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.