Detection of Alcohols and Volatile Fatty Acids by Head-Space

Transcription

1 /78/7-23$2./ JOUJRNAL OF CLINICAL MICROBIOILOGY, Jn. 1978, p Copyright ) 1978 Americn Society for Microbiology Vol. 7, No. 1 Printed in U.S.A. Detection of Alcohols nd Voltile Ftty Acids by Hed-Spce Gs Chromtogrphy in Identifiction of Anerobic Bcteri LENNART LARSSON,'* PER-ANDERS MARDH,2 AND GORAN ODHAM3 Deprtment of Medicl Microbiology,2 Lbortory of Ecologicl Chemistry,:' nd Deprtment of Technicl Anlyticl Chemistry,' University of Lund, S-22 7 Lund, Sweden Received for publiction 23 My, 1977 A hed-spce gs chromtogrphic technique for the nlysis of voltile bcteril metbolites is described. Bcteroides frgilis, Clostridium perfringens, nd Propionibcterium cnes, cultured in glucose-contining peptone yest extrct medium, were studied. The hed-spce technique ws compred with the injection of the complete liquid culture medium, nd solvent extrcts thereof, into the gs chromtogrph. Voltile ftty cids could be detected by ll three methods, wheres lcohols produced by C. perfringens nd P. cnes were detectble only in the hed-spce chromtogrms. Both FFAP nd Porpk Q were used s gs chromtogrphy sttionry phse. Porpk Q ws found more suitble thn FFAP for the seprtion of lcohols. The hed-spce technique requires minimum of preprtion before the nlysis nd is well suited to utomtion. Gs chromtogrphy (GC) is tody routinely pplied in the identifiction of nerobic bcteri. When cultured in peptone yest extrct medium supplemented with glucose (PYG medium) (8), mny nerobes produce lcohols nd free ftty cids which, fter extrction, cn be nlyzed by GC either in their nturl stte or fter being converted to more voltile derivtives. A flme ioniztion or therml conductivity GC detector is generlly used. From the qulittive nd quntittive chrcteristics in the chromtogrms so obtined, mny nerobes cn be identified s to genus nd sometimes even s to species (8). Bcteril metbolic products in broth medium cn lso be detected by GC by injection of the entire medium into the gs chromtogrph (3, 12). This technique hs been used s n lterntive to extrction methods in the identifiction of nerobes. Therml conductivity detectors cn not be stisfctorily used in such nlyses. GC of the hed-spce tmosphere hs lso been used in studies of microorgnisms. In this technique, the voltile microbil metbolites bove culture medium re smpled by gstight syringe nd injected into the gs chromtogrph (6). The hed-spce GC technique hs been pplied minly in diry reserch, e.g., for the evlution of microbil contmintion of food (1, 2, 5, 11). It hs lso been used in nlyses of fungi (1) nd in studies on bcteri of the genus Clostridium (4). In the present communiction, we describe hed-spce GC technique s developed for the identifiction of microorgnisms, prticulrly nerobic bcteri. Hed-spce chromtogrms from studies of Bcteroides frgilis, &1ostridium perfringens, nd Propionibcterium cnes re compred with those obtined from nlyses of complete culture medi tht hd been incubted with the sme orgnisms nd extrcts thereof. The usefulness of different GC sttionry phses is considered. MATERIALS AND METHODS Orgnisms. Two freshly isolted strins of ech of B. frgilis, C. perfringens, nd P. cnes were used. The strins were isolted nd identified ccording to the recommended procedures (8). Medium. PYG medium, contining 1% (wt/vol) of glucose, ws employed (8). Before incubtion, nitrogen ws bubbled through the medium t 75 C for 15 min to remove voltile compounds. The medium ws then mde up to its originl volume by dding sterile distilled wter. Stndrd solutions. In the comprtive studies, stndrd solution ws used, consisting of.5 ml of ech of ethnol nd propnol,.1 ml of ech of butnol nd myl lcohol, nd.1 ml of ech of cetic, propionic, isobutyric, butyric, isovleric, vleric, isocproic, nd cproic cid in 1 ml of distilled wter. The hed-spce technique ws lso employed when studying solution consisting of.1 ml of ech of heptnoic, octnoic, nd nonnoic cid in 1 ml of the stndrd solution described, nd solution of.1 ml of cproic cid in 1 ml of distilled wter. Culture technique nd preprtion of smples. The bcteri were inoculted into 12-ml screw-cp bottles contining 1 ml of PYG nd incubted t 37 C for 4 h. For comprison purposes, uninoculted culture medium, incubted under the sme conditions, 23 Downloded from on November 5, 218 by guest

2 24 LARSSON, MARDH, AND ODHAM ws tested. The culture medium nd 1 ml of the stndrd solution contining lcohols nd lower ftty cids were cidified to ph 2 using diluted H2SO4 (1:3 [vol/vol] in wter) nd divided into three 3-ml liquots. Ech smple ws nlyzed by one of the three GC methods described below. The cproic cid solution nd tht contining higher ftty cids were nlyzed by the hed-spce technique only. Anlysis of ether extrcts of the medium. Diethyl ether (1 ml) ws dded to one of the smples. After shking, the screw-cp bottle ws centrifuged, nd the ether phse ws trnsferred to 2-ml test tubes. Approximtely.1 g of solid nhydrous MgSO4 ws dded. After 1 min t room temperture, 2,il of the ether solution ws injected into the gs chromtogrph. Anlysis of complete broth medium. One of the smples ws centrifuged t 5, x g for 5 min to remove bcteril cells. Then, 2,ld of the superntnt ws injected. Hed-spce nlysis. The smples were trnsferred to 1-ml mpoule flsks contining 2 g of nhydrous N2SO4. The cproic cid solution ws lso nlyzed, with nd without vrying mounts of slt. The flsks were seled with rubber stoppers nd protected with Teflon sheet nd luminum crimp cps before being heted in wter bth t 75C for 1 min with occsionl shking. The cproic cid solution ws lso nlyzed fter being incubted for 2, 4, 6, nd 8 min in wter bth t 25, 35, 45, 55, 65 nd 75C. Then 2 ml of the gs tmosphere in the bottles ws withdrwn with Hmilton gs-tight syringe nd injected into the gs chromtogrph. The syringe ws heted to 8C to void condenstion of smple components during injection. GC equipment nd test conditions. Hewlett- Pckrd (model 575) nd Perkin-Elmer (model 392) gs chromtogrphs, equipped with dul flme ioniztion detectors, were used. In the cse of the Hewlett- Pckrd instrument, 2-m stinless steel column (ID 2 mm) pcked with 5% FFAP (Vrin) on Chromosorb G AW DMCS 8/1 mesh ws employed. The nitrogen crrier gs flow ws 3 ml/min. The injector nd detector tempertures were 2C, nd the temperture of the column ws 135C. When the stndrd solution contining higher ftty cids ws run, the column temperture ws 16 C. The Perkin-Elmer instrument ws equipped with 2-m glss column (ID 2 mm) pcked with Porpk Q 1/12 mesh. Here, the injector nd detector tempertures were 25C, nd tht of the column ws 195C. Nitrogen, t flow rte of 3 ml/min, ws used s crrier gs. The ttenution used ws 64 in ll nlyses. Mss spectrometry. Mss spectr were run on Vrin Mt model 112 GC-mss spectrometry combintion. The GC column consisted of 2-m stinless steel tube (ID 2 mm) pcked with Porpk Q (8/1) mesh). The crrier gs flow nd the temperture of the injector nd column were the sme s described for the Perkin-Elmer instrument. The ion source ws held t 2C, nd the electron energy ws 8 sv. RESULTS Anlysis of stndrd solutions using the three different GC techniques. Chromto- J. CLIN. MICROBIOL. grms obtined by the three GC techniques in nlyses of the stndrd solutions contining lcohols nd lower ftty cids re shown in Fig. 1. The peks representing lcohols (-d) were lrgest in the hed-spce chromtogrms. These peks were prtly hidden under the lrge solvent pek obtined when using the ether extrction technique, nd lso by the brod "solvent" pek obtined when injecting the queous stndrd solution directly into the gs chromtogrph. This brod pek lso ppered when distilled wter ws injected. The ftty cids studied (e-l) were redily detectble when using ll three smpling techniques. Lrger cetic nd propionic cid peks were obtined with the direct injection technique thn with the extrction nd hed-spce techniques. Peks representing the other cids (h-l) were, s expected, lrgest when the ether extrction technique ws used. In the hed-spce chromtogrms, the peks representing isocids were considerbly lrger thn 1 2 Retention time(min) FIG. 1. Chromtogrms of stndrd solution of lcohols nd ftty cids. Direct injection of the smple (upper pnel), injection of ether extrcts (center), nd hed-spce nlysis (lower pnel). An FFAP sttionry phse ws used. Symbols:, ethnol; b, propnol; c, n-butnol; d, n-myl lcohol; e, cetic cid; f, propionic cid; g, isobutyric cid; h, butyric cid; i, isovleric cid; j, vleric cid; k, isocproic cid; nd 1, cproic cid. Downloded from on November 5, 218 by guest

3 VOL. 7, 1978 HEAD-SPACE GAS CHROMATOGRAPHY 25 those of the corresponding stright-chin cids. Hed-spce nlysis of the cproic cid solution. The reltive pek re of cproic cid plotted ginst the temperture of the smple is shown in Fig. 2. The mount of cproic cid in the gseous phse ws lmost 1 times s lrge t 75C (used in this study) s t 45C. Temperture equilibrtion required 6 min t 75C. Addition of N2SO4 ("slting out") ws found to increse the pek heights mrkedly. From prcticl point of view, we found it dvntgeous to use such lrge mounts of N2SO4 tht sturtion ws reched. The slt used ws of regent grde nd, s indicted by chromtogrms of the pure slt, did not contin ny voltile compounds. Hed-spce nlysis of the higher ftty cids. The hed-spce chromtogrms obtined by nlysis of the solution contining heptnoic, octnoic, nd nonnoic cid displyed only smll pek of octnoic cid nd hrdly visible nonnoic cid pek. Octnoic cid ws therefore considered to be the highest stright-chin ftty cid tht could be detected under the previling conditions. Anlysis ofbroth cultures using the three different GC methods. Chromtogrms obtined by GC nlysis of PYG medium incubted with B. frgilis, C. perfringens, nd P. cnes re shown in Fig. 3. The three GC techniques gve similr chromtogrms with respect. U) S.) -i L A PYG(blnk) EBfrgilis to the ftty cids from ech species. Acetic (e), propionic (f), butyric (h), nd isovleric cid (i) produced by the bcteri studied were esily detected by ll three methods. In the hed-spce chromtogrms, however, few peks with short retention times were observed. The lrgest of these peks were identified by mss spectrometry. The mss spectr nd GC retention times of the compounds produced by C. perfringens proved identicl with those of uthentic ethnol, propnol, nd n-butnol. P. cnes produced only, 12-6 U.1 o Temperture of smple ( C) FIG. 2. Reltive pek re of cproic cid in reltion to smple temperture. Hed-spce nlysis using n FFAP sttionry phse. C.perfringens Jf Rcnes L b AAA~~~~~~~~~~~~~~~~~~~~~~~~~~~~ L Downloded from on November 5, 218 by guest 2 2O 2' o 2 Retention time (min) FIG. 3. Chromtogrms of noninoculted glucose-contining PYG medium nd cultures of B. frgilis, C. perfringens, nd P. cnes in PYG medium. Anlysis of complete medium (upper pnel) nd ether extrcts of the medium (center), nd hed-spce nlysis (lower pnel). An FFAP sttionry phse ws used. For symbols, see legend to Fig. 1.

4 26 LARSSON, MARDH, AND ODHAM propnol. The lcohol peks observed could not be identified in the chromtogrms obtined by the other two methods tested. A chromtogrm of incubted PYG medium blnks treted with nitrogen t 75 C for 15 min is lso shown in Fig. 3. Anlysis of untreted medium indicted the presence of considerble mounts of voltiles, which could be pprecibly decresed by the tretment described. The nture of these voltiles ws not further investigted. When nlyzed by the hed-spce technique, untreted PYG medium voltiles hid the ethnol nd propnol peks. However, ll the other compounds could be detected. Comprison between different sttionry phses. In Fig. 4, hed-spce chromtogrms of the stndrd solution contining lcohols nd ftty cids, using FFAP nd Porpk Q s sttionry phses, re shown together with chromtogrms of C. perfringens. Porpk Q showed superior seprtion of the lcohols. On the other hnd, the FFAP phse seprted the higher ftty cids more rpidly thn did the Porpk Q phse. DISCUSSION In the chrcteriztion of nerobic bcteri using GC nlyses, voltile ftty cids hve so fr been the principl compounds of interest. Only in cses when no voltile cids (or merely cetic cid) re detected, smples re methylted nd nlyzed with respect to nonvoltile ftty cids (8). Alcohols hve been less often used for species differentition of nerobes. However, the hed-spce technique described ppers to be highly suitble for the detection of smll mounts of lcohols nd other voltile compounds. The dditionl informtion obtined from such compounds my prove vluble for species chrcteriztion of bcteri. The ddition of n inorgnic slt, such s N2SO4, ws found to increse the yields considerbly. The slt decreses the solubility of the orgnic compounds nd hence increses the concentrtion of these compounds in the vpor bove the smple. The wter bth should be kept t constnt temperture, since vritions in temperture ffect the size of the peks obtined (Fig. 2). Stright-chin ftty cids up to octnoic cid cn be detected by the hed-spce technique described. Becuse of their greter voltility, isocids give somewht higher peks thn corresponding norml cids. One disdvntge of the hed-spce technique is tht the size of the chromtogrphic peks does not represent the true reltive proportions of the voltiles present in the smple. However, this is not criticl in GC identifiction of bcteri. J. CLIN. MICROBIOL. GC nlysis of ether extrcts of cidified broth cultures hs so fr been the technique most commonly used in the identifiction of nerobic bcteri. This method fcilittes concentrtion of the higher ftty cids (Fig. 1). Nevertheless, pprecible mounts of cetic nd propionic cid remin in the queous phse fter extrction, resulting in reltively smll peks for these cids. The lower lcohols too remin to gret extent in the queous phse. To increse the concentrtion in the ether phse, "slting out" hs been used (8). Such procedure ws, however, not performed in the present study. The solvent pek prtly hides those of the lower lcohols nd possibly lso of other highly voltile compounds. Such compounds need lower colunm tempertures (incresed retention time) or temperture progrmming, if the ether extrction technique is used. This technique is lso more lborious thn the other two methods discussed. Anlysis of n cidified culture medium by direct injection into gs chromtogrph is n esily performed method. One dvntge is tht the pek res reflect the true concentrtions of the compounds in the medium. The chromtogrphic peks obtined re smller thn those in chromtogrms obtined by the other techniques used t the sme ttenution, except for cetic nd propionic cid, due to the bsence of concentrtion step before the GC nlysis. The identity of the lrge, brod pek in the very erly prt of the chromtogrm ws not estblished. Since flme ioniztion detectors do not detect wter, this pek is ssumed to represent column mteril or collected impurities liberted by the wter injected. In hed-spce techniques, only vporized mteril is introduced into the gs chromtogrph. Mteril of high moleculr weight, e.g., extrcellulr lipids present in ether extrcts, my pper in lter nlysis. In GC nlysis of bcteril culture medi injected directly into the gs chromtogrph, nonvoltile mteril rpidly clogs the insert liner nd the first prt of the column. Consequently, these prts of the GC system must be clensed frequently nd the pcking mteril renewed. To minimize this disdvntge, only the superntnt of liquid bcteril cultures should be injected. It is even preferble tht pre-column tht dsorbs nonvoltile mteril of both low nd high moleculr weight should be instlled. FFAP is one of the sttionry phses most frequently used in GC nlysis of ftty cids nd lcohols produced by bcteri. However, other pcking mterils hve been employed, e.g., SP-122 (7). Mrshll nd Porter (9) recently described the efficient seprtion of ftty cids on glss cpillry column coted with 1, Downloded from on November 5, 218 by guest

5 VOL. 7, 1978 HEAD-SPACE GAS CHROMATOGRAPHY 27 n FFA P Porpk Q L. L. u ) C e h k b h Retention time(min) FIG. 4. Chromtogrms of stndrd solutions of lcohols (upper pnel), ftty cids (center), nd C. perfringens (lower pnel), obtined by hed-spce nlysis using FFAP nd Porpk Q s sttionry phses. For symbols, see legend to Fig pentdecnedicrboxylic cid. Porpk Q hs lso been used (12). In our experiments, this ltter phse proved suitble for nlysis of lcohols nd ftty cids. As exemplified in the present study using B. frgilis, C. perfrimigens, nd P. cnes s test orgnisns, the hed-spce technique provided more informtion thn ws obtined by the other two techniques employed. The hed-spce technique is lso less lborious thn these methods, nd is suitble for utomtion. For exmple, fter incubtion, the culture medi cn be trnsferred to mpoule flsks contining H2S4 nd N2SO, nd plced in n utomtic hed-spce nlyzer (4). After few minutes of incubtion in wter bth, the vpor phse bove the medi cn be utomticlly smpled nd injected into the gs chromtogrph. ACKNOWLEDGMENTS This study ws supported by the Swedish Ntionl Assocition Aginst Hert nd Chest Diseses. LITERATURE CITED 1. Bssette, R., nd T. J. Clydon Chrcteriztion of some bcteri by gs chromtogrphic nlysis of hed spce vpors from milk cultures. J. Diry Sci. 48: Bwdon, R. E., nd R. Bssette Differentition of Escherichi coli nd Aerobcter erogenes by gs liquid chromtogrphy. J. Diry Sci. 49: Bricknell, K. S., nd S. M. Finegold A simple, rpid method to process nd ssy ftty cids nd lcohols by gs chromtogrphy. Anl. Biochem. 51: Drsr, B. S., P. Goddrd, S. Heton, S. Pech, nd B. West Clostridi isolted from feces. J. Med. Microbiol. 9: Gurino, P. A., nd A. Krmer Gs chromtogrphic nlysis of hed-spce vpors to identify microorgnisms in foods. J. Food. Sci. 34: Hchenberg, H., nd A. P. Schmidt Gs chromtogrphic hedspce nlysis. Heyden & Son Ltd., London. 7. Huser, K. J., nd R. J. Zbrnsky Modifiction of the gs-liquid chromtogrphy procedure nd evlution of new column pcking mteril for the identifiction of nerobic bcteri. J. Clin. Microbiol. 2: Holdemn, L. V., nd W. E. C. Moore (ed.) Anerobe lbortory mnul, 3rd ed. The Virgini Polytechnic Institute nd Stte University, Blcksburg. 9. Mrshll, J. L., nd D. A. Porter Investigtions into the preprtion of glss open tube gs chromtogrphy columns. J. Chromtogr. 122: Norrmn, J Production of voltile orgnic compounds by the yest fungus Dipodscus ggregtus. Arch. Microbiol. 68: Piermi, R. M., nd K. E. Stevenson Detection of metbolites produced by psychrotrophic bcteri growing in milk. J. Diry Sci. 59: Yoshiok, M., M. Kitmur, nd Z. Tmur Rpid gs chromtogrphic nlysis of microbil voltile metbolites. Jpn. J. Microbiol. 13: Downloded from on November 5, 218 by guest