February, 1935 I N D U S T R I A L A N I ) E N G I N E E R I N G C H E M I S T R Y 195

point increased regularly and quite markedly, and the vis- cosity increased rapidly. The portion of the asphaltic com- pound soluble in carbon disulfide, designated “total bitumen” for convenience, varied between 99.6 and 99.9 per cent without apparent congruity. Figures 4 to 9 give the in- dividual variations among the three materials for most of the characteristics under (observation.

The results, with one exception, were found to be in agreement with the preliminary expectations concerning the type of change that would be apt to be brought about by continued heating. 1.n accordance with general belief, it was thought that the acid number of the materials, particu- larly the two asphalts, would tend to increase over :t period of time at elevated temperatures. Nevertheless, the re- sults revealed the opposite trend, after initial increases for the asphalts, and the authors are uncertain as to the cor- rect explanation of this phenomenon. However, it is pos- sible that acidic substances were produced initially upon heating and that they were volatilized later and escaped continuously from the oven throughout the duration of the test, especially a t the time the doors were opened for sampling. It may be, also, that asphaltic materials . which are heated for long periods in the presence of sufficient air, or sunlight, will show a subsequent rise in acidity due to the generation of more actively acidic materials through oxidation or photochemical effects.

It is hoped that other laboratories will be interested in examining typical asphalts that are commercially available with a view toward augmenting the data here reported, or for the purpose of ascertaining the direction and magnitude of changes in such other properties of the materials as may be of interest. It would be especially valuable to determine what changes in acidity would occur in asphalts heated con- tinuously in the presence of the optimum amounts of air and sunlight.

ACKNOWLEDQMENT The authors are indebted to the Western Electric Company

for facilities and encouragement while carrying out this in- vestigation and for permission to publish the data obtained.

LITERATURE CITED (1) Abraham, Herbert, “Asphalts and Allied Substances,” 3rd ed.,

n. 44. h-ew York. D. Van Nostrand Co.. 1929. (2) C&er,’L. E.,Olson,J. W., Hardlng, C. F., and Shreve, R. K.,

(3) Hixon, A. W., and Hands, H. E., J. I ~ D . ENG. CHEM., 9, 651-5, Eng. Bull. Purdue Univ., 16, No. 6 (h-ov., 1932).

esp. 653 (1917).

54, 58-68, 75, New York, D. Van Nostrand Co., 1925. (4) Spielmann, P. E., “Bituminous Substances,” Chap. IV, esp. pp.

RECEIVED October 8, 1934. A paper under this title was presented before the Division of Industrial and Engineering Chemistry a t the 86th Meeting of the American Chemical Society, Chicago, Ill., September 10 to 15, 1933.

Utilization of Agricultural Wastes I. Lignin and Microbial Decomposition

MAX LEVINE, G. H. NELSON, D. Q. ANDERSON, AND P. B. JACOBS Agricultural By-products Laboratory, Bureau of Chemistry and Soils, United States Department

of Agriculture, and Engineering Experiment Station, Iowa State College, Ames, Iowa

N THE following discussion both natural and prepared lignins are considered, and it should be borne in mind that I the results obtained with prepared lignin are not neces-

sarily applicable to natural lignin. The fate of l i g i n when exposed to the action of bacteria

has attracted the attention of numerous investigators, but their observations, as reported in the literature, are in dis- agreement. It is not the intention here to review in detail the literature on this subject, but the following excerpts may serve to outline the present status of the problem.

Schrader in 1921 (cited by Phillips, 7 ) reported that bac- teria were incapable of breaking down prepared lignin (Will- st5itter method) in 25 days.

Phillips, Weihe, and Smith in 1930 (8) concluded that “under proper conditions soil organisms are capable of de- composing lignin as found in plant materials. Under suitable conditions the rate of decomposition of the lignin may be as great as that of the cellulose (Cross and Bevan) and pento- sans.” Their experiments were made under aerobic condi-

Waksman and Tenney in 1926 (IO) at- tested to the extreme refractory nature of lignins: “Lignins are not decomposed in the soil, a t least within the experimental period of 32 to 35 days. If they are decomposed a t all, the amount of decomposition is only insignificant in comparison with the decom- position of the other constituents of natural organic matter.” In 1929 Waksman and Stevens (15) declared that “under anaerobic conditions lignins do not decompose a t all, or only in mere traces, owing to the ab- sence of specif ic organisms, while under aerobic conditions the lignins are slowly decomposed, but here as well they are found to be the most r e s i s t a n t group of plant constituents.”

I n the course of studies on the utilization qf f a r m wastes, especially the production of fuel gas by fermentation, attempts to develop a spe- cific anaerobic lignin-digesting jlora were unsuccessful.

Alkali lignin, when added to an actively digesting sludge, produced practically no gas, even under optimum conditions; furthermore, when such alkali lignin was used in conjunction with .fermenting cornstalk jlour or packing-house sludge, the gasification of the latter materials was markedly inhibited. This depressitre effect is apparently not due to a toxic action of the lignin on the bacterial jlora, but is presumably due to chemical combination, with the possible production of complexes very resistant to microbial decompos it ion.

A considerable portion of the reported losses in lignin, attributed io microbial decomposition, may be explained by the technic of selection and preparation of the sample fo r lignin analysis.

196 I N D U S T R I A L A N D E N G I N E E R I N G C H E M I S T R Y Vol. 27, No. 2

GAS PRODUCTION FROM PREPARED LIGNIN Schrader had previously reported that lignin prepared by

the Willstiitter method (acid lignin) was refractory to bac- terial action (7'). In the experiments reported by Buswell, where gas was obtained from cornstalks, the reaction of the medium was maintained slightly alkaline. As some relatively pure lignin substratum is indispensable for enrichment and isolation of lignin-destroying bacteria, alkali lignin was chosen as a test material in the present experiments.

The procedure, briefly, was to develop an active fermentation, in which lignin was presumably being decomposed, and then add only li nin to the mixture. Thus, by restricting the food su ply to frestly added lignin, those bacteria incapable of attacfing this compound would be inhibited, whereas the lignin flora would become enriched. Cornstalk flour was added to an active packing- house sewage sludge in a large Pyrex bottle equipped with the necessary outlets and apparatus to collect the gases evolved. A vigorous methane fermentation ensued. Fresh additions of cornstalk flour were made from time to time, together with sufficient ammonium salts to maintain a concentration of 400 to 500 p. p. m., thus developing an active seed in which lignin was presumably being decomposed, with gas formation. This seed was then divided into three parts. To one part was added a

. fresh batch of cornstalk flour (this served as a control on the activity of the seed), to another part was added some alkali lignin (kindly furnished by Max Phillips); and the third portion was kept as a control for gas evolution from the seed proper. Observations on each of the portions were made with two batches of seed, one developed at a high temperature (50" to 55" C.) and the other at 28" to 30" C. The results are shown in Table I and Figure 1.

TABLE I. ANAEROBIC DECOMPOSITION OF ALKALI LIQNIN~ AND CORNSTALK FLOUR

(Experiment started January 12, 1932) Digestion temp., O C . 4 0 Experiment No. T-21 T-20 T-25 R-20 RZ RX

to 55O C.- 7 2 8 ' ' to 30' C.-

8- - D/GfST/ON 47 28-JO'C. -X-*-D/GPS 74N AT SO-5S'C.

C.C. O f GAS PfR GRAM Of fRf - - - -- - * CORNSTALK fLOUR

(Sil98G. YO1 . SOLIDS ADOfUl

-a - - ~ ~ ( D / G ~ u F o AT rn-5x"c) - I' I' 28-JODCJ

S 6 9 /Z /I /6 I / 24 2 7 SO 35 36 39 42 9J- 48 T/ME (INDAYS)

Figure 2

FIGURES 1 AND 2. GAS PRODUCED FROM ALKALI LIGNIN AND CORNSTALK FLOUR

tions, and they specifically state that their conclusions may not apply to anaerobic decomposition or to prepared lignin.

Boruff and Buswell in 1930 (1) put forth the thesis that lignin in cornstalks is not only decomposed anaerobically, apparently as readily as the pentosans therein, but that methane and carbon dioxide constitute important end prod- ucts; they state that "the anaerobic flora and digestion condi- tions present were capable of fermenting a t least part of the lignin to gaseous end products. This fact may find com- mercial use in the gasification of the enormous amounts of lignin now thrown to waste."

I n the course of investigations on the utilization of farm wastes, one of the projects under study was concerned with the possible isolation of organisms capable of decomposing lignin under anaerobic conditions, the assumption being that if lignin is decomposed as readily as reported, some specific bacteria would probably be associated with the phenomenon. The value resulting from the detection of such specific organ- isms is apparent; if they exist, are isolated, and are found to be really capable of destroying lignin (and not cellulose, for example), then a possible means of recovering cellulose from cornstalks, free of the troublesome lignin may be visualized. Even if such organisms were found to be not specific, but capable of decomposing other cellular constituents, the claim that methane and carbon dioxide are important end products of lignin degradation would justify research for ascertaining the optimum conditions for such decomposition. It was incidental to such a search for specific lignin-destroying anaerobic bacteria that the phenomena briefly described were encountered.

Material sub- Lignin Corn- Seed Lignin Corn- Seed jected t o fer- stalk control stalk control mentation flour flour

Digestion vol., liters 3 . 0 3.0 3.0 2.5 3.0 3.0 Days incubated 93 20 99 89 29 90 Material added,

grams volatile solids 43.59 43.33 , . . 34.87 40.60 . . .

Seed present, grams volatile sqlids 49.47 49.47 49.47 41.48 41.48 41.48

Ratio fresh volatile solids to volatile solids inseed 0.88:l

Gas recovery at 60' F. (15.6' C.) and 760 mm., cc. 600

Vol. of pa!, cc./gram volatile solids added:

Uncor. forseed 13.75 Cor.forseed -189.0

0.87:l

24,120

556 352

8870

. . . . . .

0.84:l

1610

46.17 -79.0

0.98:l

24,540

576 496

. . .

4360

. . . . . . 7.8 7.4 7.8 7.0 6.9 7.1 PH

Lignin supplied by Max Phillips of the United States Department of Agriculture.

In 20 to 29 days more than 24,000 cc. of gas were produced from the cornstalk flour a t each of the temperatures employed, whereas only 600 cc. of gas were produced in the lignin series a t 50" to 55" C., and 1600 cc. a t 28" to 30" C. in 90 days. Gas productions of 556 to 575 cc. per gram of fresh volatile solids, added as cornstalks, were reduced to 13.75 and 46.17 cc. per gram on the basis of volatile solids added as alkali lignin. These gas figures are not corrected for gas evolved from the seed alone. Were such a correction made, negative gas productions would be encountered on the basis of the volatile solids added as lignin, for the gas evolved from the lignin series was considerably less than that produced from the "seeds" themselves.

Such a correction may be made by subtracting the amount of gas produced by the seed control from the amount produced from the lignin or the cornstalk flour series. The amount of seed used in these experiments was the same as that used in the seed control. In the case of the lignin experiments this correction gives a negative quantity of total gas and, when

February, 1935 I N D U S T R I A L A N I ) E N G I N E E R I N G C H E M I S T R Y 197

divided by the fresh volatile solids added, gives a negative quantity of gas per gram of fresh volatile solids added. This indicates a marked restrictive action and interference with the course of the fermentation on the part of the alkali lignin.

In order to ascertain whether this behavior of alkali lignin was peculiar to the particular batch amployed, a new batch of alkali lignin was prepared as follows: Broken corncobs were soaked, under pressure, in 4 per cent alkali for several hours and filtered to remove insolubles, and the lignin in the filtrate was precipitated with acid, washed, and dried.

The compositions of this lignin and the lignin previously used are shown in Table 11. The freshly prepared batch, as used, was much higher in ash and pentosans and lower in lignin content, some of the salts formed in the neutralization of the acid remaining in the material.

TABLE 11. COMPOSITION OF ALKALI LIGNIN SAMPLES CONSTITUBINTS LIQNIN A' LIQNIN Bb

Moisture 3.07 4.33 Total solids (oven-dry weight) 96.93 95,67 Volatile solids 96.87 90 01 Ash 0.06 5.66 Lignin (72% H2SOd method) 87.37 77.50 Pentoaana 0.76 7.94 Kjeldahl nitrogen 0.58 0.60

a Furnished by Max Phillips. b Prepared at Iowa State College; the method was dewribed by Phillips

in 1928 (7), but 4 per cent sodlum hydroxide was used.

The results of an experiment similar to that already de- scribed, using the freshly prepared alkali lignin, are shown in Table I11 and Figure 2. They corroborate the previous observations. Thus, 491 cc. of gas per gram of volat,ile solids added as cornstalk flour were produced in 32 days a t 50' to 55"C., but only 48 cc. per gram of volatile solids, added as prepared lignin, were produced under the same conditions. At the lower temperature the volumes of gas evolved in 57 days were 642 and 14 cc. per gram of volatile matter added as cornstalk flour and lignin, respectively. Here it was again observed that less gas was produced on addition of prepared lignin than was formed from the seed itself. The seeds used in this experiment were not identical with the seeds employed in the previous experiment.

TABLE 111. ANAEROBIC: DIGESTION OF ALKALI LIGNIN4 AND

Digestion temp , e C. -50° to 55O C. --- -28O to 30° C.- Experiment No. T-41 T-40 T-46 R-41 R-40 R-46 Materialsub- Lignin Corn- Seed Lignin Corn- Seed

CORNSTALK FLOUR

iected to fer- etalk control stalk control mentation flour flour

Digestion vol., liters Days incubated Material added,

gr+nls volatile

4.0 4.0 4.0 4.0 4.0 4.0 32 32 32 57 57 57

57.74 57.74 ... 68.70 68.90 ... 52.49 52.49 52.49 68.50 68.50 68.50

1.1:l 1.1:l ... 1:l 1:l . , ,

2760 28,425 3055 965 44,165 7500 Vol. of gas, cc./gram

volatile d i d s added: Uncor. for seed 48 491 . . . 14.0 642 ... Cor. for seed -5.1 442 . . . -95.2 532 . . .

PH 7.8 7.9 8.2 7.8 7.7 8.0 4 Lignin prepared from corncobs at Iowa State College.

Obviously a lignin-fermenting flora was not likely to be developed along the lines of investigation chosen, but, in view of the results observed, further experiments were carried on to determine the effect (on gas production) of adding alkali lignin together with other materials to an active gasifying seed or sludge.

EFFECT OF ALKALI LIGNIN ON GASIFICATION OF CORNSTALK nom AND PACKINGHOUSE SLUDGE

Actively fermenting seeds were prepared a t thermophilic (50' to 55' C.) and mesophilic (25" to 30' C.) conditions by

repeated additions of cornstalk flour. The ammonia content was adjusted to about 500 p. p. m. by addition of a mixture of ammonium salts (phosphate, carbonate, and hydroxide) and the seeds were divided into six portions of 3 liters each. The following materials, suspended in a liter of distilled water, were then added to respective batches of seed.

1. Alkali lignin 2. 3. Packinf-house sludge 4. a. Cornstalk flour 6 . No additions (seed control)

Alkali lignin together with packing-house sludge

Alkali ignin together with cornstalk flour

The ratio of fresh volatile solids added to that in the seed was in each instance about 1 to 1.1, and, where lignin was added together with other materials, lignin represented half the fresh volatile solids added.

The analyses of the cornstalk flour and packing-house sludge employed are shown in Table IV, the results of the experiments under thermophilic conditions in Table V-A and Figure 3, and those under mesophilic conditions in Table V-B and Figure 4.

The detrimental effect of added alkali lignin on anaerobic gasification of packing-house wastes and cornstalk flour is evident. The experiment a t high temperatures showed a

/ / - L / G N / N z -L XNIN + CORMSJAL x Ff a UR 9-f/GN/N *fACK. HOW€ SLUDGE 4;CORNSTAf N fLOUR I'PACK.HOUSE SLUDG€

I r'

Figure 3. Thermophilic digestion at 50' to 55' C.

CORNSTALK FLOUR

C.C.GAS PLR GRAM M€SU

2-L/6NlN+CORNSTAlt FOUU 3,-flGNlN+P. H. SfUDGP 4:CORNJJALX FLOUR I. PACK HOW€ SLUDGE

Figure 4. Mesophilic digeation at 28O t o 30' C.

FIGURES 3 AND 4. EFFECT OF LIGNIN ON GASIFICATION OF PACKING-HOUSE SLUDGE AND CORNSTALK FLOUR

Vol. 27, No. 2 198 I N D U S T R I A L A N D E N G I N E E R I N G C H E M I S T R Y

yield of 580 cc. of gas per gram of packing-house volatile solids added, as compared with 112 cc. per gram of volatile solids added as lignin together with packing-house wastes; or disregarding the lignin added-i. e., considering it as an inert material-the relative volumes of gas would be 580 and 224 cc. (per gram of volatile packing-house solids added) in the absence and presence of alkali lignin, respectively (a decrease of more than 60 per cent in gasification of the packing-house sludge). Similar results were observed with the cornstalk flour. Thus, 491 cc. of gas were produced per gram of vola- tile solids added as cornstalks, as compared with 112 cc. per gram of cornstalk lignin mixture added, or 224 cc. per gram on the basis of cornstalks (volatile solids) in the presence of alkali lignin (a reduction in gasification from cornstalk con- stituents of more than 50 per cent).

TABLE IV. COMPOSITION OF PACKING-HOUSE SLUDQE AND CORNSTALK FLOUR

PACKINQ-HOUSE CORNSTALK SLUDQE FLOUR

S eoific gravity 1.0223 . . . d’oisture,, % 94.11 6.50 Total solids % 5.89 93.50

Volatile sblids, % 4.69 81.89 Ash, % 1.20 11.61

Dry basis: Volatile solids, % 79.55 Ash 9’ 20.45 Kjeidatl N, % 8.66

8 7 . 5 7 12.43 0.37

The observations a t 28” to 30“ C. also show clearly that the addition of alkali lignin arrested gasification markedly. Thus there were produced in this experiment 836 cc. per gram of volatile solids added as packing-house wastes, and only 292 cc. per gram of volatile solids from a mixture of lignin and packing-house waste. This amount was equivalent to 584 cc. per gram on the basis of packing-house waste added (consider- ing the alkali lignin present to be inert material), a decrease in gas production from the packing-house waste (assuming only packing-house sludge gasified) of 30 per cent. For the corn- stalk flour series, the quantities of gas evolved per gram were 642, 129, and 259 cc. on the basis of volatile solids added, as (1) cornstalks, (2) cornstalks with lignin, or (3) cornstalks in the presence of lignin, respectively (a decrease of 59 per cent in gasification of the cornstalk flour constituents, assuming that all the gas was evolved therefrom-i. e., calculated on the basis that all the gas produced came from the volatile solids added as cornstalks-and that the lignin remained inert).

Since these experiments have been under way, Waksman and Iyer ( I S ) have published a detailed study of the influence of addition of alkali and acid lignin on the decomposition of protein under favorable aerobic conditions. Using the production of ammonia and carbon dioxide as measures of

decomposition, they state: “A mere mechanical admixture of lignin and protein had, in many cases, a marked depressing effect upon ammonia formation. Even the mixed soil microbial population liberated only about half as much nitrogen, from the decomposition of the proteins, in the presence of lignin as in its absence.” Later they state that “the injurious effect of lignin upon protein decomposition holds true only for native proteins.’’

Their view is that lignin-protein complexes are formed which are extremely resistant to microbial decomposition. This theory would serve to explain the results with packing- house wastes presented here, but, in view of the fact that a similar depressive effect was noted when the extremely low- protein-containing cornstalk flour was employed, the indica- tion seems to be that factors other than production of lignin- protein complexes are also involved.

DISTRIBUTION OF INHIBITORY SUBSTANCE Having demonstrated that prepared lignin exerts an in-

hibitory action on gasification, the question as to whether this inhibitory agent was in the liquid or solid phase of the fer- menting mixture was investigated. The following experi- ment was designed to ascertain this point :

A batch of material to which lignin had been added, and which was consequently not gasifying, was separated by decantation into its liquid and solid phases. (These will be referred to as the lignin liquid and solid phases, respectively.) Similarly an ac- tively fermenting batch of material to which cornstalk flour had been repeatedly added was separated into its liquid and solid phases. (These will be referred to as the cornstalk liquid and solid phases, respectively.)

To the liquid phase from the lignin was added the solid phase from the cornstalk Row, and to the solid phase of the lignin was added the liquid phase of the cornstalk flour materials. To each of these mixtures (serving as seeds) were added known amounts of cornstalk flour, the ammonia content was adjusted, and the amount of gas evolved was ascertained. This, it was hoped, would give information as to whether the liquid or solid phase of a fermenting mixture, which had been arrested by the addition of lignin, exerts an inhibitory effect when mixed with solid and liquid phases, respectively, of a very actively fermenting corn- stalk flour sludge, on subsequent addition of fresh fermentable materials.

As controls, the liquid and solid phases of the lignin series, a8 well as the liquid and solid phases of the cornstalk flour series, were respectively combined, and their efficiency as seed for the decomposition of cornstalk flour was determined.

The results are shown in Table V I and Figure 5. It is evident that the supernatant liquid from a batch of sludge which had practically ceased gasifying (as a result of addition of alkali lignin) did not contain any appreciable quantity of inhibitory substances, for, when mixed with the solid phase of

TABLE v. EFFECT OF LIQNIN5 ON ANAEROBIC DIQESTION (WITH GAS PRODUCTION) OF PACBINQ-HOUSE SLUDQE AND CORNSTALK FLOUR

A. THERMOPHILIC DIQESTION AT 50’ TO 55’ C. B. MESOPHILIC DIQESTION AT 2 8 O TO 30’ C. Experiment No. T-41 T-44 T-45 T-43 T-40 T-46 R-41 R-44 R-45 R-43 R-40 R-46 Material subjected to fermentation Lignin Lignin + Packing- Lignin + Corn- Seed Lignin Lignin + Packing- Lignin + Corn- Seed

packing- house corn- stalk control packing- house corn- stalk control house sludge stalk flour house sludge stalk flour sludge flour sludge flour

4.0 4.0 4.12 4.0 4.0 3.0 4.0 4.0 4.0 4.0 4.0 4.0

57.54 28.88 28.88 . . . . . . 6S.70 34.38 34.38 . . . . . . 58 58 57 58 58 Digestion vol., liters

Material added, grams volatile solids: Days incubated 32 32 32 32 32 32 57

. . . . . , 34.98 7o:io Lignin

Cornstalk flour Total added 57:54 58:00 57168 57.86 57.98

Packing-house sludge . . . 29.72 57:6S . . . 34136 68:90 . . . 28:98 5?:98 . . .

6 8 : 7 0 69136 7 0 : l O 68.74 68.90 Seed present rams volatile solids 52.49 52.49 52.49 52.49 52.49 52:49 68.50 68.50 68.50 68.50 6S.50 68:50 Ratio fresh Gokatile solids to volatile

Gas recovery at 60° F. (15.6O C.) and

Vol. of gas, cc./gram fresh volatile

solids in seed 1.1:l 1 . 1 2 : l 1 . l : l 1.1:l 1.1:l . . . 1:l 1 : l 1.02:l 1: l 1:l . . . 965 20,310 58,740 8,910 44,165 7,500 760 mm., cc. 2,760 6,560 34,345 6,450 28,425 3,055

solids added: Uncor. for seed Cor. for seed

14.0 29’1 836 129.4 642 . . . 48.0 112 580 112 491 , , . -5.1 59.8 542 58.7 442 . . . -95.2 185 730 20.5 532 . . . 7.7 7.8 8 . 1 7.9 7.9 8.2 7.8 7.7 7.8 7.6 i . 7 8.0 P H

0 Lignin prepared at Iowa State College.

February, 1935 I N D U S T R I A L A N D E N G I N E E R I N G C H E M I S T R Y 199

a normal sludge, the mixture served as a suitable seed for the gasification of cornstalk flour. The solid phase, mentioned previously, arrested fermentation when mixed with the liquid phase of a normal sludge and produced a marked inhibition of gasification, This would indicate that the inhibitory effects observed are associated directly with the lignin and not with any toxic soluble substance extracted from

5 5 0 2 O f GAS- CORNSTALK FLOUR (SWD + f / Q U ' D )

ADD€D

400- L/GN/N i / Q U / D PL UJ 4/. 2 G. KS, (COUN5TALX FLOUR1 M O € D boo- //' I

CORNSTALK f /OU/D RU5 i / G N / N SOL105 ff US JO 446. KS. A 5 CORMST FLOUR

nMf (IN 44 YJ I

FIGURE 5. EFFECT OF LIGNIN (SOLID AND Ex- TRACT) ON DIGESTION OF CORNSTALK FLOUR

the alkali lignin-a result in line with the observation of Waksman and Iyer in 1932. Plate counts did not disclose any evidence of a disinfecting action effected by the addition of lignin. TABLE VI. EFFECT OF LIQNIN (SOLIDS AND EXTRACT) ON

DIQESTION OF CORNSTALK FLOUR (Digestion temperature, 28' to 30' C )

Experiment N o R-53 R-51 R-52 R 50 Seed material

Digestion vol., liters Days incubated Solids present from lignin digestion,

liquid phases, volatile solids, grams

Solid phases, volatile solids. grams Solids present from cornstalk flour

digestion, liquid phase, volatile uolids, grams

Solid phase, volatile solids, grams Total volatile solids in seed prams Cornstalk flour added as,vdlatile solids Ratio fresh volatlle sollds t o volatile

Gas recovery a t 60° F. (15.6' C.) and

Vol. of gas, & . / g r a m of fresh volatile

Lignin in supernatant liquid

solids present in seed

760mm. cc.

solids added

Lignin liquid + corn- stalk flour solid

phase 2 . 0

47

12.25 . . .

28,'93 41.18 41.27

1:l

20,935

508 2.43

Corn- Lignin stalk solid + flour liquid-

liquid + phases lignin control solid

phase 2.0 2.05

47 47

12.25 46:62 46.02

4.48 . . . 56:AO 58:a7 50.44 5S..31

1:l 1:1

7115 7300

141 125 1.16 1 4 9

Corn- stalk flour

solid + liquid- phasea control

1.95 47

. . . . . .

4.48 28.93 33.41 33.49

1:1

17,875

534 1 .38

PH 7 . 3 7 .2 7 1 7 . 3

Examination of the evaporated supernatant liquid from these experiments disclosed from 1.16 to 2.43 grams of ma- terial insoluble in 72 per cent sulfuric acid and reported as lignin. In view of the procedures reported in the literature- namely, discarding the supernatant liquid and carefully washing the residue before determination of lignin-a few determinations were made to ascertain whether this practice might account in part, a t least, for the losses of "lignin" attributed to bacterial action.

EFFECT OF HISTORY OF SAMPLE AKD EXTRACTION ON LIGNIN DETERMINATIOK

Lignin was determined on four samples of cornstalk flour by the 72 per cent sulfuric acid method of Bray in 1928 (W), as modified by Peterson, Walde, and Hixon in 1932 (B), before and after a 5-gram portion had been extracted six times with 100-cc. portions of cold water. No significant differences in the results were observed, as may be seen from Table VII.

TABLE VII. EFFECT OF EXTRACTION ON LIQNIN CONTENT OF CORNSTALK FLOUR

PER CENT OF LIGNIN (DRY Basrs) Difference

MATERIAL TREATXENT GIVEN Cold-water by ANALYZED BEFORE ANALYSIS Unextracted extracted extraction

Cornstalk flour Untreated 20.25 20.01 -1.19 16 .15 16 .78 4-3.96 16 .38 16.77 +2.38 17 .41 17.22 -1.09

Cornstalk flour Autoclaved a t 151b. 18.02 16.67 - 7.75 18.73 16.69 -10.9 18.85 17.23 - 8 . 6 18.39 16.57 - 9 . 9

Similar determinations of lignin were made on samples of cornstalk flour (50 grams) which had been suspended in distilled water (1000 cc.) and autoclaved a t 120" C. for 15 minutes, followed by evaporation of the whole mass to dry- ness. The results given in Table VI1 show that extracting these samples before determining lignin resulted in losses of 7.75 to 10.9 per cent of lignin (i. e., materials insoluble in 72 per cent sulfuric acid) found by analysis, a loss which cer- tainly cannot be attributed to biological action, since the autoclaved samples were proved to be sterile. This may be due to a change in physical or chemical state by which the lignin complex was rendered partly extractable.

The following experiment was performed to ascertain whether extraction with cold water affected the determination of lignin in cornstalk flour previously eqposed to biological (anaerobic) action.

Cornstalk flour was added to a ripe digesting seed, and the fermentation was allowed to proceed to completion. The whole mass was evaporated to dryness. Determinations of lignin, with and without extractions, were made on the corn- stalk flour employed, the seed, the mixture after fermentation, and the control seed, with the results indicated in Table VIII.

OF CORNSTALK FLOUR SUBJECTED TO ANAEROBIC BIOLOQICAL ACTION

TABLE VIII. EFFECT OF EXTRACTION ON LIQNIN CONTENT

(Temperature of incubation 28' to 30' C.) Lignin analysis on: EXTRACTED UNEXTRACTED

SAMPLE SAYPLE INITIAL LIQNIN C O N T E N T

Cornstalk flour added, grams Seed added, grams Lignin present (total), grams

6 . 8 0 6 .68 7 . 6 1 9 .23

14.41 15.91 LIONIN CONTENT A T END O F EXPERIUENT (57 DAYS)

Total lignin recovered, grams 11.97 15 .05 Lignin remaining in seed control, grams 7 .70 9.00 Lignin on cornstalk dour residue (by Ifference),

4.27 6 05 of loss of lignin of cornstalk flour 37 .2 Y.4 trams total loss in ligmn 17.0 5 . 4

It will be again noted that on the fresh cornstalk flour no significant difference was observed, but that the seed showed a decrease of 17.5 per cent in "lignin" (materials insoluble in 72 per cent sulfuric acid) when determined after extraction. Attention is also called to the fact that there was apparently no loss of lignin in the control sample of seed. Any losses in lignin in the fermenting mixture are therefore attributed to changes in the cornstalk flour subjected to fermentation.

The total amount of lignin a t the beginning of the experi- ment was 15.91 and at the end 15.05 grams when determined directly (without extraction), or a loss of 5.4 per cent. The quantities of lignin present a t the beginning and at the end of the experiment, when determined after cold water extraction, were 14.41 and 11.97 grams, respectively, or a loss of 17 per cent.

Since the lignin in the seed was apparently unaltered, any changes in lignin content may be considered as having oc- curred in the cornstalk flour. On the basis of determination before extraction there was a loss of 0.63 gram or 9.4 per cent of the lignin added, whereas on the basis of determination after extraction the loss was 2.5 grams or 37 per cent of lignin introduced with the cornstalk flour.

200 I N D U S T R I A L A N D E N G I N E E R I N G C H E M I S T R Y Vol. 27, No. 2

The loss due to extraction, as carried out in these experi- ments, is comparable to what would be observed with the reported procedures of other investigators of discarding the supernatant liquid and washing and extracting the residue before determining the lignin. Obviously such reported losses cannot properly be attributed entirely to bacterial decomposition. It is evident that a change in the physical or chemical state of part of the lignin, rendering it extractable or colloidal, was effected. Whether the loss in lignin observed by determination without water extraction (9.4 per cent) (assuming that this quantity is beyond the experimental error in lignin determination) was due directly to bacterial action on lignin or to chemical reaction of the lignin with end products of bacterial decomposition of other materials, form- ing some compounds which may be acid-soluble, is left for future inquiry. The experiment here reported was not designed to throw light on these points. It indicates merely that a considerable part of the reported loss in lignin content of plant materials subjected to anaerobic bacterial action, a loss that has been attributed to microbial decomposition, may be explained by the technic of selection and preparation of the sample for lignin analysis.

Although the evidence is definite that alkali lignin is not gasified under the conditions of these experiments, and that the addition of such prepared lignin exerts a marked depres- sive effect on the anaerobic gasification of other plant ma- terials, the conclusion should not be drawn that natural lig- nins, as encountered in plants, would necessarily be equally resistant or inhibitory. That natural lignins are not so inhibitory as alkali lignin is indicated by the fact that repeated addition of cornstalk flour, with its relatively high lignin content, to a batch of fermenting material was not associated with any considerable decrease in the amounts of gas pro- duced.

Unpublished data secured in this laboratory seem to indi- cate definitely that natural lignin is the most resistant of the plant constituents when subjected to anaerobic microbial ac- tion. The claims that under anaerobic conditions lignin decom- position proceeds as rapidly as that of pentosans and cellu- lose are not substantiated by the findings in this laboratory.

SUMMARY AND CONCLUSIONS

1. Attempts to isolate specific bacteria capable of de- composing alkali lignin anaerobically were not successful.

2. Gas was not formed when alkali lignin was added to a very active sludge producing methane and carbon dioxide; on the contrary, the progress of the fermentation of the sludge was markedly arrested.

3 . When alkali lignin and packing-house waste were added simultaneously to a methane-producing sludge, the quantity of gas evolved from the packing-house waste was reduced more than 50 per cent. Similarly, if cornstalk flour was used in place of packing-house waste, a like reduction in gasification was observed. 4. The depressive effect of alkali lignin noted cannot

logically be attributed to a soluble toxic substance, since there was no evidence of disinfecting action (there being no de- crease in aerobic or anaerobic plate count), and no decrease in gas production from cornstalk flour was noticed when the liquid phase of a seed, to which a large amount of lignin had been previously added (and fermentation consequently stopped), was mixed with the solid phase of an active seed, and cornstalk flour added to the mixture.

I n view of the fact that the depressive effects of alkali lignin on decomposition and gasification were observed with the low-protein cornstalk flour as well as with the high- protein? packing-house waste, it is believed that other factors, in addition to the production of stable lignin-protein com-

5.

plexes as suggested by Waksman and Iyer (IS), are associated with the decrease in gas production.

6. The liquid portion of a batch of cornstalk flour which had been subjected to anaerobic microbial action contained substances which were presumably lignin (i. e., insoluble in 72 per cent sulfuric acid as indicated by analysis). The practice of discarding such liquid portions, washing the resi- due, and extracting before lignin analysis, introduces a serious error into the analytical determination of the fate of lignin or lignin-like complexes.

7 . A loss of 9.4 per cent of lignin in cornstalk flour sub- jected to anaerobic digestion (determined by the Bray method as modified by Peterson, Walde, and Hixon) was observed when determinations were made on unextracted samples, and this loss rose to 37 per cent if lignin determina- tions were made after cold-water extraction of the test materials. Whether these losses are due directly to bacterial decomposition of lignin or to chemical reactions of lignin with the end products of bacterial decomposition of other ma- terials, with the formation of lignin compounds which are colloidal, or soluble in water or strong acids, needs to be defi- nitely ascertained.

8. The data here presented do not warrant any final conclusions as to the relative resistance of natural lignins to microbial decomposition, but that natural lignins are not so inhibitory as alkali lignin is indicated by the fact that re- peated additions of the relatively high-lignin cornstalk flour to a batch of fermenting material was not associated with any considerable decreases in the amounts of gas produced. The report in the literature that under anaerobic conditions micro- bial decomposition and possibly gasification of lignin is a5 rapid as that of cellulose or pentosan is not substantiated by the observations in this study.

BIBLIOGRAPHY Boruff, C. S., and Buswell, A. M., IND. ENO. CHEM., 22, 931-3

Bray, M. W., Paper Trade J., 87,59-68 (1928). Buswell, A. M., IND. ENQ. CHEM., 22, 1168-72 (1930). Buswell, A. M., and Boruff, C. W., Sewage Works J., 4, 454-60

Peterson, C. J., and Hixon, R. M., IND. ENQ. CHEX., Anal. Ed.,

(1930).

(1932).

1.65-7 (1929). Peterson, ’C.-J, Walde, A. W., and Hixon, R. M., Ibid. , 4,

Phillips, Max, J. Am. Chem. SOC., 50, 1986 (1928). Phillips, Max, Weihe, H. D., and Smith, N. R., Soil Sci., 30,

216-17 (1932).

383-90 (1930). Tenney, F. G.. and Waksman, S. A., Ibid. , 28, 55-84 (1929). Zbid., 30, 143-60 (1930). Waksman, S. A,, Arch. Microbiol., 2, 136-54 (1931). Waksman, S. A., and Gerretsen, F. C., Ecology. 12,33-60 (1931). Waksman, S. A., and Iyer. K. R. N., Soil Sn’., 34.43-79 (1932). Waksman, 8. A,, and Nissen, W., Am. J . Botany, 19, 514-37

Waksman, S. A., and Stevens, K. R., Soil. Sci.. 28, 315-40

Waksman, S. A,, and Tenney, F. G., Ibid., 22, 395-406 (1926).

(1932).

(1929).

RECBIVED September 8, 1934. Presented before the Division of Cellulose Chemistry at the 86th Meeting of the American Chemical Society, Chicago, Ill., September 10 to 15, 1933.

Correction In the paper on “Action of Urea on Calcium Orthophosphates,”

appearing in the December, 1934, issue of INDUSTRIAL AND ENQI- NEERINQ CHEMISTRY, a mistake was made which it is important to correct.

On page 1309, the sixth line of the last paragraph in the first column should read: “Table V shows further that dicalcium phosphate dihydrate, in the presence of urea phosphate, gives up its water of crystallization almost completely, etc.” In the article as it appeared, monocalcium phosphate monohydrate was inad- vertently substituted for dicalcium phosphate dihydrate.