R. W. Hemingway and H. Greaves

A;S early as ~931,. Ebeling (1) recog- Abstract: The sodium salts of resin acids were readily degraded by microflora nlzed that resIn acIds were among the from two types of river water and from an activated sewagesludge. A lag phase most harmful componentsof pulp mill with little or no resin acid salt degradationbut rapid bacterial developmentoc- wastewater to fISh, causingboth death Curled which was greatly extended by a decreasein incubation temperature. and contaminationof the tissues.Hag- After this initial lag phase,the resin acid salts were rapidly decomposedwith ei- m~ (2), in 1936, showed that resin ther of the three natural inocula. Sodium salts of levopimaric/palustric and dehy- acIds at concentrations exceeding 1 droabietic acids were most readily degraded,followed by abietic and neoabietic ppm were toxic to fish. Later, in 1950, acid salts; pimaric and isopimaric acid salts were most resistant to biodegrada- Van Horn, Anderson, and Katz (3) tion. Addition of 10% of neutralized spent bisulfite liquor did not affect cell showedthat sodium salts of resin and growth or resin acid salt degradationbut addition of acidic liquor restricted deg- fatty acids were toxic to minnows at 1 radationby activatedsludge until the pH had increasedto 7.3. and 5 ppm and to Daphnia at concen- trations of 3 and 1 ppm, respectively. Keywords: Resin acids. Resin acid sodium salts. . Waste water. Toxicity Maenpaa, Hynninen, and Tikka (4) Bacteria. Biodegradation.Activated sludge. Microorganisms.Spent sulfite liquors found that resin acid salts weretoxic to Daphnia at concentrationsof 1 ppm and that the resin acids were toxic to Daphnia at concentrationsexceeding 6 ... . . ppm. When the toxicity range of the spent . ~Isulflte lIquor, and. addltlo~al dium salts of palmitic, oleic, and a resin acids and their salts is compared quantitIes ~ere removedWIth washIDg mixture of resin acids.l Of the wood- with that of the organic sulfides from and screenID~~aste ~ater (10). W.aste inhabiting bacteria screened, 54% were kraft pulping operations (0.5 to 10.0 wate~. con~alDlDgtoXIC concen~atlons completely inhibited by the highest ppm) (5-7) or the chlorinated phenolic of resID acId salts ~ not res;trlcted to concentration (ca. 40 ppm) of fatty and compoundssuspected to be in bleach e.ffiuentsfrom che~l~al pulpID~ ope~a- resin acid salts in gradient plates. Few wastewaters (0.25to 3.0 ppm) (8), it is tlons. Large quantitIes .of resID acIds of the bacteria tested appeared to uti- clear that the resin acids and their are .u.sed by the pape~ ID?USt;Y as an lize the sodium salts of palmitic, oleic, salts are potentially very hazardous a~dltlve and a. proportIon IS dIScharged or the mixture of resin acids as their compounds to aquatic animals. WIth paper mill waste water (4). Row major carbon source. A major source of resin acid salts in and Cook (11) have rePOrte:dthat some The bacteria which showed some po- water is the waste from kraft pulping waste wa~rs from debarking, ground- tential for utilizing resin acid saltS of resinous woods. Bergstrom (9) has ~ood pulp~, and. fiberboard prod';lc- were: BaciUus Lemus; Bacillus estimated that a typical 40,000 tonl tlon are tc:'XICto f~h ~ause of high pantothenticus; Bacillus pulvifaciens; year kraft mill in Sweden discharges concentrations of resID acIds: . Bacillus pumilus; Bacillus polymyxa; about 90 tons of resin each year when We have foun~ no publIShed .htera- BaciUus subtilis; Escherichia coli; Fla= satisfactory scouring of waste water is ture conceme? WI~h parameters !~por- vobacterium sp.; Pseudomonas sp.; practiced to minimize losses. The tan.t to ~he blolo~cal decomposItion of Unidentified G-187; the Unidentified brown stock wash water from kraft resID acIds or theIr salts. Therefore, we G-l95. Solutions of the resin acid salt pulping of pine wood in Finland has have conducte~ a series of experiments mixtures at a concentration of 40 ppm been shown to contain resin acid salts to derIDe the ~mportance of the source in Dubos buffer (pH 7.4) were inocu- in concentrations as high as 100-400 of mIcroorganISms, temperature, pres- lated with the previously mentioned ppm (4). Resin acids are also dis- ence of s~nt bisulfi.te liquor, ~nd P!l bacterial isolates and incubated at charged with water when resinous on the blodegra?atlon of. resID acId 27°C for periods up to 6 days while woods are pulped by the sulfite pro- salts and have bne.fly e~amIDed the ex- resin acid salt concentration and cell cesses.About half of the resin in Pinus tent of degradatIon of these com- growth were measured periodically. radiata wood was removed with the pounds. There was no significant change in resin acid salt concentration with any of these bacteria. Examination of cell .. RESULTS AND DISCUSSION counts gave inconclusive results. ~~::::;:;:;:;!;;;'[,';;':':~,.: ~ :...;~ R. W. HemiDCWay. ForestProducts Utilization Preliminary Screening of Bacterial It is well known that isolated pure Research.Southern Forest Experiment Station. Isolates Forest Service.USDA, 2500Shreveport Highway. Pineville. La. 71360.H. Greav.. Forest Products A total of 69 bacteria, isolated from Laoorat;ary. CSIRO. South Melbourne. Victoria. wood sources, were screened for their 1 A compilation of the bacterial iaolatea screened and their rea~ to addition of fatty and resin Australia. d b' l o t OI O th . ~~~~-- tolerancean a Ilty to U llze e so- acidsalts can beobtained from H. Greaves.

Reprinted from Tappi, The Journal of the Technical Association of the Pulp and Pa~r Industry, Vol. 56, No. 12, December 1973. Copyright, 1973 by TAPPI, and reprinted by permission of the copyright ownel culture systemsare often unable to de- Bacterial cell populations of the degradation began, the incubation pe- grade compounds,while the same mi- three microbial sources developed at riod req~ired to degradethe resin acid croorganismsin mixed culture systems similar rates, the total viable cell count salts below measurableconcentrations may rapidly decomposethem (12, 13). increasingby about 10°times within 20 increased to 20 hr with activated Therefore, the inability of these iso- hr of incubation at 27°C (Fig. 2). Dur- sludge,to 25 hr with Yarra River, and lates to degraderesin acid salts does ing this time there was no significant to about 30 hr with North Pine River not prove that of the 69 bacteria change in the concentration of resin inocula. The differencesin degradation screened,none may be involved in the acid salts. When marked degradation rates of individual resin acid salts degradationof these compoundswhen occurred, the bacterial population in- which were common to the three the bacteria occur in mixed natural creaseda further 108times. Total cell sourcesof microorganismswere more microflora. Raynaud and co-workers developmentduring the full incubation evident at this lower incubation tem- (14) have isolated two bacterial period with 40 ppm of resin acid salts perature. There was considerabledeg- species, capable of utilizing rosin as wasabout 1012_1018times (Fig. 2). radation of the levopimaric/palustric the sole carbon source, which were To examine the degradationrate of and dehydroabietic acid salts before named Flavobacterium resinovorum resin acid salts by these microorga- there was much degradationof abietic and Pseudomonasresinovorans. Both a nisms under conditions similar to their and neoabietic acid salts. Pimaric and Flavobacterium and a Pseudomonas natural environment, solutions were isopimaric acid salts were very resis- sp. examined in our studies appeared incubated at 12°C (Fig. 1). The lag tant to microbial attack; on severaloc- to utilize the resin acid salts when ex- phasewhen there was little or no deg- casions traces of these latter com- amined on agar plates, but this could radation of resin acid salts increasedto poundswere found after 164hr of incu- not be verified in liquid culture stud- 80 hr with activated sludge, to 100 hr bation at 12°C. ies. with the Yarra River, and to 140 hr While there was a long period of lit- with the North Pine River inoculum. tIe or no resin acid salt degradationat Loweringthe temperaturehad more ef- 12°C, there was considerablebacterial Microbial Activity of River Waters and fect on this initial period of cell growth developIDentafter 16 hr of incubation an Activated Sludge than on the subsequent degradation and the viable cell-count increasedby rate when decompositionof the resin about 109 times before there was ap- Solutions containing 40 ppm of resin acid salts became appreciable. Mter preciable resin acid salt degradation acid salts in Dubos buffer (pH 7.4) (Fig. 2). The viable bacterial cell pop- were inoculated with samples from the ulation at the point when resin acid Yarra River at South Melbourne, Vic- salt degradation became significant toria, from the North Pine River at was about 10. times greaterat 12°than .11001 -==-..~- ---~ at 27°C. Petri, Queensland, and from an acti- ~ 80 60 vated sludge at the Heatherton sewage \ '\\ g 40 - Activated II~ \\ works in Victoria. When the resin acid , Y R., \ \ \ \ salts were incubated at 27°C with the ,~ ,-- .-fa lver \ Effect of Spent Bisulfite Liquor I \ \\ activated sludge or Yarra River inocu- z 20 \ NorttIPine iver ' ib It was of interest to know what effect lum, all resin acid salts were decom- 1&1 \ 2rc ~ 101 \ \1TC the presence of a spent liquor from bi- posed to concentrations Below measur- -I able levels within 12 to 15 hr, after an -< sulfite pulping of Pinus radiata wood I- might have on the rate of resin acid initial lag phase of 18 to 20 hr during I-0 ,_-:-LL_, ~--:- ,L~'- - -;--;~ which little or no change in resin acid salt degradation with the three sources salt concentration occurred (Fig. 1). 20 40 60 80 100 120 140 160 of microorganjsms. The spent liquor Inoculation with the North Pine River was adjusted to pH 7.1 with NaOH INCUBATION PERIOD, hr and added to the solution of resin acid sample required about 28 hr before Fig. 1. Effect of temperature and source degradation of resin acid salts oc- salts in Dubos buffer at a concentra- of microfiora on rate of resin acid .alt deg- tion corresponding to 10CY0of the origi- curred, and they were degraded to con- radation. centrations below measurable levels nalliquor. after a total of 40 hr at 27°C. There Addition of the neutralized liquor was little difference in the rate of deg- caused marked alterations in the com- radation between the three sources of ~ition of the resin acids. However, microorganisms after the decomposi- - Activated sludge substantial quantities of recognizable Yarra River tion became appreciable. Differences in - - resin acids remained. Compounds mea- 4c J degradation rates of individual resin ~ 1°1 North PiM River sured in these cultures containing QI @ Liquor a acid salts were common to all sources .Q $pent liquor were: dehydroabietic, e No liquor~ .- abietic, pimaric, and material with the of microorganisms; those resin acid z' Q relative retention time of levopimaric / salts present in greateSt proportions, t- ~T/ o( ~ palustric acid methyl ester which was levopimaric/palustric2 and dehydroa- ..J bietic, were decomposed more rapidly ~ n probably methyl palustrate. Addition Go .; than abietic and neoabietic acid salts; )J'::; I. I of the equivalent of 10CY0of neutralized ~ bisulfite spent liquor had no significant pimaric and isopimaric acid sodium ..J ..J ;/ e /~/? effect on the rate of resin acid salt deg- salts were the most resistant to degra- W q U l:"'-""-::; radation when incubated with the acti- dation. When incubated with North W /j'~ ..J f ,. e vated sludge sample at either 12° or Pine River water, neoabietic acid was ~ . .'/" much more resistant to degradation 0( 27°C or with the microorganisms from than was abietic, and its stability was 5: ~~~. either of the river waters when incu- similar to that of pimaric acid. bated at 12°C (Fig. 3). The neutralized spent liquor had no influence on the 0 5 10 15 20 25 30 relative rates of decom~ition of indi- I NCU8ATION PERIOD,hr vidual resin acid salts; dehydroabietic 2 Levopimaric acid and palustric acid methyl es- FIg. 2. Effect 01 temperature and source acid salt was degraded m~t rapidly, ters were not separated using DEGS columns 80 while pimaric acid salt was the most the sum peak area was measured and reported as 01 mlcrollora and neutralized bisulfite spent levopimaric/pla1J8tric acids. liquor on rate 01 cell devleopment. resistant.

190 Vol. 56, No. 12 December1973/ Tapp; The initial rate of bacterial cell de- resin acid salts were incubated at 27°C There was substantial bacterial de- velopment was not influenced by the with the North Pine River sample.The velopment during the initial 30 hr presence of neutralized spent liquor amount of CO2liberated, as well as ra- when no significant change in resin (pH 7.1), but bacterial growth in- dioactivity of the BaCO3 recovered, acid salt concentrationoccuned. Large creasedbeyond measurable levels when was measuredat intervals throughout quantities of CO2were liberated during resin acid salt degradationbecame sig- the incubation period. A control incu- this period, but the BaCOa recovered nificant (Fig. 2). During the period bation of unlabeledresin acid salts was was not radioactive. The absenceof ra- when rapid resin acid salt degradation used to obtain information on the rate dioactivity in the CO2 liberated during occurred,there was a darkeningof the of resin acid salt degradationand bac- this initial phase lends support to the color of the culture liquor and the pH terial cell development. Results are thesis that bacterial growth in this increasedfrom 7.1to 8.1. summarizedin Fig. 5. stageof incubation is supportedby nu- While there was no effect on culture trients in the inoculum. Contaminants systemswhen 10%of neutralizedspent in the resin acid salt substrate such as liquor was added (pH 7.1), the addi- small amounts of fatty acids or their tion of the acidic liquor to the resin ~ 10080 ' ,'" esterswould be radioactive. acid salts in Dubos buffer (pH 4.7) . 60 '" Degradation of the resin acid salts prevented any significant change in e 40 \ '\ e was appreciable between28 and 40 hr (J , resin acid salt concentrationafter 50 hr c( 20 - - ActivatedSlu~ C ,.' c of incubation, and there was a marked of incubation at 27°C with the activat- ~ - --Yarra River &a increasein the amount of CO2 liberat- \ ' ed sludgesample. The viable cell count :. --- North Pine Riwr\ .\ ed during the incubation period from did not increasewhen the pH of the g: 10 E> LiqUOl'added ", 30 to 50 hr. The BaCOarecovered dur- ~ e NoliquOl' ing this period was radioactive. When culture liquid wasso low. ... ' f) \.. 0 ' the cumulative amount of radioactivity ... ti ~~'~ ;W_- I recoveredas BaCOa is comparedwith 20 40 60 80 100 120 140 160 the amount of radioactivity addedwith Influence of pH the resin acid salts, it is apparent that INCUBATION PERIOD, hr the resin acid salts were rapidly me- To gain more information on the im- Fig. 3. Effect of neutralizedspent bisulfite tabolized to CO2 as about 80% of the portanceof pH on the degradationrate liquor on rate of resin acid salt degrada- added radioactivity was recovered as of resin acids in the presenceof spent tion. CO2after 90 hr of incubation. bisulfite liquor, the liquor was back- titrated with NaOH and added to the resin acid salts in Dubos buffer at con- 6.0 EXPERIMENTAL centration equivalent to 100/0of the original liquor with initial pH levels of I ;gH ', 7.5"0 ResinAcids i.., .5 ,._-,~- , " 5.5, 6.0, and 6.5. During the courseof 100 ' .- I , -- ~ I ~ Resin acids were obtained by precip- incubation with the activated sludgeat 80 DH I l7\ii7.\\pH.. 'H" 7.0 ;;: itation of their cyclohexylamine salts 27°C, the solution becameincreasingly ~. 60-7,.9/ ~ from acetone extracts of the wood of ! 6.0../&.5 dark and the pH increasedto 7.5 to ~ 401 6.5 c Pinus pinaster and wood of a Pinus 8.1, depending upon the amount of u 6 radiata tree which had been grown for < NaOH used to back-titrate the spent z 20 6.0 Z a period in 1~O2 atmosphere(15). To liquor (Fig. 4). in w insure removal of all of the amine, the There was no significant change in ~ 10 0.5 resin acids were washed thoroughly the concentration of resin acid salts -I with a large excessof boric acid. Sodi- while the pH was below 7.2. However, ~ 0 um resinateswere salted from solutions after the pH had reached about 7.3, to. of the resin acids in a slight excessof there was a rapid degradationof the warm sodium hydroxide. resin acid salts in cultures at all three L.,. '..'~ initial pH levels (Fig. 4). The effect of 0 20 40 60 80 100 120 initial pH on the degradationrate of INCUBATION PERIOD, hr Screening of Bacteria resin acid salts was associatedalmost Fig. 4. Effect of initial pH on the rate of entirely with the time required for the resinacid salt degradation. pH to increaseto 7.3. Neither the rate A total of 69 wood-inhabiting bacte- of total resin acid salt degradationnor ria were screened for their ability to the relative rates of degradationof in- utilize fatty or resin acid sodium salts dividual resin acid salts was influenced 10 using diffusion gradient plates pre- much by the initial pH of the solution. 8 Initial pared with a Dubas mineral salt solu- It would appear that microbial attack * 6 radioactivity, % tion agar as the basal medium (16). on lignosulfonic acids present in the ~ 4 The second wedge contained in addi- spent liquor is a most important factor (3 75 tion to mineral salts and agar, filter c( ,~~~~~-! sterilized solutions of the sodium salts controlling biodegradationof resin acid z ~ of palmitic, oleic, or the mixture of salts in solutions containing spent bi- Iii w Ii' sulfite liquors at mildly acidic pH con- a: 1 50 resin acids obtained above. Plates were -' ~'IBa CO3 incubated at 25°C for 24 br except ditions. c( t- Recovered(mg) 125 where thermophilic microorganisms ~ were involved in which case the tem- perature was 45°C. In addition, the Extent of Resin Acid Salt 0 20 40 60 80 100 s.ame scree~ing experiments as men- Degradation tioned preVIously were made, except INCUBATIONPERIOD, hr that the agar medium also contained To obtain a conceptof the extent of Fig. 5. Extent or 1.C labeled resin acid the following nutrients: peptone.. 1.0%; resin acid salt degradationwith micro- sa" degradation, North Pine River water- casamino acids, 0.5%; and glucose, flora from natural waters, 1.C labeled 27°, pH 7.5, no spent bisulfiteliquor. 0.5"10.

Tapp; I December 1973 Vol. 56, No. 12 191

~ hr, colonies were counted on plates ick, R. E., Tappi 35 (12): 545 (1952). showingbetween 30 and 300colonies. 6. Seppovaara, D. and Hyiminen, P., Paperi ja Pitu 6 (1): 11 (1970). Liquid shake cultures were made 7. Cole, A. E., J. Pharm. Exp. Therap. 54 using 5OO-mlconical flasks, each con- (4): 448 (1935). taining the basal medium of Dubossalt 8. Servisi, J. A., Werden, R. H., and Mar- tens, D. W., International Pacific Salm. buffer, 5 ml of inoculum, 49 ppm of Radioactive Experiments on Fisheri~ Commission, New West- resin acid salts, and in someinstances, minster, B. C., Progress Report 17 the equivalent of 100/0by volume of To determine the extent of degrada- (1969). spent bisulfite liquor which had been tion of the resin acid salts, radioactive 9. Bergstrom, H., Svensk Papperstid. 56 adjusted to various pH levels. Control (3): 75 (1953). resin acid salts weredissolved in Dubos 10. Nelson, P. J. and Hemingway, R. W., incubations were prepared using auto- buffer at 50 ppm and the solution inoc- Tappi 54 (6): 468 (1971). claved inoculum. After various incuba- ulated with the North Pine River 11. Row, R. and Cook, R. H., Pulp Paper tion periods, samples were withdrawn sample. Carbon dioxide free air was Mag. Can. 12 (6): 38 (1971). aseptically from the cultures and the i2. Gottlieb, S. and Pelczar, M. J., Bact. passed through the incubation flask, Rev. 15: 55 (1~1). bacteria removed by centrifugation. two Ba(OH)2traps, and through a flow 13. Kleinert, J. M. and Joyce,C.S., Svensk The supernatant was pipetted off, the meter at 10 ml/min. The BaCOa was Papperstid. 62: 37 (1959). cells washed with Dubos buffer, and collected at intervals, weighed,"and its 14. Rayi1aud, M., Daste, P., Groesin, F., Biellmann, J. F., Wenning, R., Ann. the bacteria centrifugeddown a second radioactivity determined with a geiger lnst. Pasteur 115(104): 731 (1968). time. The pH of the first supernatant tube. A parallel incubation with the 15. Shain, L. and Hillis, W. E., unpub- was determined, the two supernatants sameinoculum was made to determine lishedwork. combined,and then stored at 3°C until the rate of cell developJ:Dentand resin 16. Dubos, R. J., J. Bact. 15 (4): 223 (1928). analyzedfor resin acid salt content. acid salt degradation. 17. H.en;tingw~, R. W:' Nelson, P. J., and The supernatantliquors were treated Hill1s, W.E., Tapp~54(1):95(1971). with a saturated boric acid solution, the resin acids extracted into diethyl Received for review March 19, 1973. ether, methylated with etheral diazo- Accepted May 29, 1973. LITERATURE CITED The authon are indebted to the Public Health methane,and the methyl estersdiluted Laboratory, South MeltMlume, for a sample of ac- to 3 or 5 ml with acetone.Duplicate tivated sludge, the Australian Paper Manufactur- analyse$were made of these solutions 1. Ebeling, G., Von Wasser5 (11): 192 en for water sam~ea of the North Pine River, Drs. (1931). L. Shain and W. E. Hillia for a ~ dt of ra- of resin acid methyl estersby gas-liq- 2. Hagman, N., Finnish Paper Timber J. dioactive P. radiato wood, and Mi8 L. E. Straw- uid chromatography using conditions 18 (1): 32 (1936). bridge, Miaa L. Nimmo, and Mr. L. S. Lau for 3. Van Horn, W. M., Anderson, J. B., and their excellent _istance with experimental work.. described previously (17). Viable cell The helpful sugestions of E. W. B. De Coeta and populationsin 1 ml aliquots were esti- Katz, M., Tappi 33 (5): 209 (1950). L. Shain are gratefully acknowiedced. Mr. Heming- 4. Mienpi4, R;I.Hynninen, P., and Tikka, way is particularly indebted to Dr. W. E. Hillis and mated using a seriesof 10-folddilution J., Papenja l"1£U4(.): 145 (1968). the Forest PrOOuct& Laboratory, CSIRO, for pro- plates. After incubation at 2S°Cfor 24 5. Haydu, E. P., Amber, H. R., and Dim. viding the opportunity to conduct this reaearch.

192 Vol. 56, No. 12 December1973 I Tapp;