Wiesław Barabasz, Maria J. Chmiel & Marek Ostafin

MICROBIOLOGICAL CHARACTERISTICS OF QUATERNARY

SEDIMENTS AT STARUNIA PALAEONTOLOGICAL SITE

AND VICINITY (CARPATHIAN REGION, UKRAINE)

De part ment of Mi cro bi ol ogy, Uni ver sity of Ag ri cul ture, Al. Mick iewicza 24/28, 30- 059, Kraków, Po land; e- mail: rrba raba@cyf- kr.edu.pl

Ba ra basz, W., Chmiel, M. J. & Ostafin, M., 2009. Mi cro bio logi cal char ac ter is tics of Qua ter nary sedi ments at Sta runia pa lae on to logi cal site and vi cin ity (Car pa thian re gion, Ukraine). An nales So cie ta tis Ge olo go rum Po lo niae, 79: 439–446.

Ab stract: The mi cro bio logi cal re search on the area of the pa lae on to logi cal site in Sta runia (Ukraine) re veals the de tails of bio logi cal ac tiv ity of the near- surface lay ers and Qua ter nary sedi ments. In Sta runia area rem nants of a mam moth and three woolly rhi noc er oses, and one al most com pletely pre served rhi noc eros car cass were found in 1907 and 1929. The gained quan ti ta tive re sults re gard ing the oc cur rence of dif fer ent physio logi cal groups of mi cro or gan isms show that their number var ied sig nifi cantly de pend ing on the sam pling place, sam pling depth, pH, hu mid ity and the or ganic mat ter con tent. The amount and dif fer en tia tion of the tested groups of mi cro or gan isms typi cally de creased with the depth. In sev eral deep sampling lo ca tions there was in crease in the mi cro -or gan isms, es pe cially with meth ano gens and metha no trophs. The me tano gens oc curred mainly in Pleis to cene sedi ments, com prised of clayey mud and peat, while sap ro phytic mi cro or gan isms (bac te ria, fungi and actino my ce tes) oc cur in Holo cene sedi ments com prised of clayey mud, peat and peat mud. The quan tity of mi cro -or gan isms in se lected b-ore holes was re lated to high con cen tra tion of the -or ganic mat ter (mainly peat and peat mud) and cor re lated with meth ane oc cur rence.

Key words: mi cro or gan isms, meth ano gens, Holo cene, Pleis to cene, Sta runia pa lae on to logi cal site, Car pa thian re gion, Ukraine.

Manu script re ceived 22 May 2009, ac cepted 29 September 2009

IN TRO DUC TION

The area of the aban doned ozo ke rite mine in Sta runia near Ivano- Frankivsk is one of the most in ter est ing places in Ukraine re gard ing the geo logi cal struc ture and oc cur rence of oil and ozo ke rite as well as spe cific Pleis to cene flora and fauna (Ko tarba, 2009). In the Sta runia area rem nants of a mam moth and three woolly rhi noc er oses, and one al most com pletely pre served rhi noc eros car cass were found in 1907 and 1929. Sci en tists con sider the pos si bil ity of find ing more fauna ex am ples and con duct re search to lo cate oc cur -rences of an other rhi noc eros or other rep re sen ta tives of Qua ter nary fauna. Gen eral in for ma tion cov er ing the his tory of the area and de tails about ge ol ogy and re sults of the ear -lier re search works con ducted in the area are de scribed in a spe cial mono graph de voted to Sta runia (Ko tarba, ed., 2005) and more re cent works (Ko tarba et al., 2008; Ko tarba & Stachowicz- Rybka, 2008; Soko³owski et al., 2009; Soko-³owski & Stachowicz- Rybka, 2009; Stachowicz- Rybka et

al., 2009).

Spe cial at ten tion should be paid to mi cro bio logi cal in -di ca tors as one of the pa rame ters which may in -di cate the

pres ence of a large amount of or ganic mat ter and their bio -trans for ma tion prod ucts in the soil en vi ron ment.

All bio logi cal growth and de vel op ment pro cesses are re lated to the most im por tant bio genic com pounds, car bon, ni tro gen, sul phur and phos pho rus use. The trans for ma tion of these ele ments mostly regu lated by the bi otic pro cesses (Al ex an der, 1975; Ba ra basz, 1991; Ba ra basz, 1992; Ba ra -basz & Voøišek, 2002; Smyk, 1995). Mi cro or gan isms with a high meta bolic rate, in flu ence the dy nam ics of many bio chemi cal and bio geo chemi cal pro cesses in soils. Bio chemi -cal pro cesses re lated to the trans for ma tion of or ganic mat ter and syn the sis of in or ganic and or ganic com pounds as well as bio logi cally ac tive sub stances de pend on the mi cro or gan -isms (Ba ran et al., 1993; Lynch & Poole, 1979; Met ting, 1993; Mc Ar thur, 2006; Stewart & Carl ton, 1986; Trevors & van El sas, 1995).

Mi crobes, take part in many biogeochemical pro cesses and gen er ate many char ac ter is tic prod ucts, and while do so, greatly in crease their own num bers. Mi cro bi o log i cal meth ods can be used to de tect spe cific groups of mi cro or gan

-isms, which will sug gest cer tain pro cesses. By es ti mat ing the quan tity of mi cro or gan isms or the ac tiv ity of some pro -cesses, the places of the high est in ten sity of these changes can be de fined (Al ex an der, 1975; Buckley & Schmidt, 2002; Degenes et al., 2001; Tate, 1986; Torsvik et al., 1990; Torsvik & vree¯s, 2002). Such places, in which large groups of spe cific mi cro or gan isms oc cur as the typ i cal ones, or as anom a lies, should re ceive spe cial at ten tion. High amounts of mi cro or gan isms, ac tive as the or ganic car bon

(cellulolytic bac te ria, methanogenic and methanotrophic), ni tro gen (ammonifiers, nitrifiers, denitrifiers) and phos pho -rus com pounds bio-trans form ers, will show the pro cesses of or ganic mat ter min er al iza tion which oc cur in aer o bic as well as in an aer o bic con di tions. Mi cro bi o log i cal or ganic mat ter min er al iza tion may re sult in the oc cur rence of some dif fer ent prod ucts de pend ing on the en vi ron men tal con di tions. An aer o bic con di tions, which oc curred in the nearsur -face lay ers of the Qua ter nary sed i ments, will pro mote the

Fig. 1. Sketch map of the Starunia palaeontological site and sur round ing area (Carpathian re gion, Ukraine) show ing the lo ca tion of sam pling bore holes for mi cro bi o log i cal study

fer men ta tion pro cesses, dry rot ting and methanogenesis, whereas the aer o bic con di tions will pro mote the ox i da tion and rot ting pro cesses (Fenchel et al., 1998; Kirk et al., 2004; Stew art & Carlton, 1986).

Groups of mi cro or gan isms change their bio mass, meta bolic ac tiv ity and the mi cro bio ceno tic com po si tion (bio di ver sity) as a re sponse to nu mer ous stress fac tors and stimu la tors (Ehr lich, 1996; Met ting, 1993; Tate, 1986). Mi cro or -gan isms play an im por tant role in the al ter ing of vari ous sub stances in the en vi ron ment as well as in the soil. The re -sult of their ac tiv ity is not only the de cay and min er ali za tion of the or ganic com pounds, but also the ac ti va tion of many min er als, which play a fun da men tal role in higher plants’ growth. Nu mer ous soil and pedo gene sis pro cesses like de -com po si tion of sili cates, alu mi no sili cates, apa tites and other min eral com pounds of soils, soil struc ture crea tion, syn the -sis and de com po si tion of hu mus, free ni tro gen fixa tion etc. are highly de pend ent on higher plants (Ba ra basz & Voøišek, 2002; Smyk, 1984). It may be eas ily sug gested that in ter re -la tions be tween popu -la tions of soil mi cro or gan isms and the mi cro bio logi cal suc ces sion in flu ences the en tire soil me -tabo lism. Elimi na tion or limi ta tion of ac tiv ity of any of the mi cro bio logi cal chain in the soil en vi ron ment breaks or at least dis rupts the whole food chain (Pep per et al., 1995; Mc Ar thur, 2006).

The main aim of the re search was the char ac teri za tion of the mi cro bio logi cal ac tiv ity in the gen eral en vi ron ment of Pleis to cene and Holo cene sedi ments and in the mine dump. In ad di tion an ob jec tive was locating any anoma lies re lated to the quan tity or bio di ver sity of the so called mi cro -bio ceno tic com po si tion of groups of mi cro or gan isms, which had been selecteded as in di ca tor.

SAM PLING AND METH ODS

Sedi ment core sam ples were taken from the bore holes in 2007 and 2008. Fourty-six sam ples from vari ous depths of 15 bore holes were used for mi cro bio logi cal analy ses (Fig. 1). The bore holes were made in places in which natu ral anoma lies had been iden ti fied dur ing the pre vi ous analy ses. Mi cro bio logi cal analy ses were fo cused on mi cro or gan -isms as so ci ated with car bon trans for ma tions. These mi cro-bes were cho sen as in di ca tors to evalu ate mi cro bio ceno tic trans for ma tions in flu enced by natu ral en vi ron mental chan-ges and by hu man ac tiv ity e.g. dur ing ozo ke rite exploita-tion.

About 500 grams of sedi ment from each sam ple were col lected dur ing drill ing and put into con tain ers, pre serv ing the mi cro bio logi cal ste ril ity. Im me di ately af ter sam pling the pH was evalu ated in the field (pH in H2O). The sam ples were stored at 4oC un til ready for analy sis. Mi cro bio logi cal analy ses were car ried out us ing the se rial di lu tions method to de ter mine the quan tity of bac te ria, spore form ing bac te ria, actino my ces, fungi and me ty lotrophic as well as meth -ano genic bac te ria on sev eral spe cific me dia, com monly used in mi cro bio logi cal labo ra tory.

Be cause of huge dif fer ences be tween the hu mid ity of sam ples, dry mass was also de ter mined; the re sults were cal -cu lated and pre sented as col ony form ing units (cfu) per 1

gram of the soils’ dry mass (Alef & Nan nipieri, 1995; At las & Parks, 1997; Tun lid & White, 1991; Paul & Clark, 2000). All mi cro bio logi cal analy ses were car ried out at the labo ra tory of De part ment of Mi cro bi ol ogy, Uni ver sity of Ag ri cul -ture in Kraków.

RE SULTS AND DICUSSION

The mi cro bio logi cal part of the analy ses of sedi ment sam ples aimed at show ing the prob able changes in the quan tity of soil mi cro or gan isms in the bore holes on each geo logi cal level. The re sults, pre sented in (Tab. 1, Fig. 2) show very large dif fer ences in the popu la tion of the tested groups of mi crobes. The graphs (Fig. 2) show quan ti ta tive changes in the mi cro or gan isms’ com po si tion de pend ing on the depth and the pH re ac tion.

The re sults, re lated to the oc cur rence of dif fer ent physio logi cal groups of mi crobes (Table 1), show that their quan tity var ied con sid era bly, de pend ing on the sam pling site, sam pling depth and or ganic mat ter con tent. The de -tailed mi cro bio logi cal analy ses in di cated that to gether with the sam pling depth, the quan tity and di ver sity of mi cro or -gan isms are lower. A defi nitely higher quan tity of the tested mi crobes, es pe cially bac te ria and actino my ces and a very high number of fungi which take part in cel lu lose de com po -si tion, sug gest ing that the near- surface level with aero bic con di tions, pro motes the pro cesses of or ganic mat ter min er -ali za tion in the tested Qua ter nary sedi ments.

The pH of the tested sam ples was mostly equal to about 6, fre quently rang ing be tween 6.5 and 7.5. This sig ni fies that the sam ples were neu tral or slightly acidic. The bore -hole No. 5N with pH from 6.36 to 7.54 and 33 in which pH was al ka line, from 8.07 to 9.4 were mini mal and maxi mal (Table 2, Fig. 2). Simi larly, in the tested ma te rial high dif -fer ences in hu mid ity were ob served (30 to 55%), some times it was even over 80%, e.g. sam ples from bore holes Nos 8, 23, 30N, 32N, 33, 42 and 43 (Table 2).

The near surface level, with in tense or ganic mat ter bio -trans for ma tion pro cesses, sup plied the sedi ment with much nutrient- rich sub stances which are fa vour able to chemical con di tions. These pro cesses also im proved the en -vi ron mental con di tions for the growth and de vel op ment of dif fer ent groups of mi cro or gan isms in clud ing the tested groups of mi crobes. The mi cro bio logi cal analy ses of the bio di ver sity sug gest that this lo ca tion has spe cific soil mi -cro flora, typi cal for grass land eco sys tems, rich in or ganic mat ter and with neu tral or slightly acidic pH. Af ter ana lys -ing the mi cro bio ceno tic com po si tion in each test -ing point it may be as cer tained that the tested groups un der went a high dif fer en tia tion de pend ing on the sam pling depth, es pe cially for bac te ria, actino my ces and fungi.

How ever in some bore holes at great depths where less or ganic mat ter oc curred, the number of mi cro or gan isms was ob served to in crease. This was par ticu larly in the case of the sam ples con tain ing or ganic mud at greater depths. For ex am ple, in the Holo cene pro files of bore holes Nos 5N, 8 and 32N (Fig. 2) where peat pre domi nated, a sig nifi cant in crease of the fungi – es pe cially yeasts – count was ob -served, most likely be cause they cope with the an aero bic

Ta ble 1

Num ber of se lected groups of mi cro or gan isms in core sam ples Bore-hole Depth (m) Dry mass (%) pH Strati-graphy Number of bacteria 103 (cfu g-1) Number of sporulating bacteria 103 (cfu g-1) Number of fungi 103 (cfu g-1) Number of actinomycetes 103 (cfu g-1) Number of methylotrofic bacteria (cfu g-1) Number of methanogenic bacteria (cfu g-1) 4' 2.0 45.9 6.98 H 3050 261 17 1702 120 0 4.0 62.6 6.92 P 2891 48 38 1240 350 170 4N 1.4 73.3 8.00 H 392906 82 6 68921 0 0 2.5 67.1 7.21 H 125186 63 1.5 51431 0 0 4.15 70.5 7.07 H 34043 79 1.1 790 0 0 6.3 69.5 7.41 P 3108 46 1.0 9 0 0 5N 2.1 72.8 7.54 H 6923 41 5 4321 0 0 2.7 67.3 6.64 H 6315 1204 4 1405 0 0 4.7 60.9 6.36 H 77 805 3 1.2 30 60 6.5 78.9 6.57 H 5678 253 0.4 0.1 30 40 7 5.2 66.5 7.31 H 481 0.3 0.9 320 0 0 6.55 64.2 6.63 H 436 0.3 0.6 281 20 40 8 0.5 83.9 7.61 H 38141 13 5 12387 0 0 1.5 77.2 8.04 H 43523 39 0.8 3609 0 0 2.5 78.0 7.82 H 14564 3 6 2808 0 0 3.5 71.8 6.80 H 42507 0.5 91 5201 0 0 3.8 72.6 7.78 H 26446 1.4 0.3 490 0 0 15 2.0 61.8 7.36 P 4693 178 11 2030 0 0 4.0 72.5 7.37 P 566 856 0.6 43 0 20 6.0 73.8 7.45 P 1.5 163 0.4 0.07 0 0 23 2.0 84.0 7.95 P 4524 30 0.7 3302 0 0 4.0 74.0 7.09 P 25730 23 0.2 2100 0 10 27' 8.0 76.9 6.96 MD 23927 26 10 4270 0 0 30N 2.9 73.8 6.39 P 126 7 0.5 491 40 0 5.0 75.0 6.85 P 5.3 27 0 54 210 360 7.3 84.1 8.25 P 108 6 0 2 150 270 8.2 77.8 7.90 P 129 9 0.2 57 80 60 32N 1.0 82.0 7.27 H 11098 73 6 9644 0 0 2.0 68.7 6.87 H 34798 2169 0.3 8632 20 0 3.0 69.3 7.16 H 7619 22 0.5 8501 0 20 4.0 76.5 7.41 H 10876 16 0 3470 60 70 5.0 86.0 7.58 H 12047 34 0 6322 80 80 33 1.8 77.3 9.36 H 298 16 0.02 980 0 0 4.0 67.9 9.29 P 943 6 0.2 410 0 0 6.0 83.4 8.80 P 432 8 0.08 1.0 0 20 8.1 70.9 8.07 P 17 2 0.05 0.2 0 0 36N 0.6 64.1 7.23 H 468 17 0.2 320 0 10 4.0 76.1 7.58 P 946 53 0.4 307 110 170 6.0 78.0 7.71 P 179 38 0.1 4 120 90 37 6.0 73.8 7.23 P 47 1.5 0.2 50 0 0 42 3.8 76.1 6.74 H 118 0.5 0.9 890 20 30 4.6 82.5 7.07 H 1091 5 1.1 62 60 40 6.45 84.4 7.90 P 341 2 0.5 0.3 30 40 43 3.3 70.8 7.33 MD 33898 0.7 0.2 1279 0 0 4.5 73.8 6.60 MD 23848 0.9 0.08 1310 0 0 6.7 82.3 7.80 MD 5103 194 0.04 16 0 10

con di tions due to their fer men ta tive me tabo lism. A higher number of actino my ces was also ob served. These con sti tute the most nu mer ous mi cro or gan isms in Pleis to cene (pro file No. 23) and Holo cene (pro files Nos 5N, 8 and 32N) peats (Fig. 2). Our ob ser va tions in di cate that the main sap ro phytic mi cro or gan isms (bac te ria, fungi and actino my ce tes) oc -curred in Holo cene sedi ments con tain ing clayey mud, peat and peat mud.

On the other hand a great number of meth ano gens were ob served in bore holes in which en coun tered Pleis to cene

sedi ments (Table 2). The high est amount of meth ano genic bac te ria was found in bore holes Nos 30N, 4’ and 36N, which con tain clayey mud and peat.

From the eco log i cal point of view the iso la tion of methanogenic and methylotrophic bac te ria from the tested sed i ments es pe cially from the Pleis to cene lay ers was very in ter est ing. These bac te ria use CO2 and ac e tates which orig -i nated from an aer o b-ic fer men ta t-ion of the or gan-ic mat ter. The abun dance of methanogenic bac te ria at greater depths of the tested bore holes may in di cate the mi cro bial meth ane

orig i na tion pro cess. The data show that bore holes Nos 30N, 36N, 5N, 7, 32N and 42 (Table 2) had the high est amounts of methanogenic bac te ria. Oc ca sion ally methanogens were found in the sam ples from bore holes Nos 15, 33, and 43 (Table 2), which con tained large amounts of or ganic mat ter at greater depths. The place where the num ber of methano-genic bac te ria oc curred in vary ing amounts is marked with a cir cle in Fig. 3. Try ing to es ti mate the quan tity, it can be stated that amounts of methanogenic bac te ria in the se lected bore holes were re lated to high con cen tra tion of the or ganic mat ter and were cor re lated with meth ane oc cur rence, which is marked in Fig. 3. This was doc u mented in the nearsur -face gas re search Dzieniewicz et al. (2009), Kotarba et al. (2005, 2009) and Sechman et al. (2009).

CON CLU SIONS

Mi cro bi o log i cal anal y ses of the Ho lo cene and Pleis to cene sed i ment sam ples in di cate high di ver sity of the mi cro or gan isms, de pend ing on the lo ca tion of the bore hole, sam -pling depth, pH re ac tion and sam ple humidity as well as lithology.

Mi cro bi o log i cal char ac ter is tic of the tested Qua ter nary sed i ments showed that they are ex am ples of typ i cal soil microflora of the grass land eco sys tems rich in or ganic mat -ter and with slightly acid to neutral reaction.

Methanogens oc cur ring in places are in dic a tive of an -aer o bic pro cesses of the or ganic mat ter de com po si tion. Metanogens oc curred mainly in Pleis to cene layer which con tain ing mainly clayey mud and peat.

The quan tity of mi cro or gan isms in se lected bore holes was re lated to high con cen tra tion of the or ganic mat ter (mainly peat and peat mud) and cor re lated with meth ane occurrence.

Main groups of saprophytic mi cro or gan isms (bac te ria, fungi and actinomycetes) oc curred in Ho lo cene lay ers con -tain ing clayey muds, peats and peat muds.

Ac knowl edge ments

Fi nan cial sup port from the Min is try of Sci ence and Higher Ed u ca tion (grant No. 139/UKR/2006/01) is kindly ac knowl edged. Re view com ments and sug ges tions by Jan Kucharski of the Uni -ver sity of Warmia and Mazury in Olsztyn and Andrzej Nowak of the West Pom er a nian Uni ver sity of Tech nol ogy in Szczecin were Ta ble 2

Bore holes with oc cur rence of metanogens in var i ous lithostratigraphic units

Borehole Depth (m) Dry mass (%) pH Stratigraphy Lithology Number of methylotrofic bacteria (cfu g-1) Number of methanogenic bacteria (cfu g-1) 4' 2.0 45.9 6.98 H clayey mud 120 0 4.0 62.6 6.92 P clayey mud 350 170 5N 4.7 60.9 6.36 H clayey mud 30 60 6.5 78.9 6.57 H clayey mud 30 40 7 6.55 64.2 6.63 H biogenic mud 20 40 15 4.0 72.5 7.37 P clayey mud 0 20 30N 2.9 73.8 6.39 P clayey mud 40 0 5.0 75.0 6.85 P clayey mud 210 360 7.3 84.1 8.25 P clayey mud 150 270 8.2 77.8 7.90 P peat 80 60 32N 3.0 69.3 7.16 H peat 0 20 4.0 76.5 7.41 H peat mud 60 70 5.0 86.0 7.58 H peat mud 80 80 33 6.0 83.4 8.80 P clayey mud 0 20 36N 0.6 64.1 7.23 H clayey mud 0 10 4.0 76.1 7.58 P clayey mud 110 170 6.0 78.0 7.71 P clayey mud 120 90 42 3.8 76.1 6.74 H clayey mud 20 30 4.6 82.5 7.07 H clayey mud 60 40 6.45 84.4 7.90 P clayey mud 30 40 43 6.7 82.3 7.80 MD mine dump 0 10

very help ful. We would like to ex press our grat i tude to Mark Pawlewicz of U.S. Geo log i cal Sur vey in Den ver for his crit i cal com ments, which im proved this pa per.

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