Selection and characterization of Clostridium bifermentans strains from natural environment capable of producing 1 , 3-propanediol under microaerophilic conditions

In this work, we aimed to select from natural environment non-pathogenic strains of Clostridium spp. capable of producing 1,3-propanediol (1,3-PD). As a result we isolated 2256 of Clostridium spp., including 10 strains from the genus Clostridium bifermentans. It occurred that all isolates of this species were able to synthesis 1,3-PD on the level of ca. 10 g/L. Additionally, they synthetized metabolites in microaerophilic conditions, which is very profitable from the industrial point of view. We characterized morphological and physiological properties of all isolated strains of C. bifermentans. These tests demonstrated significant dissimilarity among all isolates of C. bifermentans species.


INTRODUCTION
Conversion of renewable biomass to solvents, acids, and fuels is a good alternative to chemically synthesized products.Nowadays, there are numerous possibilities for replacing chemical techniques with biotechnological methods based on renewable resources (Willke and Vorlop, 2004).For this aim, microbial strains from the ATTC or other collections are often used.Indigenous bacteria in the natural environment, however, can produce a wide range of metabolites more efficiently (Lopez et al., 2004;Leja et al., 2011).An important group of industrially useful microorganisms is Clostridium spp.These species are able to metabolize an extremely wide range of organic molecules, among others carbohydrates, organic acids, alcohols, aromatic compounds, amino acids, amines, purines, and pyrimidine.They can be used in the production of solvents (butane, acetone, and ethanol) and acids (succinic, acetic, and butyric) (Ishii et al., 2000;Mitchell, 2001).Some Clostridium spp.such as Clostridium butyricum (Colin and Bories, 2000), Clostridium pasteurianum (Biebl et al., 1992;Dabrock et al., 1992), Clostridium diolis, Clostridium acetobutylicum, Clostridium butylicum, Clostridium perfingens, (Youngleson et al., 1998;Hao et al., 2008) are also able to biosynthesis 1,3-propanediol (1,3-PD).However, in the literature there is no information about the production of 1,3-PD by Clostridium bifermentans.In addition, there is no information that 1,3-PD might also be produced from glycerol by Clostridium strains in microaerophilic conditions.Kawasaki et al. (1998) stated that C. butyricum possesses the ability to consume oxygen and the ability to grow when oxygen is present but there is no information whatsoever as to the metabolite production in oxygen conditions.
The 1,3-PD, a typical product of glycerol fermentation, is one of the most interesting raw materials for chemical industries as it demonstrates a wide range of use in different fields, for example, it is a valuable chemical intermediate applied in organic synthesis.It is also used as a monomer for the production of biodegradable polymers (polyesters, polyether, polyurethanes, etc.), cosmetics, lubricants, medicines, and as an intermediate for the synthesis of heterocyclic compounds (Regan and Crawford, 1994;Menzel et al., 1997;Katrlík et al., 2007;Biebl et al., 1999;Leja et al., 2011).Recently, 1,3-PD has also been used as a monomer to synthesize a new type of polyester -polytrimethylene terephthalate (Biebl et al., 1999;Zeng and Biebl, 2002;Liu et al., 2007;Zhang et al., 2007).
In our work, we selected from the natural environment aerotolerant C. bifermentans strains that is able to produce 1,3-PD.C. bifermentans which was first isolated by Tissier andMartelly in 1902 (Brooks andEpps, 1958).The original name of this strain was Bacillus bifermentans sporogenes (Tissier and Martelly, 1902) and later it was re-named as B. bifermentans (Weinberg and Seguin, 1918), in accordance with the principle of binominal nomenclature.C. bifermentans is described as an anaerobe, catalase-negative, Gram-positive, endospore forming and motile strain (Regan and Crawford, 1994).However, according to Brooks and Epps (1958) C. bifermentans is non-motile, and only some young cultures are motile.These incoherent data probably resulted from a huge bio-diversity of the genus of Clostridium.Strains which were isolated and characterized during the investigation described above are a good evidence of this hypothesis.

Microorganisms, growth media, and cultivation conditions
Three thousand eight hundred and twenty-seven (3827) samples from excrements of animals and composts, and silages, samples from biogas works, soils, active sludge, rivers' sludge and wastes from food industry were collected in the region of the Wielkopolska District, Poland, during the period of January -July 2011.These samples were collected in sterile plastic jars and stored in refrigerator until experimentations (no longer than one month).
Samples were pre-cultured, in 10 mL test tubes, on modified PY medium (peptone-yeast medium) according to Biebl and Spöer (2002).This medium consisted of (g/L): BactoPeptone 10; yeast extract 10; glycerol 50; CaCl2, MgSO4 × 7H2O 0.96; K2HPO4 2; NaHCO3 20, and NaCl 4. The pre-cultivation step was conducted in anaerostats in two variants -under microaerophilic condition (95% CO2 and 5% O2) at 30°C for 7 days, according to the method described in Myszka et al. (2012), and in anaerobic conditions with CO2 flux only.The microanaerobic conditions in flask were maintained by CampyGen Kit (Oxoid, UK), while anaerobic by Gas Generating Kit (Oxoid, UK).After incubation, the samples were pasteurized (80°C, 10 min.),diluted with sterile solution of 0.85% sodium chloride and then spread onto TSC agar plates (Tryptose Sulfite Cycloserine Agar Base) (BD, USA).The plates were incubated for 24 h at temperature of 30°C.Isolated colonies (5 colonies per 1 plate) were screened on the basis of their morphological character (black colonies or black ones with a 2-4 mm opaque white zone surrounding the colonies as a result of lecithinase activity).To make pure culture and maintain culture conditions for the bacteria, screened colonies were transferred on both TSC agar plates (BD, USA) and RCM broth (Reinforced Clostridial Medium) (BD, USA).Isolated bacterial strains were allowed to grow in modified PY medium for 7 days at 30°C in 2 mL eppendorf tubes.After incubation, the broths were centrifuged (3000 rpm, 10 min).The cell free supernatants were collected and used for estimation of 1,3-PD production.For this purpose high performance liquid chromatography (HPLC) analyses were done.Hewlett Packard system consisted of auto sampler and pump, and a refractive index detector was carried out.The analyses were performed isocratically at a flow rate 0.6 ml/min at 65°C, on column Aminex HPX-87H 300x7.8(BIO-RAD).0.5 mM H2SO4 was used as a mobile phase.Standards were applied to identify peaks in chromatograms, and peak areas were used to determine samples concentration.This procedure was conducted by computer integration (ChemStation, Agilent) operated in the mode of external standards.The chromatography analyses were done duplicate for each sample.In order to identify 10 selected isolated strains with the highest level of 1,3-PD synthesis, the sequencing and phylogenetic analyses were done.Next, all strains were characterized by physiological and biochemical methods.

Sequencing and phylogenetic analysis
Total DNA from bacteria was extracted with the use of Genomic Mini AX Bacteria Kit (A&A Biotechnology, Gdańsk, Poland) after an initial incubation in 50 mg/mL lysozyme (Sigma) for 1 h at 37ºC.Sequences encoding small subunit of rRNA were amplified in PCR reaction using primers SDBact0008aS20 and SUniv1492bA21 (41).PCR products were purified using Clean-up Kit (A&A Biotechnology, Gdańsk, Poland) and sequenced at Genomed Co. Warsaw, Poland with primers used for PCR, and additionally for inner sequence with GTGCCAGCMGCCGCCCTAA primer.PCR was performed in the total volume of 100 L containing 1xPCR buffer (10 mM KCl, 10 mM (NH4)2SO4, 20 mM Tris-HCl (pH 8.5), 2 mM MgSO4, 0,1% Triton X-100), 25 ng DNA template, 0.44 M concentration of each primer, 200 M dNTP, 2,5U Run DNA Polymerase (A&A Biotechnology).PCR was carried out in the Biometra T Gradient thermocycler.The amplification of 16S rDNA consisted of 15 cycles: 1 min denaturation step at 94ºC, 1 min annealing step at 48ºC, and 2 min extension at 72ºC.The size of the amplified fragments was equal 1 500 base pairs.The sequences obtained were arranged into contigs and identified in BLAST service of the GenBank database (Altschul et al., 1990;Suau et al., 1999).

Morphological tests
All the strains were grown on agar plates containing TSC medium with 1.5% of agar (BD, US).To describe the morphology of the colonies, inoculated plates were incubated for 24 h.

Motility test
The motility test medium was used to demonstrate whether or not the cells can swim in a semisolid medium (PY with 1% of agar).A semisolid medium was inoculated with the bacteria in a straight-line stab with a needle.After incubation bacterial growth can be observed away from the line of the stab, which is evidence of the fact the bacteria were able to swim through the medium.

Urease production test
To check the urease production ability, the cultures were grown for 24 and 48 h at 37°C in 20 ml amounts of PY medium.A few strains from each group were also grown in 100 ml amounts of a similar medium and tested daily for 4 days.The following reagents were used: KH2PO4 (0.1 g), K2HPO4 (0.1 g), NaCl (0.5 g), urea (2.0 g)., 95% (v/v) ethanol in water (1.0 ml), distilled water (100 ml), Universal Indicator (5.0 ml), sufficient 0.1N -HCl give an orange colour (about pH 6.0).A 2.5 ml sample of the culture under testing was transferred to a test tube (80 mm × 8.0 mm) and centrifuged.The supernatant fluid was discarded and the sediment washed once in 1.0 (v/w) saline and finally resuspended in 1.0 ml distilled water.One milliliter of urease reagent was added and, after thorough mixing, the test was incubated at 37°C (8).

Indole production test
Twenty four hour cultures in peptone broth were examined for indole production by means of Ehrlich's reagent and by the vanillin test (Sprayr, 1936).

Sugar assimilation and fermentation tests
In sugar assimilation tests, glycerol was replaced in PY medium by such saccharides as glucose, fructose, arabinose, maltose, mannitol, glycerol, sorbitol, rhamnose, and xylose.Additionally, phenol red was used (1 mg/ml) as an indicator.The pH of the medium was adjusted to 8.6.The medium was dispensed into 10 mL tubes, sterilized by autoclaving, and then bacteria were introduced to the medium (the size of bacterial inoculum was equal 10% v/v).The pure culture of the isolates was incubated at 36°C for 24 h in 10mL test tubes, while the result was indicated by a change of color from red to yellow (Shrestha and Sharma, 1995).Control tubes were used in each set to monitor contamination.
In sugar fermentation tests, glucose, fructose, arabinose, maltose, mannose, mannitol, glycerol, sorbitol, rhamnose, and xylose were added to the PY medium.To analyze their fermentation, the technique of high liquid chromatography was applied.The analysis were performed isocratically at a flow rate 0,6 ml/min.at 65°C, on column Aminex HPX-87H 300x7.8(BIO-RAD).0.5 mN H2SO4 as a mobile phase was used.Standards were applied to identify peaks in chromatograms, and peak areas were used to determine samples concentration.It was conducted by computer integration (Chem-Station, Agilent) operated in the mode of external standards.As a control probe, the PY medium with glycerol was used.All experiments were done in duplicate.

Antibiotic sensitivity test
To characterize antibiotic sensitivities of the clostridial isolates they were tested against batteries of both traditional and non-traditional anti-clostridial antibiotics, using a rotary test method.Bacteria strains were spread onto plate dishes and the TSC medium was used.Then, the rotaries with different antibiotics were put onto plate dishes.After 24 h, in case of the strains which were sensitive to the antibiotic, a bright zone around the rotary was observed.All tests were done in duplicate.

The oxidoreduction potential of isolated strains
In this experiment the flow cytometry technique was applied.The redox potential in bacteria cells cultivated without oxygen was compared with the potential in bacteria cells cultivated with a small amount of oxygen (5%).The high value of the redox potential means that the cells were alive and the metabolic activity of these cells was high..
The samples analyzed comprised growth cultures of 10 C. bifermentans strains.Bacteria were cultivating for 24 h in microaerophilic (5% O2 and 95% CO2) and in strictly anaerobic conditions.Flow cytometric analysis of microbial cells' vitality and metabolic activity with redox potential as a relevant parameter were evaluated using BacLight Redox Sensor Green Vitality Kit from Invitrogen Company.For analysis, 1 ml of each culture was collected by centrifugation, resuspended in 1% PBS and prepared according to manufacturer's manual.Prior to the analysis, an optimization step was involved in order to assess the reagents appropriate staining concentrations.Sample analysis was performed using BD FACS Aria™III (Becton Dickinson) flow cytometer (cell sorter), equipped with four lasers (375, 405, 488 and 633 nm), 11 fluorescence detectors, forward scatter (FSC) and side scatter (SSC) detectors.The primary sample line was fitted in initial 50 μm-pore-size filter, preventing flow arrest in case of samples including particles capable to block the light of sample line or a nozzle.The instrument setup (optical alignment), stability and performance tests were made using CST system (Cytometer Setup and Tracking) from Becton Dickinson company.FACSFlow solution (Becton Dickinson) was used as sheath fluid.The configuration of the flow cytometer was as follows: 70 μm nozzle and 70 psi sheath fluid pressure.The cells were characterized by two non-fluorescent parameters: forward scatter (FSC) and side scatter (SSC), and two fluorescent parameters: green fluorescence (FL1) from RedoxSensor™ Green reagent collected using 530/30 band pass filter and red fluorescence (FL2) from propidium iodide (PI) reagent collected using 616/23 band pass filter.For excitation of both fluorescent reagents, 488 nm laser was employed.The flow cytometry analyses were performed by using logarithmic gains and specific detectors settings.The threshold was set on the FSC signal.Data were acquired in a four-decade logarithmic scale as area signals (FSC-A, SSC-A, FL1-A and FL2-A) and analyzed with FACS DIVA software (Becton Dickinson).The analysis of fluorescence signals from both fluorochromes preceded doublets discrimination procedure with the use of height versus width scatter signals measurement, in order to discriminate single events from conglomerates.The populations were then defined by gating in the dot plots of green fluorescence (FL1) versus red fluorescence (FL2).Each sample was analyzed in triplicate.The estimation of

Isolation of Clostridium strains and phylogenetic identification
In our study, PY medium proposed by Biebl and Spöer (2002) was tested for its ability to isolate bacteria of the Clostridium genus from natural samples.The aim of this work was to isolate bacteria strains that is able to produce 1,3-PD.Accordingly, Clostridium spp was isolated from all tested natural samples (Figure 1).The highest number of isolates of the genus of Clostridium was obtained from excrements of animals (1,022 isolates) and composts or silages (1,799 isolates).In addition, in our study all bacterial strains were tested for their ability to grow with glycerol in the medium and to converse this substrate to 1,3-PD.Of the total number of 3,827 isolates tested nearly 56% (2,256 isolates) fermented glycerol to 1,3-PD.Excrements of animals as well as compost and silages contained the highest numbers of isolates able to produce 1,3-PD (Figure 1).The 12 strains obtained from soil and composts, which synthetized most 1,3-PD, were identified by sequencing as C. bifermentans and C. butyricum.Interestingly, the glycerol fermentation by C. bifermentans species has not been investigated yet.The results of C. bifermentans identification are presented in Table 1.

The ability of production of 1,3-PD by isolated strains
In our experiments we investigated the ability to produce 1,3-PD, in both anaerobic and microaerophilic conditions, by 10 strains identified previously as a C. bifermentans.The yield of 1,3-PD production in anaerobic and microaerophilic condition (95% C0 2 and 5% O 2 ) via glycerol fermentation by examined strains of C. bifermentans is presented in Figure 2. The metabolite profile was the same independent of cultivation conditions.The range of 1,3-PD production was between 9.78 and 14.89 [g/L] (Y p/s =0.20-0.30;P a =0.06-0.09).During these fermentations other metabolites were also obtained.The amount of acids synthetized from glycerol fermentation by selected microflora is presented in Table 2.The metabolic pathway in all 10 strains was similar but not the same (efor example, strains C. bifermentans 371, 540, 541 were not able to synthetize ethanol, C. bifermentans 549 did not produce succinic acid, and C. bifermentans 546 was not able to synthesis formic acid).As a reference culture C. bifermentans (ATCC 638T) obtained from the American Type Culture Collection was used.The reference cultures were also grown on the PY medium in the same microaerophilic and anaerobic conditions.The

Oxidoreductive potential
During our work tolerance of investigated strains on oxygen was observed.It is noteworthy that in the literature there is information that C. bifermentans is an obligate anaerobe (Regan and Crawford, 1994).Thus, we decided to analyze the redox potential using the flow cytometry method.The aim of this experiment was to compare the metabolic activity of bacteria cultivated in the presence of small amount of oxygen and without it.
In order to assess the metabolic activity of bacterial strains analyzed, each sample was divided into three tubes, corresponding to unstained control, negative control and positive.Negative control comprised tubes with a CCCP reagent added, which acts as an electron transport chain uncoupler.After incubation, RedoxSensor™ Green reagent and propidium iodide were added to negative control and positive tubes.Thus the negative control represents a reference sample for normalization of green fluorescence signals from cells with stimulated reduction of redox potential (CCCP treated) and cells non-treated with an electron chain uncoupler, allowing us to estimate the metabolic activity of the cells.The difference in medians of green fluorescence signals corres-ponds to redox potential of the cells analyzed.This permits a strain comparison for selection of the ones with highest redox potential relevant to highest metabolic activity.This method turned out a valuable tool for assessment of metabolic activity of cells in anaerobic and microaerophilic conditions, enabling us to select strains with desired activity in microaerophilic and anaerobic processes or make combinations of both features.The values for the redox potential of investigated 10 C. bifermentans strains are presented in Figure 3.All strains demonstrated high redox potentials -both in anaerobic and microaerophilic conditions.It means that the cells are able to survive in the presence of oxygen.Additionally, in majority of the investigated strains the redox potential is similar for both types of cultivating, the presence of oxygen does not decrease the metabolic activity of cells.From the industrial point of view, it is an important property of C. bifermentans because work with an obligate anaerobe is difficult on a large scale, while this feature limits the possibilities of application of strictly anaerobic strains.

Morphological and physiological properties of isolated strains
All experiments were done in microanaerobic conditions.All of 10 obtained C. bifermentans strains cultivated on TSC medium formed opaque, black, circular, low convex colonies with entirely to slightly undulated margins.Spores are oval, central to subterminal.The biochemical features such as the motility of these strains, urease and indole production, as well as results of sugar assimilation and fermentation tests are presented in Table 3.As we can see, these strains have different features.Some of them are able to motile (C.bifermentans 371, 376, 546, and 549), while others are not.All investigated isolates were not able to produce indole and they were ureasenegative.One strain, C. bifermentans 540, showed a number of different features in comparison with others, such as inability to assimilate arabinose, mannose, and mannitol while others are able to assimilate these sugars.In our work we also checked the ability of sugar fermentation in all 10 strains (glucose, fructose, maltose, sorbitol, arabinose, mannose, mannitol, raffinose, and xylose).Table 3 shows also the results from these experiments.However, when another carbon source, different than glycerol, was used, there was no 1,3-PD production.

Antibiotic sensitivity test
Antibiotic sensitivity testing aims to determine the susceptibility of an isolate to a range of potential therapeu-  4. All 10 strains are susceptible to erythromycin, penicillin, ampicillin, tetracycline, and chloramphenicol.Our results show that all 10 strains are resistant to streptomycin, three (Clostridium bifermentans 537, 546, and 549) are resistant to gentamycin, and only three (Clostridium bifermentans 540, 541, and 543) are resistant to ortho-tetracycline.
In our work we examined the natural environment in search for strains which are able to produce 1,3-PD.On the whole the natural environment is a good source of industrially useful strains.Additionally, there is a huge probability that even anaerobic strains isolated from this source have a natural tolerance to small amounts of oxygen.Thus we selected 10 strains of C. bifermentans from silage and forest soil.In the literature there are only a few papers describing the isolation of C. bifermentans from the natural environment, namely from California desert tortoise (Dezfulian et al., 1994;Chamkha et al., 2001) but there is no data about the ability of C. bifermentans to produce 1,3-PD.C. bifermentans are described as strictly anaerobic species (Lewis et al., 1996;Chang et al., 2000;Zhao et al., 2003).During our experiments it occurred that these strains are able to grow and maintain their metabolic activity in the presence of a small amount of oxygen (5%).From the industrially useful point of view, such a feature is very profitable because maintenance of strictly anaerobic conditions on a large scale is difficult and expensive.The metabolism and physiology of C. bifermentans species is not quite known (for example, there is no unequivocal information about the glycerol pathway in C. bifermentans cells).Thus, we decided to investigate this phenomenon.During a glycerol fermentation test we found out that our isolates produced more than 9 g/L of 1,3-PD without any optimization processes.Similar results are presented in the preliminary studies by Mu et al. (2006) on K. pneumoniae DSM 2026 isolated from a garden pond (9.4 g/L of 1,3-PD).In the glycerol fermentation by C. bifermentans 1,3-PD is a main metabolite, it is produced in reductive branch of glycerol metabolism pathway.Nevertheless, our isolates are also able to form other products, such as organic acids (acetic, lactic, formic, and succinic acid) and alcohol (ethanol) (Leja et al., 2011).These by-product products are synthetized in the oxidative branch of glycerol metabolic pathway (Zeng and Biebl, 2002).The Clostridium strains growing on glycerol typically produce a variety of metabolic end-products, such as n-butanol, 1,3-PD, ethanol, acetic acid, butyric acid, and succinic acid (Biebl et al., 1999;Biebl, 2001;Biebl et al., 2002;Liu et al., 2007;Zhang et al., 2007;Kubiak et al., 2012).All these by-products are associated with a loss in 1,3-PD relative to acetic acid, in particular ethanol and butanol, which do not contribute to the NADH 2 pool at all (Zeng and Biebl, 2002).It is an example of a redox-balanced process in this pathway.Although the pathways for succinate and ethanol are equivalent regarding the overall redox balance, the energetic contribution of the ethanologenic pathway is much higher, as 1 ATP is produced per each molecule of glycerol converted into ethanol, while the production of energy in the succinate pathway is limited to the potential generation of a proton motive force by fumarate reductase (da Silva et al., 2009).An important by-product in this fermentation is acetic acid.The yield of 1,3-PD depends on the combination and stoichiometry of the reductive and oxidative pathways.It is proved that the combination of 1,3-PD generation with acetic acid as the sole by-product of the oxidative pathway results in the maximum yield of 1,3-PD (Shrestha and Sharma, 1995).Thus, the acetic acid is necessary for 1,3-PD production.The results of our experiments confirmed this fact: all 10 investigated strains are able to produce 1,3-PD and also synthesiz acetic acid.Concluding: the production of metabolites is necessary for bacteria strains.Especially important is the conversion of glycerol to 1,3-PD because it provides energy for cell growth (Biebl et al., 1999).The production of organic acids by microorganisms plays a key role in the process of controlling of appearance of other genus of bacteria in particular environments (Prevot and Malgras, 1950).Siragusa and Dickson (1992) stated that a small amount of short-chain organic acids resulted in the reduced growth of coexisting non sporogenes microflora.
Moreover, physiological properties of C. bifermentans are not well investigated.In the literature there is incoherent data about such properties of C. bifermentans as the motility of these species, the ability of indole and urease production, the fermentation of saccharides and resistance to antibiotics.Regan and Crawford (1994) found out that C. bifermentans strains are able to motile.The same kind of data is presented in the work of Prevot and Malgras (1950).However, Brooks and Epps (1958) inform us that some strains of C. bifermentans are motile while some are non-motile.The results of our work thus confirm the observations of Brooks and Epps (1858).Among our ten strains, four were motile and six were not.Regan and Crawford (1994) and Nachman et al. (1989) described C. bifermentans strains as indole positive.Brooks and Epps' (1958) results remain doubtful.They described this result as weakly positive.All our isolates were indole-positive and urease-negative, which is a finding corresponding to the data presented in papers by Brooks et al. (1969) and Brooks and Epps (1958).Brooks and Epps (1958) also investigated the ability of C. bifermentans to saccharide fermentation.They stated that their strains fermented glucose, fructose, maltose, glycerol, and sorbitol.All our strains were able to carry out these saccharide fermentations.The strains isolated by Chamkha et al. (2003) from oil mill wastewaters were also able to ferment glucose, fructose, mannose, maltose, sorbitol, and additionally myo-inositol and ribose.Nachman et al. (1989) and Regan and Crawford (1994) investigated also the sensitivity of C. bifermentans strains to antibiotics.The strains investigated by Nachman et al. (1989) were susceptible to erythromycin, penicillin, ampicillin, and tetracycline.The strains described in the work of Regan and Crawford (1994) were similarly susceptible to penicillin, ampicillin, and additionally to chloramphenicol.On the other hand, these strains were resistant to tetracycline.Other antibiotics have not been tested yet.Similar results were observed in our experiments: all ten isolates were susceptible to erythromycin, penicillin, ampicillin, chloramphenicol and also tetracycline.Probably, the incoherence in properties of C. bifermentans in different research results is a symptom of a great variety of isolated C. bifermentans strains.It may also be connected with different sources of their isolation.

Conclusions
The natural environment is a good source of industrially useful strains, such as C. bifermentans, which in our work was isolated from forest soil and sewage.C. bifermentans so far are not well known bacteria, and the predominant part of work on these species was carried out in the early 1950'.The results obtained by different scientists are not congruent.Thus, when it turned out that C. bifermentans is capable of 1,3-PD production (a feature which has not been described by any other scientists before), we decided to investigate physiological and biochemical properties of all the obtained isolates.Additionally, during this work it was found out that these bacteria are able to survive in the presence of oxygen, maintaining their ability to metabolites production (including 1,3-PD).Now, because of other interesting properties of these strains, such as effective production of lactic acid from mannitol, we are continuing our research on C. bifermentans.
PO IG 01.01.02-00-074/09, co-funded by The European Union from The European Regional Development Fund within the framework of the Innovative Economy Operational Programme 2007-2013.

Figure 3 .
Figure 3.The redox potential of C. bifermentans strains cultivated both in microaerophilic and anaerobic conditions (IF -fluorescence intensivity).

Table 4 .
The results of antibiotics susceptible test.Because in the available literature there is many incoherent data about the sensitivity of C. bifermentans to some antibiotics, in this work the antibiotic sensitivity test was done for all 10 new isolated C. bifermentans strains.The results of antibiotics susceptible test are presented in Table