Department of Biochemical Technology, School of Chemical Technology

Slovak Technical University, Bratislava

Bratislava 1999

Department of Biochemical Technology

School of Chemical Technology

Slovak Technical University

Bratislava

Research Report (Contract for Work No. 69/99)

Theoretical and Experimental Studies of Piešťany Mud and River Sediment

Microbial Community

Responsible Compilor: Ing. Mária Šturdíková, PhD.

Cooperating Compilors: Ing. Katarína Mináriková

Bratislava 1999

Contents

Introduction

1 Literary Overview of Knowledge on Microbial Community of River, Sodden

and Sea Sediments 2

1.1 Extremophillic microorganism 2

1.1.1 Thermophills and Hyperthermophills 2

1.1.2 Metabolism of Saccharides in Extremophillic Microorganisms 5

1.1.3 Metabolites of Extremophillic Microorganisms 9

1.1.4 Utilisation of Inorganic and Organic Sulphur Substances by

Extremophillic Bacteria 9

1.1.5 Characteristics of Sulphur Thermal Ecosystem 12

1.1.6 Assimilation of Organic Sulphur Substances 13

1.1.7 Thermophillic bacteria of Bacillus strain 15

2 Material and Methods 18

2.1 Chemicals and Tested Substances 18

2.2 Used Instruments 18

2.3 Microorganisms and Cultivation Conditions 19

2.4 Measurement of Respiration Using Oxygen Electrode 20

2.5 Antifungal Activity Tests 21

2.6 Enzyme Activity Determination 21

3 Results and Discussion 23

3.1 Thermophillic Bacteria Bacillus sp. SU-2 and SU-3 Utilising Sulphur

Substances 23

3.1.1 Impact of Added Sulphur Substances on Growth and

Respiratory Activity of Thermophiles SU-2 and SU-3 23

3.1.2 Products of Sulphur Substances Conversion by Thermophiles

SU-2 and SU-3 with Inhibitory Effect on Elastase Activity 29

3.1.3 Amino Acids as Substrates and Products of Thermophillic

Bacteria SU-3 Cultivation on Medium Enriched by

Sodium Sulphide 32

3.2 Characteristics of Mud from the Bypass Arm Location 34

3.2.1 Changes in Mud pH Values Under the Impact of Temperature 34

3.2.2 Biological Activity of Mud from a New Location of Bypass

Arm and Comparatory Samples from the Maturing Pool and

Mud Kitchen 39

4 Summary 41

References 43

Introduction

Sea, river and sodden sediments are the source of a varied range of

microorganisms. Physical-chemical characteristics of these biotopes especially

represent high temperatures, extreme pH, possibly high salts concentrations.

Microbial populations found in extreme environments are able to adapt to

various conditions and often produce a large number of organic soluble

substances playing the role of microbial cells protectives enabling growth,

reproduction and biochemical processes under stressful conditions. Studies of

extremophiles try to clarify the still “limited” knowledge on their metabolisms.

The use of mud and sulphur thermal water for therapeutic purposes

forms part of large international study projects on extremophillic

microorganisms. Results included in these projects also point to the possibility

of new bioprocess and microbial products generation use in economy.

Extremophillic microorganisms represent potential producers of a large

number of thermostable enzymes and secondary metabolites with significant

antibiotic features, especially interesting in pharmacology. Crucial advances

have been made in the development of effective cultivations methods for

thermophiles (dialyse bioreactors, continually aerated fermenters with cell

recycling).

Research focusing around the possibilities of microorganisms therapeutic

use include the search for their secondary metabolism products (enzyme

inhibitors, antibiotics) and study of their effective mechanism.

This study was initiated in cooperation between Slovenské liečebné

kúpele, a.s. Piešťany and the staff of Biochemical Technology Department of

Chemical Technology School at the Slovak Technical University in Bratislava. It

aimed to study the impact of sulphur substances on growth and respiratory

activity of thermophillic bacteria Bacillus sp. SU-2 and Bacillus sp. SU-3 isolated

from Piešťany mud in the previous period.

Further, it aimed to study the effect of these sulphur substances on

elastase activity, model enzyme for the assessment of mud and thermal water

therapeutical effects. This study also assessed the quality of mud from a new

location of bypass arm.

1 Literary Overview of Knowledge on Microbial Community of River, Sodden

and Sea Sediments

1.1 Extremophillic Microorganism

Extremophillic microorganisms represent a valuable source for the implementation of

unusuall biotechnological processes and the construction of unified models for the

examination of biomolecules stability under extreme conditions [1,2].

Multidisciplinary research takes place at three levels: isolation/taxonomy,

physiology/biochemy, molecule biology/genetics [3]. Many unusual microorganisms growing

in extreme biotopes, sucha s hypersalt lakes, solfatara fields and hydrothermal areas show

unique characteristics. Many of them were isolated, identified and characterised in detail.

European Commission recognised extremophiles as a priority research topic in various

biotechnological programs and also stimulated and supported research and experimental

acitivities in this field, starting from the first European Union Biotechnological Program in

1982. The program of extremophillic microorganisms research is centred around groups

Archae and Bacteria and is takes place in three principal levels: microorganisms able to live

in environment a) with extreme temperatures (thermophiles and psychrophiles), b) with

extreme pH (alcalophiles and acidophiles), c) with high salt concentrations (halophiles) [4].

1.1.1 Thermophills and Hyperthermophills

Thermophillic and hyperthermophillic Archae and Bacteria were isolated from

theothermal and hydrothermal sources with temperature exceeding 60°C. Group of these

extremophiles includes anaerobe and aerobe, chemolitoatutrophic and heterotrophic

microorganisms growing in neutral and acid pH. There is a great variance in environmental

temperature tolerance by hyperthermophiles groups. It is deducted from the heterogeneity

of 16 S rRNA and from unusual physiological characteristics. In their biotopes,

hyperthermophiles are primary producers or consumers of organic substance. Based on

excellent resistance to higher temperatures, they are an interesting object for general

research and biotechnology.

There are 54 known types of hyperthermophiles Archae and Bacteria. They differ in

phylogenesis and physiological characteristics [5, 6]. Based on 16 S rRNA analysis a

phylogenetic tree was constructed (Fig. 1.1).

Fig. 1.1 Phylogenesis of Archae and Bacteria hyperthermophiles [7]

Hyperthermophiles adapt easily to various biotopes, they grow at high temperatures and

extreme pH, redox potentials and high salt concentrations [7].

Extremely acidophilic hyperthermophiles include kinds Sulfolobus, Metallosphaera,

Acidianus and Stygiolobus [8]. They grow in aerobe, or strictly anaerobe conditions, in acid

pH (pH = 3.0). Sulfolobus kinds are strictly aerobe, their growth is autotrophic, they oxide S0,

S2- and H2 under the generation of sulphuric acid and water (Chart 1.1). certain types are

facultative or obligatory heterotrophes growing on saccharides, yeasty extract and peptone.

Chart 1.1 Energy development reactions of chemolitoatotrophic hyperthermophiles [8]

4H2 + CO2 → CH4 + 2H2O Methanopyrus, Methanotermus, Methanococcus

H2 + S0 → H2S Pyrodictium, Thermoproteus, Pyrobaculum, Acidianus, Stygiolobus

4H2 + H2SO4 → H2S + 4H2O Archaeoglobus

H2 + HNO3 → HNO2 + H2O Pyrobaculum, Aquifex

H2 + ½ O2 → H2O Pyrobaculum, Aquifex, Sulfolobus, Acidianus, Metallosphaera

2 S0 + 3O2 + 2H2O → 2H2SO4 Aquifex, Sulfolobus, Acidianus, Metallosphaera

(FeS2 + 7O2 + 2H2O → 2FeSO4 + 2H2 SO4)

Neutrophillic and slightly acidophillic hyperthermophiles are mostly strictly anaerobe,

found in the soil of solfatara areas, in undersea hydrothermal systems and in deep oil

reservoirs [9]. Thermoproteus neutrophilus, Thermoproteus tenax and Pyrobaculum

islandicum grow chemolitoautotrophically, obtain energy through fermentation of peptides,

amino acids and saccharides under the formation of fatty acids, CO2 and H2. Hydrogen

formation has a growth inhibitory effect. However, this may be removed through the

addition of S0 under the generation of H2S [10].

1.1.2 Metabolism of Saccharides in Extremophillic Microorganisms

Analysis of extremophillic microorganisms central metabolism enables the detection

of adaptability to extreme conditions and the obtaining of information on evolution. They

are mostly obligatory anaerobe heterotrophs reducing elementary sulphur to H2S. Best

developed are studies on biochemical essence of Archae Pyrococcus futiosus and Bacteria

Thermotoga maritime. In Pyrococcus furiousus, pyruvate is transformed through acetyl-CoA

to acetate, with the use of ADP forming acetyl-CoA synthetase (Fig. 1.2), which we do not

normally encounter in bacteria [11]. Pyrococcus furiousus ferments pyruvate under the

formation of acetate, H2 and CO2. As a source of nitrogen, it does not utilize ammonia or free

amino acids, but peptides and proteins (yeaty extract, tryptone, casein). Conversion of

saccharides to end acetate, H2 and CO2 takes place along the modified Entner-Doudoroff

route independent from nicotinamide nucleotids and includes new types of ferredoxine

binding oxidoreductases [12].

Of extremophillic microorganisms, enzyme glucose-ferredoxine oxidoreductase (Glu

OR) was isolated. Ferredoxine plays the role of physiological electron donor for hydrogenase,

enzyme responsible for H2 catalytic production. Glu OR oxidates glucoses to gluconic acid.

Another unusual enzyme is aldehyde ferredoxine oxidoreductase (AOR) catalyzing

oxidation of aldehydes to the respective acids, does not oxidate glucoses or aldehyde

phosphates and the reaction takes place under low potential (-520 mV).

In the third oxidation step of glucoses conversation to acetate, the reaction of

pyruvate decarboxylation to acetyl-CoA is catalysed by pyruvate ferredoxine oxidoreductase

(POR). POR is highly specific for pyruvate as substrate [13].

All three enzymes of glucose, glyceraldehydes and pyruvate oxidation use

ferredoxine as electron carrier (Fig. 1.2). Formation of glucose-6-phosphate from pyruvate

may take place under the use of enzymes of the Embden-Meyerhof route along with

phosphoenolpyruvate synthetase and fructose-1,6-bisphosphate fosphatase. In Pyrococcus

furiosus, gluconeogenesis takes place through Embden-Meyerhof route.

Fig. 1.2 Metabolism of saccharides in Pyrococcus furiosus [12]

Bacterial kind Thermotoga ferments various carbon sources, such as glucose, starch, xylanes

under the formation of acetate, L-lactase, H2 and CO2. For saccharides oxidation, it uses

Embden-Meyerhof route (Fig. 1.3). From the aspect of growth, the presence of sulphur (S0) is

not necessary. However, following its addition to the growth medium, it is reduced to H2S

(fig. 1.4).

Fig. 1.3 Metabolism of saccharides in Thermotaga maritima [12]

Sulphur reduction S0 is practically the universal characteristic of hyperthermophiles.

Sulphur reductase (sulphur-reducing ferredoxine oxidoreductase) is an enzyme localized in

cytoplasma. It uses polysulphides as electron donors. Hydrogenase is an enzyme generating

H2. This bifunctional enzyme is designated as sulphhydrogenase and reduces protons to H2

and forms H2S in the presence of S0 (Fig. 1.4).

Fig. 1.4 Coupling mechanism of carbon sources oxidation by ferredoxine with the effect of

enzyme sulphydrogenase in Thermotoga species [12]

1.1.3 Metabolites of Extremophillic Microorganisms

Carotenoids

An important characteristic of haloarchae is the synthesis of carotenoids high level,

especially of bacterioruberines with a significant role in the protection from the effects of

oxygen and light. Alternative synthetic routes from lycopene to bacteriorubines. (C50

carotenoids), or to β-caroten and retinal described in case of Halobacterium salinarium [17].

Halobacteriaceae is a family of bacteria capable of growing in salt lakes with a high

content of sodium chloride, whereby typical pink-red pigment is released into water. One of

the carotenoids is astaxantine (3.3-dihydroxy-β-β-caroten-4.4-dione), especially important in

food industry. Pink-orange carotenoid 3-hydroxy-echinenone, similar to trans-astaxantine

was found in large volumes in H. salinarium [18, 19].

Sulpholobicine

Sulfolobus islandicus, strain Hen 2P/2 produces cytotoxic sulpholobicine released into

the medium by growing cells. It has bactericide activity to Sulfolobus solfataricus PI.

Metabolite sulpholobicine has similar effect as bacteriocines. It is resistant to medicaments

with deoxyribonuclease and ribonuclease, but it is sensitive to trypsine and proteinase K

[20].

1.1.4 Utilisation of Inorganic and Organic Sulphur Substances by Extremophillic Bacteria

Sulphide production is the crucial problem related to anaerobe degradation of

sulphates and sulphites found in waste water. Sulphate is used by sulphur reducing bacteria

as electron acceptor, reaction end product is sulphide [21]. In nature, biological oxidation of

sulphides takes place along three routes: a) anaerobe oxidation by photosynthetising

bacteria, b) oxidation by denitrification microorganisms, c) oxidation by oxygen colourless

sulphur bacteria. The group of colourless sulphur bacteria includes microorganisms differing

in physiology and morphology. Colourless sulphur bacteria include kinds: Thiobacillus,

Thiomicrospira, Sulfolobus, Thermothrix, Thiovalum, Beggiatoa, Thiothrix and Thiospira.

Beggiatoa, Thiotrix and Thiospira representatives accumulate generated sulphur inside cells.

Thiobacillus is an extracellular sulphur producer. Most of the kinds grow autotrophically, but

others are mixotrophic or heterotrophic. Energy is obtained from sulphides, elementary

sulphur, sulphites, polythionates and sulphates. Sulphur and polysulphides formatkion is the

result of biologic oxidation process. Sulphur production is the speed limiting stage in

sulphides oxidation.

Elementary sulphur oxidation process in Thiobaciullus thiooxidans includes two

stages: a) bacterial cells adhesion to solid sulphur particles, b) sulphur oxidation by cells

attacking sulphur granules. Key problem in sulphur oxidation by Thiobacillus is the

mechanism of initial attack on insoluble particles. For the process of sulphur oxidation, direct

contact of cells with solid sulphur is inevitable. Bacterial cells are able of adhesion with

respect to various types of biologically inactive surfaces. Absorption of bacteria to solid

particles is a process influenced by a number of various biological and physical-chemical

factors. Elementary sulphur reacts non-enzymatically with sulphides likes Na2S,

mercaptoethanol and cysteine under the formation of hydropolysulphides.

Oxidation abilities of thiobacillus vary in the respective strains. However, almost all

oxidize sulphides, sulphur and thiosulphates [23]. Thiobacillus concretivorus, Thiobacillus

thiooxidans and Thiobacillus thioparus oxidize sulphides to sulphur using catalytic effect of

sulphoxidase enzyme.

Crucial intermediates of the inorganic sulphur compounds oxidation process are

sulphites, further oxidized to sulphates. In Thiobacillus, sulphate is oxidized to sulphite via

sulphiteoxidase under the simultaneous formation of ATP in bond to phosphorylation

transport of electrons [24].

Thiobacillus ferrooxidans is able to grow on elementary sulphur with simultaneous

reduction of mineral iron, which is the electron acceptor in the process of sulphur

compounds oxidation [26]. Sulphate ions represent anion stabilisators of hexahydrate

complex Fe2+, which is the direct substrate for enzyme system oxidizing iron. Cells T.

ferrooxidans growing on reduced sulphur compounds utilizing sulphide-Fe3+ -

oxidoreductase and sulphite-Fe3+ - oxidoreductase for oxidation of reduced sulphur

compounds and formation of sulphites. Sulphide – Fe3+ - oxidoreductase activity is inhibited

by Fe2+.

Sulphonates

As a result of biological processes and chemical synthesis, compounds called

sulphonates are found freely in nature. They contain a sulphur atom covalently bound to

carbon atom, sulphur has oxidation number +5. An example of naturally found sulphonates

are taurine, izotionate, metanosulphonate, sulpholactate, sulphonolipids, aeroginozine and

coenzyme M. Since the listed compounds are present in the environment, but do not

accumulate, it is assumed that microorganism use them for their growth, or they are

otherwise biotransformed and form part of the entire sulphur cycle [27].

Despite taurine being able to serve as an energy, carbon and nitrogen source , it may

also be used as the sole source of sulphur in Clostridium pasteurianum C1, a bacteria

isolated from soil (Fig. 1.5). Sulphoacetaldehyde and izothionate also represent utilizable

substrate. Sulphopyruvate and sulpholactate are not utilized.

Fig. 1.5 Transmission of sulphonates (taurine and cysteat) [27]

Heterocyclic Sulphur Compounds

Some thermophillic bacteria degrade dibensotiophene (DBT). Splitting place is C-C

bond, since DBT is utilized as the sole source of carbon [28].

1.1.5 Characteristics of Sulphur Thermal Ecosystem

In water sediments, sulphur is subjected to bacterial redox transformations known as

microbial sulphur cycle. Sulphates reduction to hydrogensulphides (H2S) is catalysed by

strictly anaerobe, sulphur-reducing bacteria [34].

Various natural and industrial sources of methyl sulphur compounds were identified.

They play an important role in the complete sulphur cycle [35]. From soil and sea exosystens,

especially methylmercaptane (MSH) and dimethylsulphide (DMS) are released. Except for 3-

mercaptopropionate (MPA) and methantiole, waters of shore sediments may also include

other organic thiols, such as mercaptopyruvate, mercaptoethanol and mercaptoacetate.

These substances represent characteristic components of deep sea sediments. Sea

ecosystem includes polysulphides found in high concentrations. They are able to react with

organic matter under the formation of organic polysulphides [36]. Alcylsulphides, especially

DMS, dimetyldisulphide, diethylsulphide, ethylmethylsulphide, diprophylsulphide,

dibutylsulphide and dibutyldisulphide are oxidized to various thiols, such as metanthiol,

ethanthiol, propanthiol, butanthiol [37].

In sediments, H2S is acumulated only a few centimeters beneath the surface oxidized

layer [38]. Bacterial oxidation of H2S by manganese (Mn2+) and iron (Fe2+) oxides, which

are cumulated in large volumes especially in suboxidised deep layers, is inhibited by fast

chemical reactions between the respective compounds under the formation of S0 as the key

end product [39].

In seashore sediments, various bacteria strains were isolated forming bacterial

communities in the ecosystem, especially families Bacillus and Pseudomonas. To identify the

respective prokaryote cells inside complex compounds and their genetic description, the PCR

(PI-PCR) in situ method is used. The method includes amplification of specific sequences in

nucleic acids inside intact cells and enables analysis of microbial community structure [31].

Surface oxidized sediment layer is characterized by the formation of H2S, or other

partially reduced sulphur compounds that are re-oxidized to sulphates via

chemolitoautotrophic bacteria Baggiatoa and Thiobacillus [32].

Anaerobe strains producing methylmercaptane (MSH) and dimethylsulphide (DMS)

were isolated from sea sediments, especially sulphur dependent Archae Pyrobaculum,

Thermofilum, Thermoproteus, Desulfurococcus, Pyrococcus and bacteria Thermotoga [33].

1.1.6 Assimilation of Organic Sulphur Substances

Taurine

Taurine (2-amino-ethan-sulphonate) is one of the naturally found sulphonates. In

sulphonats, sulphur may be assimilated by aerobe and fermentative bacteria, if the entire

compound cannot be utilised as an independent source of carbon and energy for growth

[27].

In various sulphonats, sulphur may be assimilated as an independent sulphur source

by many microorganisms, including strictly aerobe bacteria, possibly anaerobe bacteria,

strictly fermentative bacteria and some yeasts, details of metabolic routs for sulphonate

sulphur assimilation and enzymes participating in this metabolism have not been clarified.

The removal of taurine amino group is the fist step in case of its use as carbon and energy

source.

Dibensothiophene

Dibensothiophene (DBT) and its alkyl derivatives are representative compounds of

organosulphur heterocycles. At higher temperatures, bacterial family of Bacillus kind is able

to split the carbon-sullphur bond (C-S) in DBT molecule.

Following the atack of DBT and its methylderivatives, both growing and quiescent cells may

release sulphur atoms in the form of sulphate ions at temperatures above 60°C. Thereby,

intact monohydroxylated hydrocarbon remains remain in the molecule. Various bacteria

types degrade DBT through target bond carbon-carbon (C-C), or carbon-sulphur (C-S)

splitting [41, 42]. The use of C-S bond splitting reaction is preferred.

Families Pseudomonas degrading DBT at 55°C in the same manner, create

hydroxylated remains with an inbuilt sulphur atom. DBT degradation by these bacteria is

optimum at higher temperatures. Specific splitting place is the C-S bond [43].

The addition of DBT into growth medium, as the sole sulphur source, enables

selection of desulphurization bacteria. Biotransformations of organic substances by growing

bacterial cells are especially suitable in terms of bacterial cells growth use as indicators of

organic substances utilizations as their essential nutrition components [44].

Methanthiol and 3-mercaptopropionate

Organic thiols with low molecule weight, e.g. 3-mercaptopropionate (MPA) and

methanthiol may be found in micromolar concentrations in anoxic waters of seashore

sediments [45]. Sulphur containing amino acids and dimethylsulphoniopropionate are

degraded by bacteria under the formation of methanthiol, MPA and other organic thiols [46,

47]. Thiols are also generated in chemical addition reactions of sulphides and polysulphides

to double bonds in organic molecules, for example MPA is produced from acrylic acid.

Polysulphides

Polysulphides are formed in the process of sulphides oxidation. However, they may

be toxic. As substrate, they are used by green sulphur bacteria Chlorobium limicola and

purple bacteria Chromatium vinosum, Thiocapsa roseopersicina [48].

Unstable polysulphides (SnS2- with n=1-5) are formed at pH 8.0 – 9.0. Stable

polysulphides are tetra and pentasulphide ions [21].

Alkyl Sulphides

One of the principal components in atmospheric sulphur compounds is

dimethylsulphide (DMS). Aerobe bacteria Thiobacillus thioparus TK-m and Hyphomicrobium

family EG are able to degrade DMS along the aerobe metabolic line [49, 50]. DMS is oxidized

by NADH-dependent monooxigenase to methanthiol and formaldehyde: CH3SCH3 + O2 +

NADH + H+ = CH3SH + HCOH + H2O + NAD+. Methanthiol is then oxidized by O2-dependent

transfiormation: CH3SH + O2 + H2O = HCNO + H2S + H2O2. Hydrosulphide is oxidized to

sulphate and protection from toxic H2O2 is secured by high catalase activity of both family

types [51]. DMS and CH3SH catabolism includes initial removal of methyl groups via

transmethylation reactions. It is a mechanism, in which O2 has the function of electron

acceptor. However, it does not participate in methyl groups destruction as substrate [52].

1.1.7 Thermophillic bacteria of Bacillus strain

Various bacteria families were isolated from seashore sediments. In ecosystem, they form

bacteria communities. These are especially Bacillus and Pseudomonas. To identify

respective procaryont cells inside complex mixtures and their genetic characteristics, the

PCR (PI-PCR) in situ method is applied. It includes amplification of specific nucleic acids

sequences inside intact cells and enables analysis of microbial community structure [31].

Bacillus bacteria were isolated from thermal environments. Their growth

temperatures exceed 60°C. Extremelly thermophillic families produce industrially utilised

thermostable enzymes and substances with antifungal activity, with the possibility of

these substances use in pharmaceutical i

ndustry.

Spectrum of Substances Produced by Bacillus Bacteria

Bacillus sp. Is the producer of various extracellular and intracellular proteases. Alkali

protease (subtilizine), neutral metaloprotease and esterase are released into cultivation

medium and serine proteases are produced inside cells.

Bacillus sp family CK 11-4 produces effective fibrinolytic enzyme, which was isolated

from culture supernatant and shows thermophillic, hydrophilic and fibrinolytic activities [53].

The sequence of amino acids (14 amino acids) of the N-terminal end is identical with the

sequence of amino acids in subtilizine Carlsberg, but the efficiency of fibinolytic activity is

about 8 times higher, compared to subtilizine Carlsberg.

Extracellular α-amylase was isolated from Bacillus sp. strain. The enzyme is sensitive

to amylase inhibitor HAIM [54]. Amylase sensitivity to HAIM is similar to animal amylases,

which is the result of HAIM-amylase complex generation in molar proportion 1:1.

Except for enzymes, Bacillus strains are the producers of a large number of

substances with antibiotic characteristics, with very varied structures. Surfactine represents

a cyclic lipopeptide antibiotic, foremed by a mixture of β-hydroxy fatty acids with a chain of

13-15 carbon atoms. Main component is 3-hydroxy-13 myristic acid forming lactone circle

with anion heptaptide (Fig. 1.6). It is the carrier of antifungal and antibacterial

characteristics, it inhibits the formation of fibrin clusters, induces the formation of ion

channels in lipidic double-layer of membranes and shows antiviral and antitumor activities

[55].

Fig. 1.6 Surfactine structure [55]

Fatty acid remainder of surfactine is the part, which ancors it into membrane lipidic

double layer and integrates with biomembrane systems of bacterial protoplasts, virus

capsides or mycoplasmas. The medicament application to mammal cells contaminated with

mycoplasma bacteria stimulated the speed of proliferation and change in mammal cells

morphology. Disintegration of mycoplasma membrane depends on physical-chemical

interaction of surface active substance with external part of lipidic membrane double-layer,

which results in changes in permeability. In high concentrations, surfactine causes complete

disintegration of mycoplasma membrane system.

The possibilities of Bacillus sp. use as biologic control to patogenous funguses were

described in various studies [56].

Bacillus licheniformis strain FSJ-2 produces various perishable and non-perishable

substances with antifungal effect. In 100-fold dilution, raw FSJ-2 filtrate shows antifungal in

vitro activity towards Microsporum canis, Trichophyton mentagrophytes, Arthroderma simii

and Arthroderma benhamiae. The filtrate effect was expressed in morphological changes as

a result of chitine synthesis impairment in cell wall. The deformations take place in the

growth stage. Inhibition of fungus growth by perishable substances produced by B.

licheniformis correlates with incubation temperature, i.e. higher temperature stimulates

antifungal activity [57].

Antifungal activity and fungal growth are impacted by various factors, such as the

combination of increased bacterial metabolic activity with excellent diffusion and solubility

of perishable substances.

Antibiotic designated YM-47522 was isolated from Bacillus sp. culture YL-03709B [58]. It

shows potential in vitro antifungal activity, especially to Rhodotorula acuta and Pichia

augusta.

Mersacidine is a peptide antibiotic containing β-methyllantionine. It is classified in a

group of antibiotics produced by Bacillus sp. stains. The antibiotic is active against G+

microorganism (meticiline resistant Staphylicoccus aureus) [59].

2 Material and Methods

2.1 Chemicals and Tested Substances

N-succinyl-(Ala)3-p-nitroanilid (N-Suc(Ala)3-p-NA) (SIGMA, USA)

Tris-(hydroxymethyl) aminomethane (TRIS) and CaCl2 (FLUKA, Switzerland)

Elastase EC 3.4.21.36., from pig pancreas, 80 U.mg-1 (SIGMA, USA)

Technical and bacteriological agar (OXOID, England)

Nutrient Broth No. 2, Sabouraud soil (IMUNA, SR)

Nutrient Broth (OXOID, England)

API Broth (HI MEDIA, India)

Metionine, cysteine, serine (MERCK, Germany)

Sodium Thioglycolate (ŠUKL, CR)

Glutatione (LOBA REINCHEMIE, Austria)

Mercaptoethanol (FLUKA, Switzerland)

Mud (bypass arm, maturing pool, mud kitchen IRMA, SR)

Hexane, methanol for UV spectroscopy (MIKROCHEM, SR)

Other unnamed components of used media, other chemicals and solvents (LACHEMA, CR)

All used chemicals were of p.a. purity.

2.2 Used Instruments

Microorganisms were cultivated statically in thermostat BT 120 and TCH 100

(LABORATORNÍN PŘÍSTROJE, CR).

For incubation and permanent temperature maintenance during determination of enzyme

activity inhibition effect, thermostat UV 10 was used (MECHANIK PRUFGERäTE, Germany).

pH values of prepared media and buffers were measured with pH meter OP-211/1

(RADELKIS, Hungary).

Analytical scales (METTLER TOLEDO, Switzerland) were used in pure tested substances

weighing.

Microorganisms biomass was separated from cultivation medium on K 26 centrifugal

(JANETZKY, Poland).

Absorption of solutions in the determination of enzyme activity and assessment of growth

using turbidimetric method was measured on spectrophotometer SPECTRONIC 20 D

(MILTON ROY, USA).

Tlc PLATES – Silufol UV 254 (KAVALIER, CR). TLC plates were detected in UV range 254 to 366

nm by chromatogram viewer (DESAGA, Germany).

Respiration was measured using Klark oxygen electrode, type SOPS-31 (CHEMOPROJEKT,

CR).

To prepare lyophilised bacterial cultures, lyophilisator was used (LEYBOLD HERAEUS,

Germany).

2.3 Microorganisms and Cultivation Conditions

Unknown cultures of thermophillic, sulphur utilising bacteria were isolated from spa

mud, Piešťany. Bacterial strains were designated SU-2 and SU-3. They were identified in the

Brno collection of microorganism (CCM) and classified in Bacillus species.

To test antifungal effect of metabolites, we used the following model microorganism

strains:

Candida parapsilosis (Collection of Biochemical Technology Department CHTF STU)

Cryptococcus neoformans CCY 17-1-6

Trichosporon cutaneum CCY 30-5-10

Culture of thermophillic sulphur utilising bacteria SU-2 and SU-3 were stored on solid

cultivating medium Sulphate API Broth, hereinafter only API, with the addition of agar 25 g/l,

pH 7.4 and inoculated byweekly. They were incubated at 60°C for 3 days.

Preparation of SU-2 and SU-3 inoculum: inoculation medium Nutrient Broth,

hereinafter only NB, pH 7.4 was inoculated in a 100 ml bulb from oblique agar (API) and

cultivated statically at 60°C for 4 days.

Principal cultivation: Into 150 ml of cultivation medium, NB or cultivating broth No. 2,

we inoculated 3% (v/v) of inoculums in a 500 ml bulb and cultivated statically at 60 °C.

The cultivation served the assessment of sulphur substances effect on growth,

respiratory activity and other parameters of tested bacteria.

Testing microorganisms: Candida parapsilosis, Cryptococcus neoformans and

Trichosporon cutaneum were maintained on oblique agar (Sabouraud soil), pH 6.5.

2.4 Measurement of Respiration Using Oxygen Electrode

We assessed respiratory activity of bacterial biomass SU-2 and SU-3 from 48 hour

cultivation. Following the separation of cultivation medium, it was suspended in 5 ml of

physiological solution. Oxygen consumption was determined with the help of oxygen

electrode in a container with 9.5 ml of physiological solution, to which we added 0.5 ml of

cell suspension and 10 ml of tested sulphur substance with a concentration of 5.10-3 mol. l-1.

2.5 Antifungal Activity Tests

Biologic activity of mud extracts was studied in plate diffusion method on dermatophyte

yeast models.

Yeast strains were maintained on oblique agar. From the grown culture, we used

microbiological loop to transfer inoculum into 5 ml of liquid Sabouraud soil. They were

incubated at 28°C for 2 days.

Following cooling to 45°C, we inoculated Sabouraud soil (100 ml) with the addition of

agar (20 g/l) with 100 µl of yeast cells suspension and distributed it into Petri-bowls

(diameter of 90 mm) – 20 ml each.

On the agar medium surface, we distributed paper discs (8 mm in diameter, Whatmann

No. 2) with 50 µl of tested mud metabolites extract. Following incubation (48 hours at 28°C),

we assessed biological activity based on read inhibition zones averages representing a semi-

quantitative sensitivity picture.

2.6 Enzyme Activity Determination

Elastase

To 3 ml TRIS buffer pH 8.0 (0.05 mol.l-1 TRIS with 0.05 mol.l-1 CaCl2), we added 0.02

ml of elastase with a concentration of 2.10-6 mol.l-1. The reaction mixture was incubated in

water bath at 37°C for 30 minutes. The reaction was initiated through the addition of 0.01 ml

N-suc-(Ala)3-pNA (resulting concentration in reaction mixture: 1.10-3 mol.l-1). After 25

minutes, the reaction was stopped through the addition of 0.3 ml 50% CH3COOH. In the

reaction, elastase releases p-nitroanilide from substrate. It was determined through

spectrophotometric measurement of absorbance at 405 nm.

Inhibition activity of cultivation medium and pure substances supernatants:

To define activity of respective enzymes, we added to the reaction mixture 0.03 ml of

cultivation medium supernatant, or 0.03 ml of pure substance solution.

Results and Discussion

3.1 Thermophillic Bacteria Bacillus sp. SU-2 and SU-3 Utilising Sulphur Substances

Thermophiles are characterised by dominant sulphur metabolism, since almost any

natural environment with temperatures suitable for thermophiles is also rich in sulphur and

sulphur substances. Thermophillic microorganisms grow better in anaerobe conditions as a

result of lower oxygen solubility in water solutions at increased temperature. However,

certain extreme environmental conditions are best tolerated by organisms capable of aerobe

growth. Obligatory anaerobe thermophiles were isolated from truly varied environments.

They are characterised by larger differences between kinds, compared to aerobe organisms.

Chemoorganotrophic thermophiles grow the most intensely in the presence of S0 or

polysulphides reducing to H2S in the process of sulphur respiration or disimilatory

fermentative sulphur reduction. Thermophillic microorganism reducing sulphate are

considered partner microorganisms of fermentative anaerobes generating low energy

substances in ecosystem with the ability of further independent transformation. Partner

microorganisms in obligatory symbiotic arrangement with fermentative anaerobes utilise

their products (organic acids of primary metabolism, amino acids, alcohols).

3.1.1 Impact of Added Sulphur Substances on Growth and Respiratory Activity of

Thermophiles SU-2 and SU-3

Application of some sulphur substances into cultivation medium NM enabled the

observation of these substances effect on growth and respiratory activity of sulphur utilising

bacteria SU-2 and SU-3. We used turbidimetric method to assess growth during static

cultivation at 60°C for 168 hours. Measured values of optic density at 620 nm were

compared with values obtained from control media analysis (cultivating medium with the

addition of sulphur substance without inoculum).

Respiratory activity of bacterial cultures SU-2 and SU-3 was evaluated through

the measurement of oxygen consumption by cell suspension. Respiration efficiency was

expressed as the consumption of oxygen in µmol.l-1 during bacterial oxidation of tested

exogenous substrates.

The results of experiments studying the effect of certain sulphur substances on the

growth SU-2 and SU-3 bacteria and antielastase activity were presented in the previous

research report No. 73/98. We continued our experiments and extended the range of tested

sulphur substances. Thereby, we studied their effect not only on the growth of

microorganisms, but also looked at their respiratory activity related to the growth of

microorganism and transformation of sulphur substances. Results of experiments are

presented in Fig. 3.1, Fig. 3.2, Fig. 3.3 and Fig. 3.4.

SU-2 bacteria isolated from mud grew very intensely on cultivating medium with

bensylthiocyanate, or dibensyldisulphide (Fig. 3.1, Fig. 3.3). In a limited scope, they

stimulated growth and cysteine (CYS), glutathione (GSH), sodium sulphide (SUNa), thiorea

(TMO), dimethylsulphoxide (DMSO) and sodium thioglycolate (TGNa). Other sulphur

substrates DTN and mercaptoethanol (MOH) significantly inhibited the growth of SU-2

bacteria. Correlation between the growth of culture SU-2 and its respiratory activity was

identified in seven samples of the entire palette of added sulphur compounds (Fig. 3.3).

Increased respiratory activity of SU-2 in the presence of TMO, DMSO, DTN and serine (SER)

indicates intense transformation oxidative reactions of these sulphur substances with no or

limited utilisation by the microorganism for its growth. Similar results were reached in the

cultivation of thermophillic bacteria SU-3 isolated from mud with the addition of sulphur

substances (Fig. 3.2 and Fig. 3.4). Bacteria growth was significantly stimulated in cultivating

medium with bensylthiocyanate, dibensyldisulphide, sodium thioglycolate, tiorea and

sodium sulphide. The microorganism did not grow, if cultivating medium contained external

mecaptoethanol, glutathione and DTN. Thermophillic culture respirated intensely on sodium

sulphide, dibensyldisulphide, cysteine, glutathione and sodium dithionate (DTNa). The

highest respiratory activity was recorded in the case of Na2S application as substrate. O2

consumption represented 59.98 µmo.l-1. Dimethylsulphoxide, serine, sodium thioglycolate

and mercaptoethanol participated in bacterial oxidation conversion only with limited effect

(Fig. 3.4).

Fig. 3.1 Biomass growth time relation for Bacillus sp SU-2 bacteria cultivated at 60°C in

NM cultivating medium with the addition of sulphur compounds (5.10-3 mol.l-1),

Thermophillic culture growth was assessed turbidimetrically at 620 nm

Ab

sorb

ance

A (

62

0 n

m)

cultivation time [h]

Control

DBS

BTC

DTN Na Sulphide

Control – without the addition of sulphur compound

DTN – 5.5´-dithio-bis-(2-nitrobenzoic) acid

DBS – dibensyldisulphide

BTC – bensylthiocyanate

Fig. 3.2Biomass growth time relation for Bacillus sp SU-3 bacteria cultivated at 60°C in

NM cultivating medium with the addition of sulphur compounds (5.10-3 mol.l-1),

Thermophillic culture growth was assessed turbidimetrically at 620 nm

Control – without the addition of sulphur compound

DTN – 5.5´-dithio-bis-(2-nitrobenzoic) acid

DBS – dibensyldisulphide

BTK – bensylthiocyanate

Ab

sorb

ance

A (

62

0 n

m)

cultivation time [h]

Control

DBS

BTC

DTN Na Sulphide

Fig. 3.3 Respiratory acitivity and gwoth of culture Bacillus sp SU-2 in static cultivation in liquid

NM soil nwith the addition of sulphur compounds (5.10-3 mol.l-1)

A620 - absorbance measured at 620 nm

DBS – dibensyldisulphide BTK – bensylthiocyanate

DTN – 5.5´-dithio-bis-(2-nitrobenzoic) acid MOH - mercaptoethanol

TGNa – sodium thioglycolate TMO - thiorea

SUNa – sodium sulphide MET – methionine

DTNa – sodium dithionate DMSO – dimethylsulphoxide

CYS – cysteine GSH - glutatione

SER – serine (intermediary of msulphur KON – without sulphur substance

amino acids biosynthesis)

10

0. A

620,

resp

irat

ory

act

ivit

y [μ

mo

l O2/l

]

sulphur substance

growth

respiration

Fig. 3.4 Respiratory activity and growth of culture Bacillus sp SU-3 in static cultivation in liquid

NM soil with the addition of sulphur compounds (5.10-3 mol.l-1)

A620 - absorbance measured at 620 nm

DBS – dibensyldisulphide BTK – bensylthiocyanate

DTN – 5.5´-dithio-bis-(2-nitrobenzoic) acid MOH - mercaptoethanol

TGNa – sodium thioglycolate TMO - thiorea

SUNa – sodium sulphide MET – methionine

DTNa – sodium dithionate DMSO – dimethylsulphoxide

CYS – cysteine GSH - glutatione

SER – serine (intermediary of msulphur KON – without sulphur substance

amino acids biosynthesis)

3.1.2 Products of Sulphur Substances Conversion by Thermophiles SU-2 and SU-3 with

Inhibitory Effect on Elastase Activity

Microbial low-molecule enzyme inhibitors represent a group of secondary

metabolites with interesting therapeutical effects. These bioactive substances often are part

of metabolic pathways as natural inhibitors of enzymes active in them. inhibition effect may

be born by substances produced as secondary metabolites, often isolated from cultivation

mediums filtrates following cultivation, possible, they are products of microbial conversion

of various substances. Results of inhibition effect determination in samples from cultivating

medium filtrates following cultivation in liquid cultivating medium NM with the addition of

sulphur substances of SU-2 culture are presented in Chart 3.1. Into the reaction mixture, we

10

0. A

620,

resp

irat

ory

act

ivit

y *μ

mo

l O2/l

]

sulphur substance

growth

respiration

added solutions of selected sulphur compounds in concentration 5.10-5mol.l-1. They are

listed in order with increasing inhibition effect on pancreatic elastase activity (from 24.5 to

72.2%): DBS, BTK, DTNa and MOH. Elastase activity inhibition under the effect of these

substances was also identified in cultivating medium filtrates following 168 hour

fermentation of thermophillic bacteria SU-2. Mercaptoethanol (5.10-5 mol.l-1) significantly

inhibited activity of pancreatic elastase (72.2%). In the medium filtrate without culture, the

inhibition effect of mercaptoethanol was not as significant, probably as a result of active SH-

group masking by nutrient components in the cultivating medium. Elastase activity was also

reduced by sulphur substance DTN tested as pure substance, as well as an addition to

cultivating medium, where DTN was intensely respirated by SU-2 culture. The effect of these

sulphur substances on elastase activity was not that significant in the assessment of medium

filtrates following 168 hour cultivation of SU-3 thermophille (Chart 3.2). The highest activity

was observed in medium filtrate following mercaptoethanol addition (23.8%). Sulphur

substances MOH and DTN inhibited the growth of SU-2 and SU-3 cultures. Hence, we did not

assume that elastase activity was effected by SU-2, or SU-3 cultures metabolites. DTN

inhibition effect is probably based in oxidation products of this substance conversion, which

is supported by the measured values of respiratory activity (Fig. 3.3 and Fig. 3.4).

Low-molecule thiols are found in river and sea sediments in very low concentrations

(micromolar). They are formed in the transformation of methyl sulphur compounds and

sulphur amino acids (Chap. 1.1.6). Low-molecular thiols inhibit the activity of enzymes with

the contents of aldehyde group in substrates. In the experiment, we confirmed inhibition

effect of mercaptoethanol on elastase activity. Increased elastase activity is related to

pathological conditions, such as rheumatic, skin and respiratory diseases.

Chart 3.1 Inhibition effect of sulphur substance and filtrates following Bacillus

sp. Bacteria SU-2 cultivation on elastase activity

Sulphur substancesa Elastase activity inhibition (%)b

Pure substances Cultivation medium filtrates

Control SU-2

DBS

DTN

BTK

MOH

24.8

64.5

55.2

72.2

0.0

8.5

7.4

10.4

10.2

50.4

10.0

49.4

a- Elastase inhibition in % expressed with respect to control samples (without the

addition of tested substance)

b- Sulphur substance concentration in reaction mixture (5.10-5mol.l-1)

Control – cultivation medium filtrate without culture

DBS – dibensyldisulphide

DTN – dithio-bis-(2-ntribensoic) acid

BTK – bensylthiocyanate

MOH – mercaptoethanol

Chart 3.2 Inhibition effect of sulphur substance and filtrates following Bacillus

sp. Bacteria SU-3 cultivation on elastase activity

Sulphur substancesa Elastase activity inhibition (%)b

Pure substances Cultivation medium filtrates

Control SU-3

DBS

DTN

BTK

MOH

24.8

64.5

55.2

72.2

0.0

8.8

7.4

10.4

0.0

2.8

17.8

23.8

c- Elastase inhibition in % expressed with respect to control samples (without the

addition of tested substance)

d- Sulphur substance concentration in reaction mixture (5.10-5mol.l-1)

Control – cultivation medium filtrate without culture

DBS – dibensyldisulphide

DTN – dithio-bis-(2-ntribensoic) acid

BTK – bensylthiocyanate

MOH – mercaptoethanol

3.1.3 Amino Acids as Substrates and Products of Thermophillic Bacteria SU-3 Cultivation

on Medium Enriched by Sodium Sulphide

Knowledge on amino acids utilisation and biosynthesis by the isolates of bacteria

metabolising sulphur inorganic and organic substances may draw a picture on the pool of

free amino acids in the environment of these bacteria. Microorganisms use various amino

acids as building blocks in the process of biologically active substances biosynthesis, such

antibiotics, enzyme inhibitors, immunomodulation substances. Sulphur containing amino

acids are degraded by certain bacteria under the formation of methanthiol and other

alcylthiols, substances found in river and sea sediments.

At the end of cultivation in cultivation environment Bacillus sp. SU-3, we identified a

reduction in sulphur amino acid metionine by 15%. The remaining sulphur amino acids were

not detected in the cultivating medium. The culture activity accumulated certain amino acids

in the cultivating medium (Chart 3.3). The volume of glutamic acid increased by 319.6 %,

arginine by 217.2%, lysine by 120.5%, glycine by 117.6%. The exhaustion of alanine and

aspartic acid from the medium and increased formation of glutamic acid, arginine and lysine

suggest that carbon assimilation takes place through cyclic reductive pathway of carboxylic

acids in this organism.

Chart 3.3 Utilisation and biosynthesis of amino acids in static cultivation of bacteria SU-

3 at 60°C.

Amino acid Amino acid concentration in

medium (mg/l)

Utilisation

/%)

Biosynthesis

(%)

0 h 168 h

Glutamic acid

Glycine

Arginine

Leucine

Lysine

Tyrosine

Valine

Fenylalanine

Histidine

Isoleucine

Metionine

Aspartic acid

Alanine

42.29

78.14

44.30

71.36

58.15

106.28

62.16

95.11

86.05

41.78

43.10

33.20

66.88

185.82

169.90

140.50

128.90

128.26

127.77

121.92

116.56

72.89

50.74

36.46

0.00

0.00

--

--

--

--

--

--

--

--

15.3

--

15.4

100.0

100.0

319.6

117.6

217.2

80.6

120.5

20.2

96.1

22.6

--

21.5

--

--

--

Static cultivation in NM soil enriched by sulphide. The contents of amino acids was

determined on automatic amino acids analyser using Spockman method [51].

3.2 Characteristics of Mud from the Bypass Arm Location

Mineral springs rich in sulphur are neutral or slightly alkaline with pH values 7-9.

Sulphate reducing bacteria show maximum growth at pH 6-8. Some isolates may grow in the

conditions of slightly acidic environment, such as in cave and surface waters with pH values

ranging from 3 to 4. Such environment was found for sulphur reducing bacteria in

sediments. Both processes, sulphates and metals reductions utilise protons, which results in

increased alkalinity in the organism environment. With respect to environmental pH, it was

also identified that toxic metals solubility is lower in neutral environment, compared to

acidic pH. Activity of sulphate reducing bacteria is significant in increased pH values of

environment characterised by low concentrations of carbon source.

Certain extremde conditions are best tolerated by organisms capable of aerobe growth.

Obligatory anaerobe thermophills were isolated from very varied environments and are

characterised by increased diversity of kinds, compared to aerobe microorganisms.

Thermophillic organisms prefer anaerobe growth as a result of reduced oxygen solubility in

water solutions at lower temperature. Prevalence of sulphur metabolism in thermophiles is

not a surprise, since almost any natural environment with temperatures suitable for

thermophiles is rich in sulphur.

3.2.1 Changes in Mud pH Values Under the Impact of Temperature

Mud from the new bypass arm location was collected simultaneously with the

remaining comparison samples from maturing pool and mud kitchen (Irma). On the

collection day, all mud samples were divided into three beakers, 400 ml each. Mud was

maintained at three different temperatures – 4°C, 30 °C, or 60°C for a period of 28 days.

pH values of mud maintained at 4°C, 30°C, or 60°C were measured in 7-day intervals.

30g of mud was taken from each beaker, diluted by distilled water (15 ml) to a consistency

suitable for pH measurement. Following thorough mixing and temperature stabilisation at

the laboratory temperature (25°C), we measured pH values of prepared mud samples. The

results of measurements are presented in Fig. 3.5, Fig. 3.6 and Fig. 3.7. following four

experiment weeks, we are able to conclude that pH values of mud from bypass arm,

maturing pool and mud kitchen slightly increased through maintenance at three different

temperatures. The biggest pH change was observed in the mud sample from maturing pool

maintained at 60°C. This leads to the conclusion that the process of system adaptation to

environmental conditions change was the most dynamic under these conditions. In all three

mud samples maintained at 601C, pH kept increasing in the first 21 days. The samples

maintained at 4°C and 30°C did not show any significant pH changes following two weeks.

Figure 3.5: pH change of mud from maturing pool with temperature maintained at 4°C, 30°C

or 60°C for a period of 28 days

0 – Day of mud samples collection from the maturing pool

Time [day]

Figure 3.6: pH change of mud from bypass arm with temperature maintained at 4°C, 30°C or

60°C for a period of 28 days

0 – Day of mud samples collection from the bypass arm

Time [day]

Figure 3.7: pH change of mud from mud kitchen with temperature maintained at 4°C, 30°C

or 60°C for a period of 28 days

0 – Day of mud samples collection from the mud kitchen

3.2.2 Biological Activity of Mud from a New Location of Bypass Arm and

Comparatory Samples from the Maturing Pool and Mud Kitchen

Antifungal activity of substances isolated by hexane extraction from mud were

assessed in plate diffusion method with testing model yeast strains (Crytococcus

neoformans, Candida parapsilosis and Trichosporon cutaneum). Hexane extracts from the

bypass arm mud contained substances inhibiting growth of yeasty microorganisms (Chart

3.4). Mud from bypass arm and mud kitchen maintained at 60°C for 28 days contained more

efficient antifungal substances than identical mud samples maintained at lower

temperatures. In the samples from maturing pool, we recorded antifungal activity of

substances also at 4°C.

Partial results of biological activity assessment of mud from new bypass arm location

suggest that the quality of this mud is comparable with mud from the maturing pool and

mud kitchen and mud assessed under the project in the previous years. More detailed

conclusions on the quality of this mud may be drawn following more complex studies, such

as the mapping of sediment microbial community, production of microorganism metabolites

with inhibition effect on patophysiological enzymes activity, utilisation and transformation of

sulphur substances by microorganism isolates, etc.

Chart 3.4 Effect of mud extract substances on the growth of yeasty dermatophytes

Mud Temperature

°C

Antifungal activity – inhibition zone (mm)

CN CP TC

Maturing pool

Bypass arm

Mud kitchen

4

30

60

4

30

60

4

30

60

20

0

16

+

+

15

+

0

18

+

+

+

+

0

16

0

0

12

20

0

14

+

0

17

+

0

+

CN – Cryptococcus neoformans, CP – Candida parapsilosis, TC – Trichosporon cutaneum

Mud samples maintained at 4°C, 30°, or 60°C for a period of 28 days and extracted into

hexane.

4 Summary

In relationship to previous studies on Piešťany mud and thermal water aimed at the

uncovering of therapeutic effect principles, we continued in research activities. This project

also included exploration of facts to amend information on thermal ecosystem microbial

community, on sulphur and other substances found in river and sea sediments. We

considered it important to also collect information on the metabolism of thermophillic and

sulphur bacteria, crucial microorganisms participating in mud formation and maturing.

Previously, in the presence of sulphur substances in mud extracts and cultivating

medium filtrates of thermophillic bacteria, substances with inhibition effect on elastase

activity were reported. Therefore, we continued in experiments.

In the first part of the experimental work, we dealt with thermophillic bacteria Bacillus

sp. SU-2 and Bacillus sp. SU-3. Previously, we isolated both cultures from Piešťany mud.

Thereby, we verified and confirmed the effect of some sulphur compounds on the growth of

SU-2 and SU-3 bacteria. In this research project, we extended the palette of tested sulphur

substances (mercaptoethanol, dithio-bis-(2-nitrobenzoic) acid, bysylthiocyanate and

dimethylsulphide), as well as the parameters characterising activity and metabolic processes

of microorganisms (respiratory activity of bacteria Bacillus sp. SU-2 and Bacillus sp. SU-3,

utilisation and production of amino acids by thermophillic bacteria growing in the

environment of a sulphur substance).

In the second part of our experimental work, we assessed the characteristics of Piešťany

mud from a new bypass arm location.

We identified that sulphur substances dibensyldisulphide and bensylthiocyanate

significantly stimulated the growth of thermophillic bacteria Bacillus sp. SU-2 and Bacillus sp.

SU-3 (by 120 to 230%). Sulphur substances tested in the previous project (sodium sulphide,

thiorea, sodium thioglycolate) stimulated the growth of these bacteria by maximum 66%.

Correlation between the growth of bacteria SU-2 and SU-3 and their respiratory activity

was recorded in several samples of the entire palette of added sulphur substances (SU-2:

glytatione, cysteine, sodium sulphide, thiorea, sodium thioglycolate, bensylthiocyanate, SU-

3: sodium sulphide, thiorea and dibensyldisulphide). Increased respiratory activity of SU-2 in

the presence of thiorea, dimethylsulphoxide, dithio-bis-(2-nitrobensoic) acid, serine and of

SU-3 in the presence of glutathione, sodium dithionate and dithio-bis-(2-nitrobensoic) acid

indicates intense transformation oxidative reactions of these sulphur substances.

The identification of mercaptoethanol effect on elastase activity in in vitro experimenets

is remarkable. Mercaptoethanol is found in thermal ecosystem sediments in micromolar

concentrations. In the form of pure substance (without the effect of culture),

mercaptoethanol inhibited elastase activity by 72.2%. As a result of metabolic processes in

Bacillus sp. SU-3, its inhibition effect was reduced to 23.8% and 49.4% in SU-2. Similar effect

was observed in dithio-bis-(2-nitrobensoic) acid, benylthiocyanate and dimethyldisulphide

on elastase activity.

Building on the knowledge that the source of methanthiol, mercaptoethanol and other

alcylthiols are sulphur amino acids degraded by certain bacteria, we defined amino acids as

substrates and products of Bacillus sp. SU-3. Following cultivation, we identified reduction of

sulphur amino acids in cultivating medium and increase of glutamic acid, arginine , lysine and

glycine concentrations. Amino acids represent building blocks in biosynthesis of biologically

active substances (antibiotics, enzyme inhibitors, immunomodulators) produced by various

microorganisms.

We assessed certain characteristics of mud from new bypass arm location. By

maintaining mud at various temperatures (4°C, 30°C and 60°C), pH values increased slightly

(from 6.1 to 7.5). The biggest pH changes were recorded at 60°C, when the probably most

intense biological and chemical processes were taking place also in the samples from the

maturing pool and mud kitchen. In terms of its biological quality, qualitative values of new

mud were assessed only through the testing of inhibition effects on dermatophytes growth.

Hexane extracts of mud from the bypass arm location contained substances inhibiting the

growth of Cryptococcus neoformans, Candida parapsilosis and Trichosporon cutaneum.

Similar effects were also recorded by substances in comparison mud samples from the

maturing pool and mud kitchen IRMA.

In the assessment of Piešťany mud quality, one of the assessment criteria could also be

the identification of mud respiratory activity, or of cultures isolated from it. Results of these

studies may be applied in mud preparation technological process and contribute to the

extension of knowledge on its therapeutical effect.

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