WO1991013996A1

WO1991013996A1 - A method for obtaining an anaerobic thermophilic bacterium, thus obtainable bacterium and its use for the fermentation of carbohydrates

Info

Publication number: WO1991013996A1
Application number: PCT/NL1991/000038
Authority: WO
Inventors: Wilhelmus Jurriën DRENT; Jan Cornelis Gottschal
Original assignee: Rijksuniversiteit Groningen
Current assignee: Rijksuniversiteit Groningen
Priority date: 1990-03-14
Filing date: 1991-03-13
Publication date: 1991-09-19
Anticipated expiration: 1992-09-14
Also published as: AU7490891A

Abstract

The invention relates to a method for obtaining an anaerobic thermophilic bacterium comprising isolation of a carbohydrate fermenting bacterium capable of producing succinate at a temperature within a range of 40-80 °C using carbohydrate as a major source of carbon and energy. Bacteria obtainable according to said method and a method for the fermentation of carbohydrates with a succinate producing bacterium, at a temperature within the range of 40-80 °C and in a medium containing carbohydrates metabolisable bij said bacterium is described.

Description

A method for obtaining an anaerobic thermophilic bac¬ terium, thus obtainable bacterium and its use for the fermentation of carbohydrates.

The invention relates to a method for obtaining an anaerobic bacterium capable of producing succinate.

Methods for isolating anaerobic bacteria, which may be used in the fermentation industry, are generally known. Often samples of natural sources such as soil and mud are used for such isolation procedures comprising a so-called, well-known enrichment step. Very often, such bacteria are cultivated at a temperature below JJO'C in a medium with relatively high nutrient concentrations.

In general enrichment cultures are set up in such a way that the physico-chemical conditions are most favourable for the type of organism wanted. As a result this type of organism will then soon outnumber other types due to its higher rate of growth. It is evident that innumerable combinations of medium composition and physical conditions can be created. Temperature, pH, osmotic-value, redox potential and extent of aeration are just a few of the parameters that may be varied.

In many isolation procedures yeast extract is added to the medium. Typically the concentration in which it is added is at least 1 g/1.

Anaerobic bacteria capable of producing succinate are described in the European patent applic¬ ation 0 249 773« The use of various strains of succinate producing bacteria in the fermentation of carbohydrates such as dextrose, sucrose, fructose, lactose, soluble starches and corn syrups is described. The fermentation is conducted in an aqueous medium containing dissolved carbondioxide and nutrients and growth factors needed for reproduction and growth of the micro-organisms employed. The concentration of carbohydrate in the medium lies between 20 and 100 g/1, as a lower concentr¬ ation than 20 g/1 would yield too little succinate for practical recovery. Yeast extract is present at a con¬ centration of 10 g/1. The pH-value is maintained between 5.8-6.8. At higher pH-values the main product is lactate rather than succinate. The fermentation process is preferably carried out at a temperature of about 38^*0.

According to the present invention a method has been found for obtaining an anaerobic thermophilic bacterium comprising isolation of a carbohydrate fermenting bacterium, which is capable of producing succinate at a temperature within a range of 4θ-8θ°C, using carbohydrate as a major source of carbon and energy. By this method bacteria are obtained which may be used for improving fermentation processes, e.g. processes for the production of organic compounds by fermentation of carbohydrates. The greater part of the known succinate producing micro-organisms are not interesting from a biotechnological point of view for a number of reasons, e.g. succinic acid is formed in too low concentrations to make the recovery of the ferment- ation product economically feasible and the fermentative bacteria tend to lyse leading to an unstable production process. A production process using thermophilic bacteria may offer a number of advantages compared with the production process using mesophilic bacteria amongst others increased productivity as the reaction rate of organisms and enzymes increase and mass cultivation of thermophilic bacteria will be cheaper than that of mesophilic bacteria due to reduced contamination problems (for a complete survey of the advantages for using thermophilic bacteria in biotechnological processes see Sonnleitner, Adv. in Biochem. Eng. 28: 69~ 138).

It is preferable for the isolation method according to the invention to use a nutrient medium which contains yeast extract in the range of 0-1 wt . % , preferably 0.01-0.1 vt . % , in particular about 0.01 vt . % , based on the total weight of said medium. Yeast extract present in the medium in an amount of more than 1 vt . % results in unpractically low numbers of successful isolations of anaerobic thermophilic succinate producing carbohydrate fermenting bacteria. Although the yeast extract need not be present in the nutrient medium, a small amount thereof (preferably less than 0.1 wt . % ) appears to favour the growth of the bacteria in question. A 90 % success rate of isolation of anaerobic thermophilic succinate producing carbohydrate fermenting bacteria was found when yeast extract was present in a concentration of 0.01 wt . % , based on the total weight of said medium.

In the method according to the present invention various polysaccharide materials may be used. Preferably, such polysaccharides are soluble in the liquid nutrient medium which - in general - comprises water. Good results were obtained by using dextrans , fructans and xylans as polysaccharides, inulin or starch being examples of preferred materials. The enrichment according to the present invention, for example using inulin can be carried out in a broad pH-range, e.g. between pH 5 and 9 . This is surprising because as yet only a few inulinases with a pH optimum above pH 5 have been found, viz. Panaeolus papillonacens , Debaromyces cantarelii and Arthrobacter urafaciens (Mukherjee, K. & Sengupta, S. 1987. Purification and properties of a non-specific- fruetofuranisodase (inulinase) from the mushroom Panaeolus . Can. Journal of Microbiology 3_3_. 520-52 ; Guiraud J.P., Bernit, C. Gelzy, P., (1982), Inulinase of Debaromyces cantarellii. Folia Microbiologica 27_, 19" 24; Uchimyama, T., Niwa, S. & Tanaka, K. (1973). Purification and properties of Arthrobacter ureafaciens inulinase. Biochemica Biophysica Acta 3 . 412-420). The further conditions of the method according to the present invention will be easily estimated by persons skilled in the art. Bacteria of the type, which may be obtained according to the method of the invention, are present in many natural sources. They were e.g. isolated via en¬ richment in batch and continuous cultures, after in- oculation with samples originating from a heat-tower of a sugar factory, soil around a Jerusalem artichoke, fresh cow manure and mud from a tropical pond belonging to the Botanical Garden of the University of Groningen, the Netherlands. The cells of the novel species of Gram- positive, strictly anaerobic, thermophilic bacteria were rod-shaped and non-motile. The G+C content of the DNA was 35«8 +_ 1.0 mol % . Growth on inulin was possible between 40 and 65^*0, with an optimum around 5δ^*C.

The present invention relates to bacterium which may be obtained according to any of the methods of the invention described in the above. It will be clear that for obtaining the bacteria according to the invention further modifications of the conditions may be possible.

In particular, the present invention relates to bacteria of the species Clostridium thermosuccinogenes . The bacteria according to the invention are capable of fermenting a great number of sugars. Accordingly, the present invention also relates to a method for the fermentation of carbohydrates with a succinate producing bacterium in a medium containing carbohydrates metabol- isable by said bacterium and further nutrients required for growth, wherein said fermentation is carried out at a temperature within a range of 40-80^*0, preferably by using one of the bacteria indicated in the above. , In the method for the fermentation of carbo¬ hydrates according to the present invention the temper¬ ature preferably lies in the range of 50-75'C, corresponding to the optimum growth of the bacteria used. Mentioned in the above, a great number of poly- saccharide materials may be used as source of energy and carbon, such as xylan, dextran or fructan, e.g. inulin or starch. An especially preferred polysaccharide is material from the Jerusalem artichoke. Another useful material may be chicory.

Whereas during the method for obtaining (isol- ating) the thermophilic bacterium a rather low carbo¬ hydrate concentration is used in the growth medium during the method for fermentation of carbohydrates a higher carbohydrate concentration is used. It will be self-evident that for economical reasons the highest possible concentration of carbohydrate is preferred.

During the fermentation of carbohydrates according to the invention the same pH-values as during the method for isolating the bacteria may be used. A pH- value of about 7 is preferred. The method of the invention is well-suited for the production of succinate from inulin as higher concentrations are obtained upon comparison with the prior art.

The present invention also relates to compounds, obtained according to the fermentation processes of the invention. Furthermore, the present invention relates to the use of the specific bacteria in the fermentation industry.

Examples

Source of inulin degrading bacteria

The bacteria were enriched from fresh cow manure, beet pulp from the C.S.M. sugar refinery at

Hoogkerk in the Netherlands, soil around a Jerusalem artichoke and mud from a tropical pond belonging to the botanical garden of the University of Groningen.

Media and cultivation conditions

The bacteria were cultivated at 8^{*c fi}t a pH of 7«0 +_ 0.2 using a basal medium containing the following components (g/1): NaCl, 1.2; MgCl₂.6H₂0, 0.4; KCl, 0-3;

CaCl₂.2H₂0, 0.15; NHhCl, 0.27; KH₂P0iι , 0.205; Na₂S0jι , 0.1; bicarbonate buffer NaHCOo, 2.52 or a phosphate buffer NaH₂P0iι, 2.06 and Na₂HP0 , 2.66; Na₂S.9H₂0, 0.15; yeast extract, 0.025; casamino acids 0.025; resazurin. 0.001; trace elements solution SL6 [Pfenning, N. & Lippert, K.L. (1966). Uber das Vitamin B12 Bedurfnis phototropher Schwefelbakterien. Archives of Microbiology 5>. 245-256] (1 ml/1) and vitamin solution [Heijthuijsen, J.H.F.G. & Hansen, T.A. (1986), Interspecies hydrogen transfer in co-cultures of methanol-utilizing acidogens and sulfate-reducing or methanogenic bacteria, FEMS Microbiology Ecology 3_8. 57- 64] (1 ml/1). The mineral medium was autoclaved under a gas phase of N₂/C0₂ (802/20*) or N₂ (100X), in the case of a bicarbonate buffer and a phosphate buffer, respectively. The following stock solutions were sterilised separately and added aseptically to the mineral medium using syringes: NaHCOo (84 g/1), NaH P0|ι and Na₂HP0iι (68.6 g/1) and (157-3 g/1). respectively yeast extract (50 g/1), casamino acids (40 g/1) and vitamin solution. The vitamin and the trace elements solution were filter-sterilised, the other components were autoclaved.

Enrichments The bacteria were enriched in batch culture, using 600 ml bottles filled with 250 ml bicarbonate- buffered basal medium and 1.5 g/1 inulin as the sole source of carbon and energy. The same medium was used for enrichment in continuous culture. Pure cultures were obtained after diluting in culture tubes with a butyl rubber septum and a screw cap, and subsequently plating the highest dilution with visible growth on agar plates (medium supplemented with 2 % agar) . These plates were in a 2.5 1 jar incubated at 60^*C, to which 0.7 nil 10 mM Na S was added in order to maintain anaerobic conditions. After repeated plating the strains were isolated. Growth experiments

Growth experiments at different pH and temperature were done in culture tubes, sealed with a butyl rubber septum and a screw cap (16x225 mm). These tubes contained 9«5 ml buffered basal medium, to which 0.5 ml stock solution of the carbon and energy source was added.

The relationship between the μ_max and the pH was determined in MES-buffered medium at low pH, and Tris- HC1 at high pH. In the other experiments bicarbonate was used, all substrates were filter-sterilised (0.2 μm) .

The enzyme and product formation, during growth on inulin was studied in batch culture, using a 600 ml bottle filled with 250 ml carbonate or phosphate buffer, in which the concentration of inulin was 1.5 g/1-

Chemical analysis

Alcohols, short-chain fatty acids, both volatile and non-volatile were analyzed by gas chromatography [Laanbroek, H.J., Geerligs, H.J., Sijtsma, L. & Veldkamp, H., (1984), Competition for sulfate and ethanol among Desulfobacter, Desulfobulbus , and Desulfovibrio species isolated from interdial sediments. Applied Environmetal Microbiology 4j , 329~334], [Nanninga, H.J. & Gottschal, J.C. (1985). Amino acid fermentation and hydrogen transfer in mixed cultures. FEMS Microbiology Ecology, 3_1. 261-269]- Formate was determined according to [Lang, E. & Lang, H. (1972), Spezifische Farbreaktion zum direkten Nachweis der Ameisensaure. Zeitschrift fttr Analytische Chemie 260, 8- 10]. H₂ and C0 in the gas phase of the cultures were analyzed on a Pye Unicam 104 gas chromatograph equipped with a thermal coductivity detector [Laanbroek et al.,1984, as mentioned before]. Organic carbon was determined with a Shimadzu Carbon Analyzer (Model 500) . The sugar content of inulin was determined after complete hydrolysis with 0.3 M HCl with the method of Somogyi [Somogyi, M. (1952) , Notes on sugar determination, Journal of Biological Chemistry 195. 19- 23]-

Enzyme assay

Inulinase activity was determined by measuring the appearance of reducing sugars with the method of Somogyi using cell culture, culture supernatant and cells resuspended in 0.3 phosphate buffer after centrifugation (12,000 g ) . The reaction mixture contained 2 ml sodium phosphate buffer (30 mM, pH 6.8), inulin (6 g/1) and 1.8 ml enzyme containing sample. The cell culture was inactivated by flushing the culture with air. The formation of fermentation products could never be measured, during enzyme activity measurements. One unit of inulinase activity is defined as the amount of enzyme catalysing the liberation of 1 μmol reducing sugars min^"l. Specific enzyme activities are expressed per milligram of Cell-carbon.

Other methods

Absorbance was measured in a 1 cm cuvette in a vitatron colorimeter. A standard method described by Gerhardt et al. [Gerhardt, P., Murry, R.G.E., Costilow, Nester, E.W., Wood, W.A., Krieg, N.R. & Philips G.B. (1981), Manual of methods for general bacteriology, American Society of Microbiology, Washington, DC] was used for gram staining, taking cells from a chemostat, running at a dilution rate of 0.15 h"¹. Cells of an exponentially growing culture of Pseudomonas oxalaticus and Escherichia coli served as a control. The presence of cytochromes was investigated by recording air- oxidized versus dithionite-reduced spectra of whole cells and cell free extracts with an Aminco spectrophotometer type DW2a. The mol % C+G was determined using ultracentrifugation and buoyant density measurements [DSM, Braunschweig].

Isolated strains Four different strains were isolated, using different techniques of enrichment and sources for inoculation (Table 1).

Morphology and ultrastructure The cells of the isolated thermophilic strains were rod shaped, 2 μm to 4 μm length and 0.3 μ to 0.4μm in width. Frequently spores were observed in stationary cultures. The diameter of the spores was approximately 0.6 μm. After negative staining with uranyl acetate, it can be seen under the electron microscope that the cells of the type strain IC are peritrichously flagellated. However, no motility was observed in phase contrast microscopy.

Colony characteristics

Small, white, smooth colonies are formed within a few days of incubation in an anaerobic jar, both on nutrient broth and basal medium agar plates, supplied with inulin (diameter of the colonies 1-2 mm).

Physiological characterisation

All strains were able to grow on a large range of sugars (Table 2).

Various fermentation products were formed, when the strains were cultivated on fructose, glucose or inulin, including succinate, formate, acetate, lactate, ethanol and small amounts of C0₂ (see Table 3 for the fermentation balances). No cytochromes were present in strain IC, grown on inulin.

Growth experiments

The relationship between μ_max and temperature depended on the source of carbohydrate. Comparison of the optimum curves for growth on inulin and fructose indicated that both the optimum and maximum temperature of growth is higher when fructose is used (Fig. 1). In the pH-range from 6.1 to 9-0 growth of strain IC on inulin was possible with an optimum around 7_*6.

Enzyme production in batch culture

The general pattern of inulin hydrolysis by strain IC in batch culture is shown in Fig. 2. From this growth curve a μ_max of 0.31 h^"1 could be calculated. During growth no accumulation of sugars could be observed (<0.2 mM) . The production of inulinase in batch parallelled the change in optical density and was almost completely cell-bound. Inulinase activity in the supernatant could only be measured in the stationary phase, probably due to cell lysis. During growth experiments no significant drop of pH was observed (< 0.4 pH unit) .

Characterization of the enzyme

The cells were pregrown in an inulin limited continuous culture, run at a dilution rate of 0.05 h~ . In the chemostat part of the inulinase is found in the extracellular culture fluid. There were no differences to be found between the cell-bound and cell-free inulinase, after examining range and optimum of pH and temperature, indicating that the cell-bound and the cell-free activity might be caused by the same _^enzyme (Fig. 3 and Fig. 4). Enzyme activity was detected from approximately 30°C to 68^*0 and over a pH-range from 5-8 to 8.4, with a temperature optimum around 58°C and a pH optimum around 6.8. Table 1

Origin and enrichment-technique of the four inulin- degrading strains

Strain Source of enrichment Enrichment technique

IA soil around Jerusalem artichoke batch ID 50°C-mud from heated pond chemostat

(D=0.02 h"¹) IC heating tower of sugar factory batch IE fresh cow manure batch

Table 2 Survey of the substrates that sustained growth of the four isolated, inulin degrading strains

The inital concentration of the tested substrate is indicated between brackets. All strains used fructose, glucose, galactose, xylose, ribose, sucrose, lactose, maltose, cellobiose, raffinose, H₂/C0₂ ( 8θ%/20% ) and starch (monosaccharides , each 10 mM; disaccharide 5 mM; trisaccharide 3«33 mM and insoluble substrates (1.5 g/1). No growth was observed with methanol (both 10 mM and 30 mM tested), gluta ate, glycerol, pyruvate, citrate (each 30 mM, arabinose (10 mM) , casamino acids (5 g/1). xylan, pectin and cellulose (each 1.5 g/1). Growth on the substrate tested: < 15 % of 0D660 on 10 mM fructose (-); between 15* and 25% of 0D660 on fructose (+) ; > 2 * of 0D660 on fructose (+).

Substrate Strain IA Strain IC Strain ID Strain IE galactose (10 mM) mannitol (10 mM) yeast extract (2 g/1) sorbitol (10 mM) Table 3 Fermentation balance of strains IC and IE The strains were grown on inulin (1.5 g/1). using both a NaHCO^ buffer (30 mM, pH 6.8) and a sodium phosphate buffer buffer (30 mM, pH 6.8). To the latter a small amount of NaHCO was added (5 mM) .

Bicarbonate buffer Phosphate buffer Strain Strain Strain Strain

IC IE IC IE

Inulin fermented 8.71 8.76 9.11 8.72 mM reducing sugars

Products formed (mM)

Formate 1.24 3.14 0.94 2.65

Acetate 11.96 6.54 13.98 6.82

Lactate 1.74 0.00 0.91 0.00 Succinate 2.08 5.78 1.94 5-79

Ethanol 1.44 1.63 1.42 1.99

H₂ 0.6 0.51 1.09 0.95

C0₂ consumption ND ND -1.60 -3.01

Cell material (mM Carbon) 9.22 9.41 10.90 9.36

Recovery Carbon ND ND 94 95

Recovery COD 102 97 103 100

(Chemical Oxygen Demand)

LEGENDS

Figure 1

Effect of the temperature on specific growth rate of strain IC, using inulin (1.5 g/1) or fructose (10 mM)^' as the carbon and energy source. ( ) μ_max on inulin, ( ) μ_max on fructose.

Figure 2 Kinetics of growth and inulinase activity of strain IC in batch-culture on inulin medium (1.5 g/1). ( ) ODggQ. ( ) cell bound-inulinase activity, ( ) cell-free inulinase activity, ( ) concentration of inulin, expressed as mM fructose.

Figure 3

Effect of pH on activity of cell-free ( ) and cell- bound ( ) inulinase, expressed as percentage of maximum activity at 58"c (cell-bound 8.5 U/l, cell-free 2.78 U/l).

Figure 4

Effect of temperature on activity of cell-free ( ) and cell-bound ( ) inulinase, expressed as percentage of maximum activity at 58°C (cell-bound 9-2 U/l, cell-free 2.25 U/l) .

Claims

C L A I M S

1. A method for obtaining an anaerobic thermophilic bacterium comprising isolation of a carbohydrate fermenting bacterium, which is capable of producing succinate at a temperature within a range of 40-8o"c, using carbohydrate as a major source of carbon and energy.

2. The method according to claim 1, wherein for the isolation a nutrient medium is used which contains yeast extract in the range of 0-1 vt%, preferably 0.01-0.1 vt . % , in particular about 0.01 wt#, based on the total weight of said medium.

3- The method according to claim 1, wherein for the isolation a nutrient medium is used which contains a polysaccharide such as a xylan, a dextran or a fructan, e.g. inulin or starch, as substantially the sole source of energy and carbon.

4. The method according to claim 1, wherein the isolation comprises an enrichment which is carried out at a pH-value within. the range of about 5"8, preferably at a pH of about 7-

5. A bacterium obtainable according to any of claims 1-4.

6. Bacterium of the species Clostridium thermosuc- cinogenes corresponding or belonging to the strain IA, IC, ID or IE, deposited under Nos. DSM 58O6, DSM 5807^T, DSM 58O8, DSM 5809 respectively at the DSM Collection in Braunschweig, Federal Republic of Germany.

7. A method for the fermentation of carbohydrates with a succinate producing bacterium in a medium containing carbohydrates metabolisable by said bacterium and further nutrients required for growth, wherein said fermentation is carried out at a temperature within a range of 4θ-8θ^βC.

5

8. The method according to claim 7. wherein said bacterium is a bacterium according to any of claims 5-7«

9. The method according to claim 7. wherein said 0 fermentation is carried out at a temperature within a range of 50-75°C.

10. The method according to claim 7. wherein said medium contains a polysaccharide such as a xylan, a 5 dextran or a fructan, e.g. inulin or starch, as substantially the sole source of energy and carbon.

11. The method according to claim 10, wherein said medium contains material from the Jerusalem artichoke as 0 said polysaccharide.

12. The method according to claim 8, wherein the carbohydrate concentration of said medium is within the range of 20-100 g/1. 5

13. The method according to claim 8, wherein in the fermentation medium a pH-value within a range of about 5-9. preferably a pH-value of about 7 is maintained.

Q 14. Compounds, e.g. succinate compounds, prepared according to the method of any of claims 7-13«

15. The use of bacteria according to claim 5 or 6 in the fermentation industry. 5

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