatteras a wholewasprobablytheonemost universallyaccepted, whereastheothers, whichincludedonly a part of the soil organic matter, and frequentlyavery small part,were given lessconsideration.Without goinginto a detailedreviewof thenumerous conceptionsof humus prevalent duringthe 19thcentury, it maybe well tocallattentiontosomerecent ideas onthis subject. Ollech (789) defined"humus," in 1890, as comprising "all those substances which are formedinthedecomposition andfermentationoforganic matterof plant andanimal origin, orthroughtheactionof certainchemical agents uponthis organic matter, in the formof amorphous, non-volatile, non-odorous, more or less dark colored organic compounds." Ramann(859) spoke of humus or "humus bodies" as "colloidal complexes of-varyingcomposition, consistingof unchangedcolloids of theoriginalplant substancemixedwithcarbon-richdecompositionproducts." Inbothof these definitions therole of microorganisms intheformationof humusisdefinitelyrecognized. According toOden(781), however,humus represents "thoseyellow-browntodark-browncoloredbodiesof unknown constitution whichoriginate in nature through thedecom-position of organic substances under the influence of atmosphericagenciesor in thelaboratorythroughchemical reagents (largelyacidsoralkalies); theypossessa definiteaffinityfor waterandshow, if nottruesolutionordispersion, a distinctswelling." Inthisdefinitionnoattention is paid to the importance of microorganisms which bringabout thetransformationof the plant andanimal residuestogiverisefinallytohumus (129).Characteristics of humus. By taking into consideration the processesof humus formation, largely as a result of the activities of variousmicroorganisms, onemaysuggest thefollowingdefinitions: Humus isa complexaggregateofbrowntodarkcoloredamorphoussubstances,whichhaveoriginatedduringthedecompositionof plant andanimal6 DEVELOPMENT OF KNOWLEDGE OF HUMUSresidues bymicroorganisms, under aerobic andanaerobic conditions,usuallyinsoils, composts, peat bogs, andwater basins. Chemically,humus consists of certainconstituents of the original plant materialresistant tofurther decomposition; of substances undergoing decom-position; of complexes resulting from decomposition, either by processesof hydrolysis or by oxidation and reduction; and of various compoundssynthesizedbymicroorganisms (968). Humus is a natural body; itis a composite entity, just as are plant, animal, andmicrobial sub-stances; it is evenmuchmore complexchemically, sinceall of thesematerials contribute to its formation. Humus possesses certain specificphysical, chemical, and biological properties which make it distinctfromother naturalorganicbodies. Humus, in itself or by interactionwith certain inorganic constituents of the soil, formsacomplex colloidalsystem, the different constituents of which are held together'by surfaceforces; this systemis adaptable to changing conditions of reaction,moisture, andactionof electrolytes. Thenumerous activities of thesoil microorganisms take place in this system to alarge extent.It is now definitely recognized that humus hasresulted from thede-compositionof plant andanimal bodies, mainlythroughthe,agencyof microorganisms although the possibility of certain chemical reactionstakingplace inthe process is not excluded. Humus has, therefore,certain specific properties which distinguish it from other naturalbodies. Thesepropertiescanbebrieflysummarizedas follows:1. Humuspossessesa darkbrown toblackcolor.2. Humus is practicallyinsoluble inwater, although a part of itmaygointocolloidal solutioninpurewater. Humus dissolves toalarge extent indilutealkali solutions, especiallyonboiling, givingadark colored extract; a large part of this extract precipitates whenthe alkali solution is neutralized bymineral acids. Certainconstit-uents ofhumusmay also dissolve in acid solutions and be precipitatedat the isoelectric point, which is atapH of about4.8.3. Humus contains a somewhat larger amount of carbonthandoplant, animal, andmicrobial bodies; the carl;>oncontent of humus isusuallyabout 55to56per cent, andfrequentlyreaches58percent.4. Humus contains considerablenitrogen, usuallyabout 3to6percent. The nitrogen concentrationmaybe frequently less thanthisfigure; inthecaseof certainhighmoor peats, for example, itmaybeonly0.5-0.8per cent. It mayalsobehigher, especiallyinsubsoils,frequentlyreaching10to12percent.5. Humuscontains theelementscarbon and nitrogen in proportionsNATURE AND CHARACTERISTICS OF HUMUS 7which areclose to10: 1; thisis trueof many soils andof humus in seabottoms. This ratio varies considerably with the nature of the humus,the stage of its decomposition, the nature and depthof soil from whichitisobtained, andclimaticandotherenvironmental conditionsunderwhichit is formed.6. Humus is not in a static, but rather in adynamic,condition, sinceit isconstantly formedfrom plant and animalresidues andiscontinu-ously decomposed further by microorganisms.7. Humus serves as a source of energy for the development of variousgroupsofmicroorganisms, and, duringdecomposition, givesoffa con-tinuousstreamofcarbondioxideandammonia.8. Humusis characterizedbya highcapacityof base-exchange, ofcombiningwithvariousotherinorganicsoil constituents, ofabsorbingwater, and of swelling and by other physical and physico-chemical prop-ertieswhich make itahighly valuableconstituent of substrateswhichsupport plant andanimal life.Importance of humusinsoil processes: The functions of humus inthesoil arelargelythreefold: 1. physical, therebymodifyingthesoilcolor, texture, structure, moisture-holdingcapacity, andaeration; 2.chemical, influencingthe solubility of certainsoil minerals, formingcompounds withcertainelements, suchas iron, whichrenders themmorereadilyavailablefor plant growth, andincreasingthebufferingproperties of thesoil; 3. biological, byservingas a source of energyforthedevelopment ofmicroorganisms, aswell asbymakingthesoilabetter medium for the growth of higher plants; it also supplies aslowbutcontinuousstreamof nutrientsforplant life.Humus exerts various other important effects uponplant growthwhich are still awaitingexplanation; althoughsome of these effectsarebelievedtobeinjuriousinnature, most aredefinitelyestablishedtobehighlybeneficial. Thevariousattemptstoexplaintheseeffectsby the formation of plant toxins, on the one hand, and of "plant stimu-latingsubstances"or "auximones" (135, 715, 933, 180, 1297), ontheother, are still a matter of dispute. Of course, all referencetothe"spirit of thesoil" or the "reservesoil force" represents speculativegeneralizationsnot baseduponsoundexperimental evidence.Humus types. Although there is no doubt that because of theirr ~ l e inplant nutrition, themost important forms ofhumusarethosefound in field and garden soils, other formsof humusexist, which evenif notutilized foragricultural purposes, have important industrial andother applications. Thefunctionof humus inplant aridanimal life8 DEVELOPMENT OF KNOWLEDGE OF HUMUSintheseaandininlandwater basins is still a matter of conjecture,although its importance is well recognized. Humus in coal and inpeat as a source of fuel has been one of the major agents in the develop-mentof modern industrialcivilization. The functionof humus in theoriginofpetroleumisstill a matterofdispute (1173). Any attempt,however, to divide humus on the basis of its practical utilization wouldprovetobelargelyartificial.Sincethesuggestionof Linnaeusto separate humus into several wellrecognizedtypes, variousclassificationsof humushavebeen proposed.Anearlystudent of humus (517) recognized four distinct types orvarieties: 1. thebrown variety, foundin living vegetation, in recentlyfallenlitter, in peat, indecomposing sea-weeds upon theshores and infungi; 2. theblackvariety, usuallyfoundin an active stateofdecom-positioninthedeeper layers ofsoil, indecomposingleavesandwoodof forests, in animal manures, in peat of swamps and in muds; 3. humusin a state of transference, namely in the waters of rivers, lakes, springs,andeven in rainwater; 4. humusina fossil condition, intheformoflignite, brown-coal and other carbonaceous deposits, aswell as in manyminerals, suchashydratedoresofironandmanganese.The accumulated knowledge concerning the origin and chemistryof humus permitsamorelogical system ofclassification based entirelyupontheprocessesandconditions ofhumusformation.A. Humus types formed by decomposition of plant and animal residues underaerobic, or only partly anaerobic, conditions, in composts and in soil:I. Humusof composts:1. compostsof stable manures,2. composts of plant residues, withor without additionof in-organic salts("artificial manures").II. Humus in soil:1. plant residues decomposed under conditions of high acidity orlow temperature, or both:a. raw humus in forestsoils,b. raw humus in heathsoils,.c. alpinehumus; )2. plant residues decomposed under less acid, neutral, or alkaline,conditions: typical soil humus (chernozems, humus inchestnutsoils, serozems, forest mull humus, etc.).B. Humus types formed by decomposition of plant and animal residues underanaerobicconditions:.1. Recent formations:1. highmoor p e a t s , ~ -2. lowmoor peats,3. sedimentary peats.NATURE AND CHARACTERISTICS OF' HUMUSII. Old formations:1. softcoals(brown coals, lignites),2. hard coals(anthracites).C. Humus typesformedin water basins:I. Recent formations:1. water-solublehumus,2. humus inmarinebottoms.II. Geologic formations:1. sourcebeds of petroleum.9''''Thesedifferent types ofhumusvaryconsiderablyinchemical com-position, because of differences in the nature of the materials from whichthey originated, conditionsof decomposition, and extent ofdecomposi-tion. Someof thesetypes are, therefore, frequentlysubdividedintoseveral subtypes. This system of humusclassification is not freefromcriticism: thesedimentarypeats, for example, canreadilybe placedin group 0;however, sincethey formhumusaccumulations similar tothose of typical peats, they are classified with these. Frequently (258),soil humus is dividedonthebasis of its agricultural utilizationintotwo general types; namely, "acid humus" and "mild humus"; the latteris richin, if not saturatedwith, calcium,magnesium, andother bases,while theformer contains largelyhydrogeninits exchange complexandlittle, ifany, calcium andmagnesium. Under"acidhumus"oneusually understandshumusof highmoor peats, raw humusforest soils,andsometimeshumusofacid sandysoils. The"mild humus"repre-sentsthetypical humusof chernozems, forest mull soils, andsimilarformations. Thesetwotypes of humus are considereddifferent, notbecause they were found to be markedly different in chemical nature orbecause they were produced under different climatic, soil, or vegetationconditions, but because they differ in the nature of the bases saturatingthe exchange complex. However, the mere addition of calciumtothe"acidhumus" doesnot changeitintoa "mildhumus"; it wouldtake many yearsof decompositionbefore the formercould change intothelatter, demonstratingeventosuperficial examinationthemarkedchemical difference betweenthe two types of humus. This will bebrought out indetail inthefollowingpages.CHAPTERIITHEROLEOFHUMUSINPLANTNUTRITION"Nur an Humus Jehlte es, so meinte man Jrither, nUT an Ammoniak Jehlees, 80 meint man jetzt."-LIEBIG.Theearlierconceptionsoj the r ~ l e of humusinplant nutrition. Thenutrition of plants was considered by the Ancients, as well as during theMiddleAges, toinvolveprocesses similar tothosein animals;thenu-trientswerebelievedtobeabsorbedina preformedconditionbytheroots from the soil and to be subsequently modified in the plant tissues."Earth" was believed to be one of the essential "elements" upon whichplants feed, inadditiontotheother three elements; namely, water,air, andfire. Without knowingmuchabout theexistenceof humusinthesoil or its significanceinsoil processes, theAncients attachedconsiderable importance to its abundance. This can be recognizedby the fact that the Romans judged the quality of the soil by itscolor,although Columella (185) emphasized the injurious effect of acid humusin peat.VanHelmont (417) andother naturalists of the 17thcenturyat-tempted to show that plants withdraw only water from the soil and thatmanuresact favorablyupon plantgrowth only by supplying heat andmoisture. Accordingtothesenaturalists, thelivingpowerof animalsandplants enables themtoconstruct all tissues, includingeventhemineral constituents, fromthe atmospheric air and the soil water.Toward the end of the century, Woodward (1303) concluded thatearth, and not water, is the element essential for vegetable production.T h ~ plant physiologists of the 17th century, including such prominentinvestigatorsas Malpighi, didnot dispensewiththeideathat plantsabsorb their organic nutrients from the soil; the Aristotelian conceptionof this process was somewhat modified, toincludetheideathat theabsorbedorganiccomplexes undergovarious chemical changes withinthe planttissues, before theybecomeapartof theliving plant. Theprevalentideas concerningthenatureofthematerialsabsorbedfromthe soil were very vague and were usually veiled in various metaphysicalspeculations. Some investigators referred tothe absorbed nutrients10Re,LE OF HUMUS IN PLANT NUTRITION 11as"thejuicesof the earth,"whileothers spokeof "minute particlesofsoil." Some believed that plants, like animals,exertaselective actionupon the food particles, which explained the favorableresults obtainedfromplantrotationsin practicalcropproduction. Stahl, thefounderof thephlogistontheory, expressedtheidea thatplantmaterialshavethe same chemical composition as inorganic substances; the former werebelievedtobeproducedfromthelatter, namely, fromtheinorganicsalts andchiefly fromwater and"phlogiston"(125). In thecourseoftimetheimportanceinplant development ofsuchaninorganicsub-stanceasnitratewasestablished(348); someappreciationofthetruefunctions of leaves also developed.The period between 1630 and1750 was characterized by an industri-oussearchfor "theprincipleofvegetation"; durmgthis period, salt-peterandwater wereindividuallyproposedas theelementsessentialfor plant growth. In1730, Tull stated that"small earth-like particlesserveasnutrientsfor plants, andairandwaterhelptoextract theseparticles fromthesoil." Kiilbel (597), inhis bookonsoil fertility,emphasized that magma unguinosum, supposed to be present in humus,was the principle of plant growth and the major source of soil f ~ r t i l i t y .Wallerius, soonafterward, inhisfamous bookon"Principlesof Agri-cultural Chemistry" (1245), concluded, fromthechemical analysis ofplants, that the humusof thesoil is theessential nutritiveelement forplant growth (nutritiva) , whiletheother soil constituents onlyassistinthe process of nutritionbymixingthefoodor dissolvingit, thusbringingit intoa conditionmoresuitablefor entranceintotherootsystems oftheplants (instrumentalia). Thefavorable actionof limeon soil was -believed to consist in dissolving the "fatness" of the humus,'while the function of clay was considered as that of fixing and retainingthis fatness. These ideas enteredthe agronomic literature, andthe"fat of the land"became synonymouswith soil humus and the supplyof plant nutrients. Humus was consideredtobeof thegreatest im-po!tanceinplant nutrition.Theendofthe18th andtheearly partof the19th centurieswasaperiodof definiteprogress inknowledge of the processes involvedinthegrowthofplants, largelyas a. result oftheworkof Ingen-Housz,Senebier, and deSaussure. Ingen-Housz(476) clearly recognizedth-atleaves absorb fromtheair a gaseous constituent whichtheyuse inthepresenceoflight toproducethecombustiblebodiesintheplant.Senebier (995) suggested that theprocessofCO2-assimilation involvesreduction of this gas in the presence of light and results in the excretion12 DEVELOPMENT OF KNOWLEDGE OF HUMUSofoxygen;hebelieved, however,that theCO2necessary forthe nutri-tionof theplantsdid notcomechiefly fromtheair, butwasabsorbedby the roots from the soil and was brought to the leaves with the risingsap. Thelastconceptionwaslargelyresponsibleforthe fact that thehumusof thesoil wasforalong timeconsidered tobethetrue sourceof carbon for plant life. Only Th. de Saussure (942) succeeded inclearly establishing the importanceof thegaseousexchange in thenu-tritionof plants. He definitelydemonstratedthat plants synthesizetheirorganic matter fromcarbondioxide andwater, andgiveoffoxy-gen; theCO2isobtained entirely fromtheair, whilethenutrientsde-rived from the soil are very insignificant in amounts,as compared withthe total plant mass produced. He believedthat the nitrogen wasabsorbed from the soil or the atmosphere, in the formof vegetable andanimal extracts, asammoniagas, or in other water-soluble forms. Heemphasizedthat the inorganic substances are absorbed chieflyfromthe soil.DeSaussurewas thefirst todevelop the idea of modern plant nutri-tion, based upon careful chemical investigations. It was thus definitelyshown that theplant obtainsitsoxygenandcarbondioxidefromtheair, whilethenitrogen andvariousminerals, which make upa definitepart oftheplantconstituents, areobtainedfromthesoil throughtheroot systemof theplant. The fertilityof thesoil wasbelieved tode-pend, toacertain extent and within certain limits, on the amountandnatureof the water-soluble constituentsthat thesoil contains. Theplant, byabsorbingtheseconstituentsfromthesoil, canexhaust thesoil and render it infertile. A soil may,therefore,lose the greater partof its vegetative substance without showing any differences in its physi-calpropertiesfromthoseofafertilesoil.Inspite of these exact contributions toa better understandingofthe nutritionof plants, the idea still prevailed during theearly partofthe19th century thatsoilhumusfunctions asa sourceofcarbon. Aspecial "vital force"in the plantcellswas still believed tobring aboutthesynthesisofmineral substances. Hassenfratz (see457) definitelystated, about 1800, thatthecarbonnecessary forplant growthis ob-taineddirectlybythe roots fromthedarkcoloredsoil constituents,whicharefoundabundantly infertilesoils. Thustheprevailingideathat soil humus is the true nutrient of plants still remained. This con-ceptionoftheroleofhumuswasthemoreacceptablebecause it wasgenerallyrecognizedthat the fertility of the soil depends uponthishumus.R6LE OF HUMUS INPLANT NUTRITION 13Thaer (1139) maybeconsideredasthemost outstandingexponentof thehumustheoryofplant nutritionduringthisperiod. Thefactthat stable manureconsistslargelyoforganicsubstancesand that, ondecomposition, manuregives risetohumus, ledThaertosuggest, in1798, thatplantsthriveprincipallyuponthehumusinsolution. Hestated further, in1808, that "humus comprisesamore or lessconsider-ableportionof thesoil; fertilityof thesoil depends largelyuponit,since, besides water, humus is the onlymaterial whichsupplies thenutrients to plants." The favorable influence of certain inorganicsubstances upon plant growthwasexplained by theincrease in humusformationinsoil throughtheadditionof thesematerials. However,even Thaer had to admit that the probableeffectof humus upon vege-tation was exerted through the influence of the carbonic acid gas whichwasproduced.These ideas concerning the function of humus in plant nutritionprevailedthroughout thefirst half of the 19thcenturyandwere ac-cepted bysuch prominent chemists as Berzelius, Mitscherlich, andDavy. Thisbecomesevident fromsomeoftheconceptions ofDavy(206), who stated that oils were good manures on account of the carbonandhydrogenthattheycontain;thattheadditionoflimetothesoilhasafavorableeffect, because itdissolvesthehardvegetablematter;that putrid urine is less useful as amanure than fresh urine; that farm-yardmanureshouldnot beallowedtoferment before itisappliedtotheland. He admitted, however,that the,l1sh constituentsof thesoilareessentialforplant nutrition anddonotactmerelyasstimulants.Berzelius(113), as late as1839, still considered that humus was a plantfood, althougha fewyears previouslySprengel (1075) hadexpressedideas similar tothose of de Saussure, that plants obtainthe carbondioxidefromtheair, while the nitrogen, whichis alsoanimportantplant food, is derived from the soil.With very fewexceptions, the early agronomists, plant physiologists,andchemistsconsidered that theorganic matterof thesoil, known ashumus, wasthechiefnutrient for plants; thelatter werebelievedtoabsorb the humus directly through their roots. The presence ofhumus in thesoil waslooked upon as the primary requirement for soilfertility. The carbon of the humuswas considered to be an importantsource of carbon for plants, without necessity for its alteration previoustoabsorption. Theseideasreceivedsupport fromexperimentswhichindicatedthatplant growthincreasedwiththehumuscontent ofthesoil.14 DEVELOPMENT OF KNOWLEDGE OF HUMUS/Early students of the chemical nature of soil-organic matter observedthat humus and "humic acids"(a group of constituents of humus whichwere soonto become verypopular) are insoluble in water, andarerelativelysolubleinalkali solutions. Thisledtothesuggestionthatthepresence in thesoilofalkalies andalkali earths, whichareusuallyfoundin theashconstituentsof theplant, contribute tothe solubilityandassimilationof humus. Itwasbelieved that acontinuous streamof humusproceedsfrom" thesoil to therootsof theplants.Some of the outstanding plant physiologists of the early 19th century,suchas de Candolle, Treviranus, Megen, andothers, while carefullypursuing various problems of plant physiology, didnot always paysufficient attention tothechemicalprocessesinvolved in thenutritionof plants. Thehumustheoryofthechemists andagriculturists wasacceptedbythemuncritically. Theseprevalent ideasconcerningtheroleof humusin plantnutritionwerewell expressedbyLiebig, inhistypicallysarcasticmanner: "Vegetablephysiologists considerthefor-mationof woodyfibre fromhumus as verysimple; theysayhumushas onlytoenter intochemical combinationwithwater inorder toeffect theformationof woodyfibre, starchorsugar."Liebigandhis period. About 1840, thereappearedtwoimportantcontributionstothesubject of plant nutrition, whichdealt a severeblow to the humus theory,although they did not dispose of it entirely:Boussingault (141-2) demonstrated that plants can make normalgrowthwithnutrientsaltsaloneandwithout anyhumuswhatsoever,andLiebig(638) published hisfamousbookon"Chemistry in itsAp-plicationto Agriculture and Physiology," inwhichhe ridiculed theideathat plants derive their carbonfromthesoil andnot fromthecarbonicacidoftheair. Liebigconsideredtheabundanceofhumusin thesoiltobemorea result thana causeofsoilfertility. Hecon-cluded that only inorganic or mineralized substances are used as nutri-ents byall greenplants. Plants live oncarbon dioxide, ammonia,nitrate, water, phosphoricacid, sulfuricacid, silicicacid, lime, potas-sium, andiron, andsomeneed, inaddition, sodiumchloride. Liebigthus brought home toagronomists, to agricultural chemists, andtoplant physiologists the results of Ingen-Housz, de Saussure, and others,byemphasizingclearlyanddistinctlythe importance ofash constit-uents inplant nutritionand theroleof thecarbondioxideof theat-mosphereinthis process.AccordingtoLiebig, humus, because of its insolubility, is not ab-sorbed by plantsandwouldnever even suffice to supply thenecessaryRe>LE OF HUMUS IN PLANT NUTRITION 15carbon tothetotalvegetation. Thehumuscontent of thesoil isnotdiminishedbyplant growthbut is actuallyincreased. However, hecametorecognizelater thathumusexertsa favorableeffect onplantgrowth. This was believedtobeduetothefact that it supplies aslow andlasting streamofcarbonicacid, whichservesasasolvent ofsoil constituents necessary for plant nutrition; a part of the carbon dioxideof thesoil is diffusedintotheouter air; theplants coveringthe soilwiththeirleaves assimilatemorecarbondioxidethroughtheir leavesthandothose thatdependexclusively upon thecarbondioxideof theatmosphere.1Although Liebig's contribution was of inestimable importance indisposing of the humus theory of plant nutrition and in clearly empha-sizing the fact that plants obtain their carbon from thecarbon dioxideof the atmosphere and the minerals from the soil, his ideas on the nitro-gennutritionof plants were opentoconsiderable criticism. Hebe-lieved that ammonia was the specific source of nitrogen which the plantsutilized and thattheammoniacontentof thesoil atmospheresufficedtosupply thenitrogenneedsof theplants, justasthecarbondioxideof theair providessufficientlyfor thecarbonrequirements. Carbondioxide, ammonia, andwater werebelieved byLiebig tobethethreeessentials inplant nutrition. Inhis attempt toestablishhis theorythat plantsobtain only alkalies (potash) and phosphoricacid fromthesoil andthecarbonfromthe atmosphere, Liebigcommitteda graveerrorinconsideringtheatmospherenot onlyasthesourceof carbonbut of nitrogenas well.In spiteof theinvestigationsofdeSaussureandBoussingault, sup-portedbythebrilliant arguments of Liebig; inspite of theworkofSachs, Knop, andotherswithwatercultures, whereby itwasdemon-strated that plantscan synthesizealltheirorganiccomplexesfromin-organic substances, in thecomplete absence of organiccompounds, theideastill continuedtoprevail for someyearsthat organicsubstancesof the soil were intimatelyinvolvedinplant nutrition. As late as1872, Grandeau(361, 362, 1191) statedthat humusfurnishes carbon1 "Der Humus ernahrt die Pflanze nicht dadurch, dasz er imloslichen Zustandevonderselbenaufgenommenundals solcher assimiliert wird, sondernwei! ereine langsame und andauerndeQuelle von Kohlensauredarstellt, welche als dasLosungsmittel gewisser fUr die PflanzeunentbehrlicherBodenbestandteileundauchalsNahrungsmittel, dieWurzelnder Pflanze, solangesichimBodendieBedingungen zur Verwesung (Feuchtigkeit und Zutritt der Luft) vereinigt finden,invielfacher Weisemit Nahrungversieht."16 DEVELOPMENT OF KNOWLEDGE OF HUMUStoplants; accordingtohim, themineral nutritionof plants cantakeplace only through the agency of the black matter. Humus richsoils,such as Russian chernozems, contain a peculiar organic substancewhichbindsvariousinorganiccompounds; thesearecapableofresist-ingthe action of dilute hydrochloric acid. This substance is madesoluble byammonia water; it contains 3-6per cent nitrogenandalarge part of the phosphoric acid of thesoil. The ammonia water wasthus looked upon as the solvent of thefoodfor plants.Even deSaussure suggested, in1842,thatthefunctionofammoniaor nitrates inplant growth consists inrenderingthe humus solubleand not acting directly as nutrients. He believed that his experimentsdefinitely demonstrated the fact that "humic acid" andsubstancesextractedbywaterfrompeat areabsorbedbyplants; hefoundthatwhen humusenters therootsof the plant, as shown by the absorptionof a deeplycoloredsolutionofpotassiumhumate, it is changedtherechemicallyandassimilatedbythe plant. Johnson (507) argued, in1883, thatalthoughLiebig andhisadherentswere right inconcludingthat thefinal productsof decompositionof organicsubstancesinthesoil, namely, carbonicacid, ammonia, nitricacid, water, andtheashingredients, formthe chief nutrients of agricultural plants, the factremains that various forms of humus ("soluble humates, ulmates,erenates, andapocrenates, togetherwithothersolubleorganicmatterof thesoil") areabsorbed by theplants andassimilated. The superi-ority of stable manure over inorganic fertilizers for crop production wasbelievedtobeequallyconclusiveevidence thathumusisdirectlyuti-lized byplants (361, 152, 874, 464). Numerous other claims weremadethatplantsgrewwell withtheirrootsimmersedinsolutionsofsoil organic compounds, prepared by extraction with potassiumorammoniumhydroxide. These claims were not sufficient, however,to establish definitelythat humus is assimilated directlybyplants,although theyhave demonstratedthat it is not injurious andmayactuallybeinsomemanner beneficial toplant growth, incontradis-tinction to the earlier ideasof Liebig.The introduction of nutrient salt solutions for the cultivation ofplantsbySachsandKnopin1860openedthewayfor exact experi-mentationinthis direction. Acton (11) demonstrated, in1889, thatplantsareabletoassimilatedirectlyglucose, sucrose, andglycerol inwater cultures and utilize them for starch synthesis in the dark;glyco-gen, dextrin, andstarchcouldnot be assimilated. Similar observa-tions were madebyLaurent (614), whoshowedthat cornandpeasR6LE OF HUMUS INPLANT NUTRITION 17assimilateglucose, notonly inthedark, butalsoin thelight, both inthepresenceandintheabsenceof carbondioxide; sucrose, glycerol,and potassium humate were also utilized. Maze(685) found that cornassimilatessugars, starch, and peptone. Cailletet (165) demonstratedthat certainforest shrubs (Adiantum) arecapableof growinginsoil,at the expense of the organic compounds, by a process similar to that ofthe fungi of the soil. According to Knudson(554)when soluble carbo-hydratesareabsorbed by plants, rootgrowthis stimulated toamuchgreater extentthantopgrowth. Inthecaseofvetch, the ratio tops/roots wasreducedfrom4.6to2.0, inthepresenceof glucose, andto1.2, in the presence of sucrose.Theabilityof greenplants toassimilate organic nitrogenous com-pounds was evenmore definite. Wiley (1281) reportedin1897thatoats grown in peat soils contained 25 per cent more nitrogenthanoatsgrown inordinary mineralsoils; alarge partof thisnitrogenwasfound in the plant in the amide state and not asprotein. Hecame totheconclusionthat plantsareabletoassimilatethenitrogenpresentin peatlargelyasamide. Soon after, Lutz(661) showed that variouslower aminescan be assimilated and utilized by higher plants,without,being firstacted upon by microorganisms andtransformed to ammoniaornitrate. Othersubstances, however, likephenylamines, naphthyl-amines, andalkaloids, exert a decidedtoxiceffectupon plantgrowth,anddonot offer available sources of nitrogen, unless theyare firstdecomposedbymicroorganisms. HutchinsonandMiller (471) foundthat wheat andpeasareabletoassimilateurea, acetamide, glycerol,alloxanandpeptone. Theabsorptionbyplantsof nucleicacid, xan-thine, guanine, creatinine, arginine, asparagine, andothernitrogenouscompounds has been established by Schreiner and Skinner (986).These observations were confirmed by Brigham(155), who found, how-ever, that bettergrowthwas obtainedwhentheplant cultures wereinoculatedwithBac. 8ubtilis.Inthe' presence of light, greenplants synthesize practicallyall oftheir organic substances photosynthetically, and the presence of organicsubstancesdoesnot alter thisprocess. Even in thoseinstanceswheredirect assimilationof organic complexes byplants has beenstudied,the role of microorganisms in the process has not always been ex-cluded. Theactivitiesof theseorganismsinbringing aboutmodifica-tionsof theorganic matterdependupona numberoffactors, suchasnatureof soil, natureof plant, environmental conditions andnatureof organisms. In some cases, they may beofconsiderable importance18 DEVELOPMENT OF KNOWLEDGE OF HUMUSin theutilization of organic matter, asin thecaseof mycorrhizafungiin thenutritionofforest treesandorchids.Indiscussingthefunction of humus inplant nutrition, it maybewell toconsider theunderlyingfactors responsible for thefavorableeffects of stablemanures uponplant growth. Whetherthis effect isdue to theorganiccomplexes or to the mineral constituentsof the ma-nure is not a new question. Discussions of the problem appeared manyyears ago, evenantedatingthoseofThaerandLiebig. Noneof theproponentson either side, however, claimed thattheeffect of manureisduesolely toeither theorganicor themineral constituents. Thaerand the representatives of the "humus" group believed that it is largelyduetothehumus content, whereas Liebigandthe "mineral" groupbelieved that it is the mineral constituents which are chiefly responsiblefor thefavorable effect of themanure (772). Accordingtoa recentcontribution (771), the favorable effect of stable manure is exertedthroughits colloidal substances, whichstimulatethe root systemoftheplant.Theresults oftheinvestigationscitedhereandofnumerousothers(124, 685, 722, 1021, 898,875,91, 179)on the absorption andassimila-tionof sugars, organic acids, andnitrogenous organic compounds byplants canbesummarizedasfollows:1. Various organic compounds, such ascreatine, creatinine,arginine,histidine, guanine, xanthine, hypoxanthine, andnucleic acid, canbedirectlyassimilatedbyplantsandcanreplacenitrateinculturesolu-tions (985-6).2. Leguminous plants are able toobtaintheir nitrogenfromrootnodules in the form of organic compounds, probably amino acids(1212).3. Under sterile conditions certain plants are able to assimilatevarioussugars (1021); lecithincanbeassimilatedas asourceofphos-phorus, and cystine,asasource of sulfur(1134).4. Humusoffers a veryfavorablesourceofavailableirontoplantsand -to microorganisms, at reactionswhich are optimum for the growthof plants (163, 790, 641).5. Someplantslackingchlorophyll makeuse of organicsubstancesfromthesoil humusastheirexclusivesourcesofenergyandmaterialfor synthesis. They are usually assisted in thisprocess by mycorrhizaformations, whereby the organic matter is generally altered beforeassimilation. Othermycorrhizaassociationsappeartobeinvolvedinproviding certain organic substancesforchlorophyll bearing plants, asin the case of orchids.RLE OF HUMUS INPLANT NUTRITION 19Thespecificeffectsofcertainorganicsubstances upon plant growthcannot be interpreted as proving that organiccomplexes in general aredirectly assimilated by plants and that, therefore, theaddition of suchsubstancestothesoil is highlydesirableinplant production. Thereisno doubt thatorganicmatter isof greatimportance in thesoil, butthisis not becausesomeoftheconstituentcomplexes maybedirectlyassimilated by the plants, but principally because humus exerts impor-tant effects upon the physical and chemical properties of the soil,because it contains a considerable quantity of plant nutrients, andbecause it offers a favorable mediumfor microbial activities. Allattempts, therefore, tointroduceintopractical agriculturetheuseoforganicfertilizersonalarge scale, withclaims based entirely upon thepossiblefeedingcapacityoftheplantsuponsomeoftheorganiccon-stituents, or uponsome other mysterious action of these complexesuponplantgrowth, haveso far completely failed (218, 400, 401).Thepresenceinsoil of specificplant stimulatingsubstancesis dis-cussed in detail elsewhere(Chap. XV).Modernideasconcerningther6le of humus insoil processesandplantnutrition. Liebighada purelychemical conceptionof the processesof decompositionoforganicmatterinsoilsandincomposts. Itwashis belief that theorganicbodiescombinewithoxygenoftheair, bya process of "decay," givingverysimplecompounds; intheabsenceof oxygen, the organic substances interact, giving rise toreductionprocesses, namely, thoseof "putrefaction,"as a result ofwhichthereis a greater amount of residual organic matter thanbythe aerobicprocess. The two processes were believed to exclude one another.With such primitive ideas concerning the decomposition of organicmatterinnatureandwithout theproperrecognitionoftheactivitiesof living organisms inthese processes, it is not surprisingthat theimportance of humus in the soil was not sufficiently appreciated.Liebig's influenceuponthesubsequent development ofthescienceofplant nutritionwas so great that even at the present time manyc h e m ~ists have not freed themselves from some of his opinions on this subject.With thedevelopment of thenew scienceof microbiology, when thenumerous functionsof themicroscopic formsof life inhabiting the soil,composts, bogs, and the sea began to be moreclearly understood, newlight was thrown not onlyuponthe originand chemical nature ofhumus, but alsouponits role inplant nutrition. Althoughvariousmicrobiologists, especiallyMitscherlichin1850andPasteurin1852-1860, began to recognize the importance of microorganisms as chemicalCHAPTERIIITHECHANGINGCONCEPTIONSCONCERNING THECHEM-ICALNATUREOF"HUMUS" AND"HUMICACIDS""Manbefindetsich in Bezug der Wurdigung organischer Sto:ffe im Bodennoch in der Kindheit und dieses ersieht mandarausdeutlich, dass man siedurchgehends collektiv nimmt. ' '-MULDER."Humus"and"humicacids." Theterm"humus" has had variedusage, principallybecauseverylittlewasknownconcerningthecom-plicated processes which lead to the formation of the dark coloredorganicsubstances insoils, incomposts, andinwater basins. Someofthemost outstandingchemists of the19th, andevenof theearly20th century, consideredthat these processes were rather simple intype and were effected chiefly, if not entirely, by atmosphericagencies.Chemically, humuswasusuallybelievedtobeverysimpleincompo-sition and made up entirely of one or more substances acidic in nature;theseweregenerallyclassifiedas "humicacids." Someinvestigators(654) believed that humuswasacompound of "humic acid" and lime;in theprocessof plantnutrition, thelimewasabsorbedby theplant,leaving the"humusin thesoilashumicacid.".Theterm"humicacid" was alsousedbydifferent investigatorstodesignatedifferent preparations. Thistermwasmost frequentlyap-pliedtothatpart oftheorganicmatterofsoils, peats, andcompostswhichissolubleinalkali solutions. Insomecases, thetermwasap-pliedtoonly thatpartofthealkalisolutionwhichisprecipitatedbymineral acids. Inviewof thefact that a number of "humic acids"have been described, Oden (781) suggested that "humic acid" or"humusacid"shouldbeusedtodesignateonly thatpart oftheacidprecipitate whichis not soluble inalcohol, while the alcohol-solublepart was giventhename "hymatomelanicacid." Inmost instancesthevarious "humicacids" werenot definitechemical compoundsbutrather representedpreparations possessing certain commonphysical,chemical, and biological properties. Beginning with one compound"isolated" nearly acentury and ahalf ago, more and more names wereintroducedtodesignatenew"humicacids"; this was apparentlythe2122 DEVELOPMENT OF KNOWLEDGE OF HUMUSresult of an attempt to explain the seeming discrepancies in the natureof compounds isolatedfromdifferent sources of humus bydifferentmanipulations. Conditionsbecamemorecomplexwiththegrowthofchemistry and thedevelopmentof better techniques for the separationof organic compounds. The various chemical formulae suggestedatdifferent times for the"humicacids" were, for themost part, hypo-thetical innature.The progress of our knowledge of the organic chemistry of soils,peats, andcomposts, orthechemistryofhumus, is closelyrelatedtothe changing conceptions of the origin and chemical nature of the"humicacids"andtheirrelatedcompounds.The early period (1786-1826). Thefirst historical referencetotheisolationofa compoundsimilar to thatwhichbecamelaterknownas"humicacid" dates toAchard (8), who, in1786, extracteda brownsubstancefromsoil andpeat, bythe use of alkali solutions. Uponadding sulfuricacid to thealkali extract,heobtainedadark brown toalmost blackprecipitate. Achardalsorecordedthefact that differ-ent layersof peat arechemically different in nature, and seem to haveundergone varying processes or degrees of decomposition; differentquantitiesof material were extractable from them by alkali solution.In 1797, Vauquelin (1206) obtainedsubstancessimilarinappear-ance and behavior from the stem of an old elm tree infected with fungiand fromthe bark of other trees and frompeat. The precipitateformedwhenthealkali extract was acidifiedwas againdissolvedbymeansof alkali solutions; it also produced salts with calcium and otherbases. Vauquelin did not name the substance thus prepared; thiswasdone later byThomson (1158-9), whosuggestedthename "ulmin,"fromthelatinnameoftheelmtree (Ulmus). Klaproth(548) foundasimilar blacksubstancein thealkaliextractofthegum secreted byanoldelmtree. Incourseoftimetheterm"ulmin"cametobeap-pliedtoall preparationswhichcouldbeobtainedbymeans of alkaliextractionfromsoils, peats, coals, andevenfromplants, andwhichwereblackincolor.De Saussure(942) applied the term terreau to thedark-colored mat-terformedfromdeadplants'undertheinfluenceofairandmoisture;healsousedanothersynonymous term, "humus,"theLatinequiva-lent ofsoil, todesignatethis material. DeSaussurenotedthat thishumuswasricherincarbon andpoorerinhydrogenandoxygen thanthe plant material fromwhich itoriginated. Einhof (263) introducedthe term "acid humus"("sauereDammerde")todesignate the humus22 DEVELOPMENT OF KNOWLEDGE OF HUMUSresult of an attempt to explain the seeming discrepancies in the natureof compounds isolatedfromdifferent sources of humus bydifferentmanipulations. Conditionsbecamemorecomplexwiththegrowthofchemistry and thedevelopmentof better techniques for the separationof organic compounds. The various chemical formulae suggestedatdifferent times for the"humicacids" were, for themost part, hypo-thetical innature.The progress of our knowledge of the organic chemistry of soils,peats, andcomposts, orthechemistryofhumus, is closelyrelatedtothe changing conceptions of the origin and chemical nature of the"humicacids"andtheirrelatedcompounds.The early period (1786-1826). Thefirst historical referencetotheisolationofa compoundsimilar to thatwhichbecamelaterknownas"humicacid" dates toAchard (8), who, in1786, extracteda brownsubstancefromsoil andpeat, bythe use of alkali solutions. Uponadding sulfuricacid to thealkali extract,heobtainedadark brown toalmost blackprecipitate. Achardalsorecordedthefact that differ-ent layersof peat arechemically different in nature, and seem to haveundergone varying processes or degrees of decomposition; differentquantitiesof material were extractable from them by alkali solution.In 1797, Vauquelin (1206) obtainedsubstancessimilarinappear-ance and behavior from the stem of an old elm tree infected with fungiand fromthe bark of other trees and frompeat. The precipitateformedwhenthealkali extract was acidifiedwas againdissolvedbymeansof alkali solutions; it also produced salts with calcium and otherbases. Vauquelin did not name the substance thus prepared; thiswasdone later byThomson (1158-9), whosuggestedthename "ulmin,"fromthelatinnameoftheelmtree (Ulmus). Klaproth(548) foundasimilar blacksubstancein thealkaliextractofthegum secreted byanoldelmtree. Incourseoftimetheterm"ulmin"cametobeap-pliedtoall preparationswhichcouldbeobtainedbymeans of alkaliextractionfromsoils, peats, coals, andevenfromplants, andwhichwereblackincolor.De Saussure(942) applied the term terreau to thedark-colored mat-terformedfromdeadplants'undertheinfluenceofairandmoisture;healsousedanothersynonymous term, "humus,"theLatinequiva-lent ofsoil, todesignatethis material. DeSaussurenotedthat thishumuswasricherincarbon andpoorerinhydrogenandoxygen thanthe plant material fromwhich itoriginated. Einhof (263) introducedthe term "acid humus"("sauereDammerde")todesignate the humusCHEMICAL NATURE OF "HUMUS" 23of peatsandswamps, as distinguishedfrom"mildhumus," whichisrichinbases (1139).Theideasof deSaussureconcerning theoriginandchemical natureofsoil organic matterorsoilhumusweretooadvancedforhisperiodand were soon forgotten. Inan attempt to discover new"humicacids" andto determine their chemical nature and origin, no cleardistinction was made between complexes formed under the naturalconditions prevailing in soils, composts, and peats, and the dark-colored complexes formedbythe interactionof certainorganiccom-poundswithacidsandalkalies, usually athightemperatures. Whencarbohydrates are treated with concentrated acids, a dark coloredmass is formed whichis similar incertainproperties tothe "humicacids" of decomposingplant residues. Thisleda number ofinvesti-gators to assume that both are the same chemically and that the natu-ral complexes formed by the little understood process of "humification"aresimilar totheproductswhichresult fromthetreatment ofcarbo-hydrates withacids.In 1819, Braconnot (147)submitted starch and sucrose to the actionof hydrochloric and sulfuricacids; asaresultof thistreatmentadarkliquidwas obtained, whichgavea blackprecipitateondilutionwithwater. This precipitate, which was soluble in alkalies, Braconnotcalled "artificial ulmin," since he believedit was analogous tothe"natural ulmin" found in the diseased elmtree. Later Braconnotobtained "ulmin" on warming glucose with analkali solution. Healso extracted a substance which he believed was identical with "ulmin"fromthe rotted material foundin the rootcavitiesof anoldelm tree;this was solubleinalkalies andprecipitatedbyacids intheformofbrown-black flakes. "Ulmin"was found not only in the rotted organicmatteroftreesbut alsoinpeatandeveninlignite. Braconnotsug-gestedthat "ulmin" must doubtless beanimportant constituent of"terre d'ombre," but it could not be obtained fromcoal. He prepared"artificial humus" or "artificial ulmin,"not onlybytreatingvariousorganic substances with mineral acids, but also bytreating lignousmaterial with potassiumhydroxide. Colin (182) and Proust (852)believedthat theydemonstratedthe presence of a brownsubstancesimilar to "ulmin" in the products of dry distillation of wood.In1822, Dobereiner (225) designatedthedarkcoloredfraction ofthe soil organic matter as"humus acid"("Humussaure"). Since thenthe terms"humic acid" and "humus acid"have been used quite indis-criminately, and seldom was any differentiation made between the two;24 DEVELOPMENT OF KNOWLEDGE OF HUMUSwhendistinguished, thefirst termwas usuallyconsideredtobemoreinclusive, comprising all of the "humic acids," whereas the secondwasappliedonlytoa specificpreparationobtainedastheprecipitatefrom the alkali extract by treatment with an acid. Wiegmann(1279),inoneof theearliest paperson thechemistryof peats, referred tothesubstance thus extracted as "ulmin" or "humic acid," thus opening thewayfor a confusion thatwasbound toarisefroma lackofdifferenti-ation betweenthe brownand dark colored complexes found in de-composingwood, incomposts, insoil, inpeat, andinbrowncoal, orproduced ontreatment of carbohydrates and proteins with mineralacids or alkalies.Sprengel and Berzeliu8(1826-1839). In 1826, Sprengel(1074) under-tooka detailed study of theorigin andchemical natureof the"humicacids" and their salts. It is surprising'to find that many of Sprengel'sideas are still current in the textbooks on soil science, without duerec-ognition being given to the investigator who first conceived them.According toSprengel, "humicacid"isformedduring thedecomposi-tionofplants, wherebya largepart of thecarboncombines withat-mospheric oxygen and withsome water; this process was believedtobesimilar tothat whichtakesplacewhen"humicacid"isformedontreatment of plantresidueswithpotassiumhydroxide. Thebasesenabletheplantsubstances toremovetheoxygen fromtheair. Theplant materials are transformed inthe process into "humic acids,"forwhichall bases haveconsiderableattraction. Ifthesoilisrichinbases, theresulting"humic ~ c i d " isimmediatelybound; thesoil willthen react neutral and contain "nilld humus." Such soil showsahighdegreeoffertility. The"humicacid"maybedecomposedfurthertocarbondioxide andwater. If thesoil is poor inbases, the "humicacids" remainfree andthe soil becomes acid, because of the "acidhumus,"asin thecaseof peatsoilsformedfromhighmoor peats.Sprengel treated dry peat withdiluteacid toremovethebasesstillbound to the'.'humicacid." Theresiduewaswashedwithwater anddigested for several dayswithammonia, ina closedvessel. The darkbrownsolutionwastreatedwithhydrochloricacid, anda darkbrownprecipitate("humic acid")was obtained. This was not apure organiccompoundbut containedsome ferric hydroxide andclay. The pre-cipitate was redissolved in sodium carbonate solution and reprecipitatedin thecoldwithhydrochloricacid;heat was avoidedsoastopreventthe formation of a compound with iron oxide. The "humic acid"thus prepared had ahigh water-holding capacity,100 parts of the moistCHEMICAL NATURE OF "HUMUS" 25preparationleaving, ondrying, only5 parts of material whichcon-tainedtracesofash. The dryresidual substancehadashiningblackcolor andcould easily bepulverized, breaking upintoirregular pieces..The freshlyprepared, moist "humicacid"wasinsoluble in water aslongasit containedsomefree mineral acid, but, as soonas theacidwas completely washed out, the "humic acid" went into solution, color-ing the wash water first yellow, then brown. It dissolved more readilyinwarmthanincoldwater, 6500parts of ice coldwater andonly150-160parts of boilingwater dissolvingonepart of "humic acid."When thewaterof hydrationwasremoved bydrying, freezing, or bymeansof anelectric current, the"humicacid"again became insolublein water. The"humicacid"which had been dissolved in water couldbereprecipitatedbyall mineral acids, except phosphoric, andbyallsalts of alkali earths and heavymetals, except gold, giving rise to"humates." Precipitationcouldalsobebrought about bypulverizedcharcoal and partly bycalcium andbariumhydroxides.. The"humicacid" thus obtainedwas negativelychargedandactedas a strongeracid than carbonic, liberatingsilicic acid fromsilicates. It formedsalts, giving soluble compounds with alkalies, whereas the salts ofalkali earths and of heavy metals were insoluble. A solution of "humicacid"in potassium hydroxidetreatedwith iron sulfategaveaprecipi-tateof "ironhumate"; compounds were alsoformedwithaluminumhydroxide, from ~ h i c h the aluminumcould be separatedonly withconsiderable difficulty. The various compounds thus producedwerenot constant incomposition: some wereacid, others basicorneutralinreaction. ThemethodsdevelopedbySprengel for thepreparationof"humicacid,"forthe studyofitschemical composition andquan-titativedeterminationinsoils, weregenerallyadoptedanduseduntilrecent years.In1830, Boullay(139) madeachemical ,analysisof thedarkcoloredmaterialobtained by treatment of sugar withmineral acids. Hecal-culated that "artificialulmicacid" (having thuschangedthenameofBraconnot's "artificial ulmin") had a chemical composition ofCS()Ha0015. Malaguti (669) suggested an explanation for the trans-formationof thecarbohydrates by mineral acids intocompounds simi-lar to those produced under natural conditions in soils, peats, and com-posts. He stated that nitric acid changes sugar into oxalic acid,whereas hydrochloric and sulfuric acids give partly "humic acid,"whichissolubleinalkalies, andpartly"ulmin,"whichisinsolubleinalkalies. Concentratedacidsact moreenergetically thanweakacids.26 DEVELOPMENT OF KNOWLEDGE OF HUMUSUnder theinfluence of theacid, thesugar loses water, givingfirst acompoundofthestructureC12H2S014, whichchangesto"humicacid"and finally to formic acid. The mechanism of formation of the "humicacid"wasillustratedasfollows:C12H22011= C12H1206 +5H20Sucrose "Humicacid"Stein (1092) proposedthe formula C24HlS09for the "humicacid"obtainedbythe action of hydrochloric acidupon sugar. Berzelius(112) adopted later another formula, namelyC32H32016, forthisprepa-ration.Berzelius recognized the importance of the investigationsof Sprengelbut he was misled by the ideas of Boullay and Malaguti concerning theformation of "humic acids" fromcarbohydrates byacid and alkalitreatments. Hedistinguished two darkbrown bodies: 1. Asubstancesoluble in alkalies, which gave a dark brown precipitate when thealkaliwasneutralized by anacid(the"humicacid"ofSprengel), wasfirstcalled "mylla," then"geic acid," and finally "humic acid". 2. Theother substance, which was insoluble inalkalies (the "humus coal"ofSprengel), hedesignatedfirst as"mull coal"andlater as"humin."Berzelius recognized that the term "ulmin" was being used for avarietyof substances probably possessing marked chemical differences; thecomplexesextracted fromthesoil by theuseof analkali solution, forexample, probably have no special relation to those which are extractedfrom an elm tree or from other plants. He called attention to the futil-ity of designating by one namebodies only little investigated and pos-sessinga certain superficial similarity.In 1833, Berzelius isolated from mineral waters and later from slimymudrichinironoxide, twonewcompounds whichhe designatedas"crenic acid" and"apocrenic acid." These complexes were believedto be present also in plants and in soil. They were prepared by extract-ing the material with hot potassiumhydroxide solution; the alkalisolution was then saturated with acetic acid, and copper acetate added;the brown colored precipitate was designated "copper apocrenate."Whenthefreeaceticacidwasneutralizedwithammoniumcarbonate,a grayish green precipitate, called "copper crenate," was formed.Whenthecoppercompounds weredecomposedwithhydrogensulfide,the"freeacids" were broughtintosolution. Under theactionofair,the"crenicacid" in solution was believed to beoxidized to"apocreriicacid." The "crenic acid" preparation was soluble inwater andinCHEMICAL NATURE OF "HUMUS" 27alcohol and formed insoluble compounds with silicic acid and withaluminumhydroxide. The "apocrenic acid" preparationwas similarinitspropertiesto"crenicacid" but wasdarkcoloredandwas onlypartly soluble in water and in alcohol; it could be precipitated from itsaqueous solutionwithinorganic acids andwithammoniumchloride,andformed compounds withaluminumhydrate. Thesalts of thesecompoundswithalkalies, withmagnesium, withiron, andtosomeex-tent the salts with calciumwere soluble inwater. WhenBerzelius(113) investigatedthe "humic acid" of a rottingoaktree he couldhardly distinguish it from "apocrenic acid." He suggested that"crenic" and "apocrenic acids" are formed inthe decomposition oforganic matter ingeneral andinthe rottingof woodinparticular;fallowingof soil and cultivation giverise to"humic substances"whichare assimilable byplants; when coal is acted upon bynitric acid,"crenic" and"apocrenic acid"are produced. Different formulaeweresuggestedfor thesetwopreparations, namelyC24H12016andC24H6012.Peligot (812) was the first to demonstrate that "artificial humicacid" varies chemically from "natural humic acid," the former contain-ing62per cent carbon, andthelatter, only57per cent. However,theseanalyses werenot sufficiently' conclusivetoestablishthediffer-encebetweenthetwopreparations, since, as will beshownlater, thecarboncontent variesconsiderably inbothartificial andnatural com-plexes, depending upon the nature of the material used and the methodsemployedintheirpreparation.Mulder-Hermann(1839-1862). Beginning with1839, there appearedaseries of papers by Mulder(731-734),a pupil of Berzelius, concerningtheformationof brownandblacksubstances bytheactionof acidsupon carbohydrates. The various preparations were separated intotwo groups onthe basis of their color andsolubilityinalkalies: 1.brownbodies, or the alkali-soluble "ulmic acid" andalkali-insoluble"ulmin"; 2. blackbodies, orthealkali..soluble "humicacid" andthecorrespondingalkali-insoluble"humin." Thefirst groupwasbelievedto be somewhat richer in carbon.AccordingtoMulder, inthedecompositionof plantsubstances, theorganicn1.atter isfirst changed to"ulmic acid"and to"ulmin,"whicharepresumablyfoundindeadleaves, rottingwood, andbrownpeat.On oxidation or on boiling withacid or withalkali solutions, oxygen isabsorbedbythe "ulmicacid" andthe "ulmin"; this results intheirtransformationto "humicacid" and"humin"; onfurther oxidation,the"humic acid"is changed to"crenic acid"andfinally to"apocrenic28 DEVELOPMENT OF KNOWLEDGE OF HUMUSacid,"gradually giving rise to productscontinuously poorer in carbon."Apocrenicacid"wasreducedbynascent hydrogento"crenicacid,"and the latter was oxidized again by the oxygen of the air to "apocrenicacid." The "ulmin" and"ulmicacid" were believedtorepresent agroupofbodiesof thegeneralformula,C4oH2s012 +nH 20nbeing1, 2, 3, or higher. Muldersuggested thatthefollowingreac-tions are involved in the formation of "humic bodies" fromplantconstituents: +8 C021Cellulose "Ulmic acid"C4oH2S012 +O2= C4oH24012 +2H20"Ulmic acid" "Humic acid"C4oH24012 +"Humic acid" "Crenic acid"Thehumus preparations were driedat 140-195C., a temperaturesufficientlyhightomodifytheir 'chemical nature, hencetheformulaesuggested werepractically meaningless. Mulder believed that "humicacids"arecompoundsof carbon, hydrogen, andoxygen, the latter twoinaratioof twotooneasin water;nitrogen, whichisalwayspresentinthe "humic acid" preparations obtainedfrompeat andsoil, waslookeduponas animpuritylargelypresent intheformof ammonia.Mulder recognized later (735) that the nitrogenin "humic acid" isin the formof aprotein,which remains in the soil for along time as animpurityof thelatter. Mulderdescribeda number of newprepara-tions called "glucic acid," "apoglucic acid," "chlor-humic acid," "humo-nitricacid,"etc. These nameswere later abandoned, in preference toBerzelius' classification.The complex terminology of the "humic acids" and the lack of under-standingof the process of humus formationintroducedconsiderableconfusionduringthemiddleof thelast century. Tosummarize, itissufficient toquote fromMulder himself: "rottingwood contains, atthe beginningof the process of rotting, crenic, apocrenic andulmicacids;if freeair isadmitted, thecrenicaciddisappears and ischangedto apocrenic. The cellulose is changed to ulmin and ulmic acid,which,under the influence of air, are changedtohuminandhumic acid."These andsimilar ideas concerningthemechanismof decomposition1 No attempt is made to balance thesereactions, since they arehighly hypo-thetical.CHEMICAL NATURE OF "HUMUS" 29ofplant residues andtheir transformationintohumus couldnot butexert aninfluence of extremedisorder uponthesubsequent develop-ment ofthesubject.These ideas influenced Liebig (638), who stated that humus is formedfrom plant residues by aprocessof"eremacausis," or slow combustion,whereby the plant bodies, especially substances which contain nitrogen,absorb oxygen and are slowly converted into humus. However, Liebigclearlyrecognizedthelimitations of thestudyof "humicacids"andtheir bearing upon ourknowledge of theorigin andchemistry of humus.He stated definitely in1840 that"humicacidofchemistsisaproductof the decompositionof humus byalkalies; it does not exist inthehumusofvegetablephysiologists." BerzeliusandMulder, as well asLiebig, believed that "humicacids" arenot present ina freestateinthesoil, but that theyexist intheformof neutral bodies, largelyas"ulmin" and"humin." Whentheinsolublebodies are washedwithwater andextractedwithalkali solutionS'; theyare partlydissolved,giving rise to the corresponding "ulmic acid" and "humic acid." Mul-deremphasized that"naturalhumicacids" canbedistinguishedfrom"artificial humicacids" by the fact that the latter are totally insolubleincertainalkalisalts, whereastheformer arepartlysolubleinpotas-siumnitrateandsulfate, andcompletelysolubleinpotassiumacetatesolution. Several yearslater, S. Johnson(507) foundthat if thesoilis treated continuously with sodium carbonate solution, all the "humus"can bebroughtintosolution. ThisledJohnson toexpresshisdoubtsconcerningthe existence of "ulmin" and "humin," as distinct from"ulmic acid"and "humic acid."A most extensive study of the dark colored compounds obtained fromsoil, peat, and or prepared artificially in the laboratory, was madebyHermann (422-424). Thehumus bodies were divided intothreemain groups:1. Substancessolubleinalkaliesandprecipitatedbymineral acids:1. "Humicacids"of the typeCaoHsoOI5, insolublein sodium acetatesolutionandprecipitatedbyall acids:(a) "anitro-humic acid," freefrom nitrogen and pre-pared from sugar;(b) "sugar-humic acid," CsoHso012Ns, contaiJ;lingnitrogenandprepared from sugar;(c) "ligno-humic acid,"CSOH60012N6,prepared from rotten woodbyextractionwithalkaliesandprecipitationwithacids;(d) "meta-ligno-humicacid," C6oH60012N5, producedbyboilingfreshlyprepared"ligno-humicacid"withwater.30 DEVELOPMENT OF KNOWLEDGE OF HUMUS2. "Aprocrenic acids" containing less water and more carbon.. Thesearesolubleinalkali acetatesolutions, withtheliberationofacetic acid, and are not precipitated from alkali solutions withaceticacid;the"apocrenicacid"ofBerzeliusbelongstothisgroup. Thesecomplexeswere separatedon thebasisof theirnitrogen and oxygen content:(a) "torfic acid" and "apo-torfic acid," C2oHs406N4, foundlargely inpeat, insoil, and inbrowncoal;(b) "arvic acid" and "apo-arvic acid,"C30H4s06N12, with specialreference to the "Tula-arvic acid" and "Siberia-arvicacid" ;(c) "Porla-apocrenic acid,"obtained by Berzelius froma PorIawell (384) ..II. Humus substances soluble in water:1. "Humous extract,"C16H3207N2.. The"humous extract"was con-sidered tobea body that remains in solution, when the alkaliextract of a soil has beentreatedwithacetic acidandwithcopper acetate, toremove the bodies belongingto Group Iandthe "crenicacid." Whenthisextract was treatedwithleadnitrate andammonia, a precipitate was formed givinga "chestnut-brown, shiny transparent substance, having abitter taste.. " It was solublein80per cent alcohol andinether, andcouldbeprecipitated withvarioussaltsandacidsas a brownmass of thenature of a waxysubstance. Withbariumandcalciumit gave browncompounds, soluble withdifficulty.. It was precipitated with basic lead oxide and copperacetate, evenintheabsenceof ammonia, andwas thus dis-tinguished from"crenicacid."2. "Crenicacid" remainedinsolutionwhenthealkali extract of asoil was acidified with acetic acid and when copper acetate wasadded; when this liquidwas treatedwithanexcess of am-monia, the "crenic acid" was precipitated. Hermann dis-tinguished several crenic acids; namely, "ligno-crenicacid," ClsHlS09N2, obtained fromwood; "hurno-crenicacid,"ClsH3609N4, from a chernozem soil; "torfo-crenic acid,"C15H4S012N4, frompeat; "anitro-crenicacid," C15H24014, freefromnitrogen. Onfurther oxidation, these "acids"changedintothecorresponding"oxycrenic acids"; during thisprocessofoxidationtheywerebelieved tobeabletoabsorbnitrogenfrom theair;they were distinguished from the"crenic acids"bybeing precipitated withleadandcopper salts, eveninthepresence of a slight excess of acetic acid. Theformula for"torf-oxy-crenic acid" was given asC12H1204N4.III. Humussubstancesinsolubleinwaterandinalkali solutions:1. "Anitrohumin."2. "Nitrohumin."3. "Nitrolin."CHEMICAL NATURE OF "HUMUS" 31The first two complexesw ~ r e those previously designated by Sprengelas"humus coal" and by Berzelius and Mulder as "humin." Hermannseparated them on thebasis of their nitrogen content. "Anitrohumin"was preparedfromsugar andwas, therefore, nitrogen-free. "Nitro-humin" containednitrogen becauseit was obtainedfromsoil. Thethird complex, "nitrolin," was discovered byHermannin nitrogen-rich wood,the specific name originating from the words"nitrogenium"and"lignum,"meaningnitrogenandwoodrespectively. Therottedwoodwas groundandwashedwithwater; thematerial whichsettledinthewater was boiledwithpotassiumcarbonatesolution; the "ni-trolin" did notgo into solution; itwasinsoluble in water, alkalies, andacidsandcontained12 per centnitrogen.Onthe basis of the aforementioned complexes, Hermannreportedthe followingcomposition of peat in the vicinity of Moscow:Peat-carbon, nitrolin and plant remains 77.5 per cent"Humic acid".. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 17 per cent"Humic extract"... . . . . . . . . . . . . . . . . . . . . . . . . . . .. 4 per centAmmonia. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 0.25 per cent"Crenic acid"... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . traceAsh. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 1.25 per centAchernozemsoilyielded89.58per cent inorganic matter, 1.66percent nitrolin, etc., 1.77per cent "humic acid," 3.1 per cent "humicextract,"1.77 percent "torficacid," 2.12percent "crenicacid."All thesedesignations whichhavebeenappliedtopreparationsob-tainedfromsoil andpeat, orfromsugarsbytreatment withmineralacids, couldhave beenmultipliedadinfinitum, the limit beingonlythenumber of soilsthat onewouldcaretoextract. But evenmorefantasticthanthenames, weretheideas ofHermannconcerningtheoriginof all thesecomplexes. It is sufficient tocall attentiontothefactthathewasfirmlyconvinced thatall plants obtain their nitrogenfromtheatmosphere, andthat eventheartificiallyprepared"humicacid" and"humin" canabsorb nitrogen. Whenthe "humic acids"arechangedto"crenic"and"oxycrenic" acids, morenitrogenis ab-sorbed. Thisconceptionwasquitewrong, sincemost of thenitrogenfound in these preparationsoriginated from thenitricacid and ammo-niawhichwereusedextensivelyintheirextractionandprecipitation.Senft (996) actually suggested thatmost ofthenitrogen inthesoil ispresentlargelyasammonia, combinedwiththe"humicacids."Theseideas wereseverelycriticizedbyMulderand, althoughHer-32 DEVELOPMENT OF KNOWLEDGE OF HUMUSmann(425) soon replied, Mulder disregarded his statements completelyinhis bookonsoil chemistry (735). Inviewof the fact, however,that Mulder also disregarded the very excellent work of Sprengel,Baumann(74)accusedM u l ~ e r of overlooking the work of Sprengel andHermann, inordertoplace intheforegroundhis ownmuchinferiorwork. Baumannfurther emphasizedthefact thatin'spiteof certainrepetitionsandconfusions interminology(suchasnaming the"humicacid" of Berzelius, "torfic acid"; the "apo-crenic acid," "oxy-crenicacid" and the"ulmic"and"humicacids"ofMulder "anitro-humic" , ,and"sugar-humic" acids) inHermann's work, his manipulationsandchemical analysesshoulddeservegreaterconsiderationthanMulder'sideasof separation of the humuscomplexeson thebasisof brown anddarkcolors.In addition to the "acids" of Mulder and Hermann, numerous othershave beendescribed, including: "mudesous acid," C12HIOOS, of John-ston(508); "carbo-ulmic acid,"C4oH36016, obtained by Herz(517)fromlignite; "silico-azo-humicacids" which, accordingtoThenard (1148),areformedspontaneously in thesoil fromthe"humicacid,"fromtheammonia in rainwater, from the nitrogenof the air, and from the silicainthesoil (517).Therapidincreaseinthenumberofpreparationsisolatedfromde-composing plantresidues and from humus formationsanddescribed as"humicacids,"aswell asthegrowingconfusion in theterminologyofthesevarious preparations, was largelyduetotheinsufficient knowl-edgeof theprocessesofdecompositionof plant and animalresidues innature.Ontheotherhand, theuseof wordsandsymbolswhereideasandfactswere lacking has contributed further to the lack of understandingof the processes underlyinghumus formation, and, therefore, of thechemical natureof humus itself. Itissufficient tocitethefollowingideas of Souberian (1069) presented in 1850: "decomposed wood(Dammerde) consists of a mixture of pure 'humus,' some 'calciumhumate,' andasubstancewhichchangesintohumus, undertheinflu-ence of alkalies, in the presence of air; the same is true for soil and peat,accompaniedbya rapidabsorptionof oxygenbytheorganicmatterkept in contact withammonia. The favorable effect of bases uponacidpeat isduetothetransformationofthealkali-insoluble'humus'intoalkali-soluble'humicacid.' "The science of microbiology, whichalonecould elucidate thoseproc-esses, wasstillnon-existent. Itisinteresting tonotethateven at anCHEMICAL NATURE OF "HUMUS" 1,33earlystage of thedevelopment of our knowledge of theactivities ofmicroorganisms, suggestions weremadeconcerningtheir contributionto the processes of humus formation. Braconnot, for example, reportedin1838that thebodyof thefungus Agaricus atramentarius contains"humic substances." Similar suggestions were made for Uredosegetumby Lucas (655), whobelieved that the "humic" material in the sporesof ~ h e fungus is formedfromstarchthroughthelifeactivities of theorganisms. A specific "acid" occurring infungi upon decomposingwood has later (1088a) been describedas"polyporicacid."With an increasing understanding of the chemistry of plant productsandtheirtransformationbymicroorganisms, thechemistryofhumuswas boundtobecomebetterunderstood. Asimilarity was observedbetweentanninsanda substancedesignatedas lignoin, foundintheold bark of trees; this lignoin was found to possess most of the propertiescharacteristicof"humic acid"andwas, therefore, believed to serveasa source of "humus" innature. This ideawas confirmedbyHesse(429), who found a parallelismbetween the transformation of thequinine andcinchonine in the bark of the quininetree into lignoin andthe formationof"ulmicacid"from sugar by theuse of mineral acids.The presence of humus-like compounds inplants was demonstratedby Thenard(1149), whoextracted"humus"fromsaw-dust bymeansof ammoniumhydroxide; he further (1146) emphasized the role ofthese compounds inthecompostingof stable manure. Millon (790)isolateda similarpreparationfromwoodcarbonizedat 330C.Thesynthesisofdarkcoloredsubstancesresemblinghumusfromavariety oforganiccompoundscontinued. Berthelot (101) foundthat,whenamixtureofalcoholicalkalinehydroxideiswarmed withcarbontetrachloride in a closed vessel, substances similar to "humic acid"are obtained. According to Hardy(391), amixture of chloroform andacetone, undertheinfluenceofsodiumcarbonate, gives a brown sub-stance similar to "humic acid." This preparationwas described as"acet-ulmic acid"of thecompositionC14H1204; itwaseasily convertedto "dioxy-acet-ulmic acid,"C14HIOOS. Liebig found(638) that an alka-line solution oflactose andtannin on standing in contact with air,givesrise to"humic compounds." Lefort prepared(616)from rotting wooda substancewhichhecalled"xylylacid,"oftheformula C24Hao017.Therelationship ofnitrogentotheprocess ofhumusformationwasespeciallyconfusingtoa number of investigators, sincenitrogenwasfoundin somepreparationsandwaseitherabsent or present in smallamounts in others. Thecommon idea prevailed thatnitrogen ispres-34 DEVELOPMENT OF KNOWLEDGE OF HUMUSent in thehumusin theformof ammonia, givingasaltwith the non-nitrogenous "humic acid.'-' Thenard (1145, 1150) emphasized the role ofnitrogenous substances in the origin of humus. He suggested the name"fumic acid" for the humus present in stable manure, in whioh nitrogenisknowntoplayanimportant part; "fumicacid"wasalsoextractedfromcultivated soil, but it was then foundtocontain only little nitro-gen, much carbon, and certain amounts of hydrogen and oxygen.Mulder (735), however, believedthat "fumicacid"ismerelyanam-moniumsalt of "humicacid,"inotherwords, "ammoniumhumate."Aninsufficient knowledgeofthechemical compositionofplantandanimal residuesundergoing decomposition, atotal lackof appreciationoftheimportanceof microscopicforms oflifeintheprocesses of de-composition, andthefailuretorecognizethechangesthat takeplacein the variousconstituentsof the plant and animal residuesasaresultof decomposition, werelargelyresponsibleforthefailuretorecognizethe exact natureof humus. Theintroductionof the concept of theexistence of specific "humic acids"; the description of an ever-increasingnumber of these acids; the use of formulae based merely on elementaryanalysisandonpreconceivedideas concerningthesimplicityof thesecompounds; thegeneralizationthat darkcoloredsubstancesfoundindecomposing plant materials, in peat, and in soil are the same asthoseproducedbyheatingcarbohydrates withacids-all tendedtofurtherconfusethe subject and introducecomplications atatimewhen infor-mationonbasicprincipleswasstill lacking.Detmer-Hoppe-Seyler(1871-1889). In contradistinction to the ideasof theearlier workersconcerning themultiplicity of humussubstancessoluble in alkalies andprecipitated by acid, Detmer(219) cametotheconclusion that all these preparations represent only one such substance.This can be obtained from soil or peat andcan be prepared artificiallyfromsugar; theformula C6oH54027 wassuggestedfor this compound,whichwasdesignatedas"humicacid,"whilethename"humusacid"was reservedfor those compounds whicharesoluble inwater. Thenitrogenpresent inthe "humic acid" was still consideredtobe animpurity, andevery possibleeffortwasmadetoremoveit. Peat wasextracted with potassium carbonate solution for 48 hours; the extractedportion was precipitated with amineral acid; this process was repeatedby extractingtheprecipitateagainwithpotassiumcarbonatesolutionand by reprecipitating andwashingtheprecipitatewithboilingdiluteacidandwithhot water. Thepreparationthusobtainedwas fin