Abstract:
Methods for obtaining cellulolytic microbes with high growth rates are disclosed. For example,  C. thermocellum  colonies with growth rates higher than 0.17 hr″ 1 have been obtained by the present methods. In realizing higher growth rates, better bioprocessing efficiency can be achieved resulting in increased economy.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
       [0001]    This application claims priority to U.S. Patent Application No. 61/100,637, filed Sep. 26, 2008 and is incorporated herein by reference. 
     
    
     GOVERNMENT INTERESTS 
       [0002]    The United States government may have certain rights in the present invention as research relevant to its development was funded by The Department of Energy BioEnergy Science Center (BESC) contract number 60NANB1D0064. 
     
    
     BACKGROUND 
       [0003]    1. Field of the Invention 
         [0004]    The present invention pertains to the field of biomass processing to produce ethanol and, more specifically, to the selection and use of thermophilic organisms with high growth rates. 
         [0005]    2. Description of the Related Art 
         [0006]    Cellulosic biomass represents an inexpensive and readily available raw material from which sugars may be produced. These sugars may be used alone or fermented to produce alcohols and other products. Among bioconversion products, interest in ethanol is high because it may be used as a renewable domestic fuel. Bioconversion processes are becoming economically competitive with petroleum fuel technologies. Various reactor designs, pretreatment protocols, and separation technologies are known, for example, as shown in U.S. Pat. Nos. 5,258,293 and 5,837,506. 
         [0007]    One of the barriers impeding the establishment of a cellulosic biofuels industry is the recalcitrance of lignocellulose, that is, the difficulty of converting cellulosic biomass into soluble sugars. Various naturally cellulolytic microorganisms, such as  Clostridium thermocellum  ( C. thermocellum ) and  Clostridium cellulolyticum  ( C. cellulolyticum ), may be used to achieve this conversion. Strains of  Thermoanaerobacterium thermosaccharolyticum  and  Thermoanaerobacterium saccharolyticum , which have substrate-utilizing capabilities that compliment those of  C. thermocellum  may also be used, includings strains that have been genetically modified to produce ethanol at high yields. 
         [0008]    The rate of cellulose conversion is dependent on the growth rate of cellulolytic microorganisms. That is, the higher the growth rate of cellulolytic organisms, the faster the rate of cellulose conversion and, other things being equal, the smaller and less expensive the reaction vessel in which cellulose solubilization and fermentation of resulting sugars occurs. The growth rate for  C. thermocellum  on the cellulosic substrate Avicel, has been observed to be about 0.17 h −1  (Lynd, L. R., P. J. Weimer, W. H. van Zyl, I. S. Pretorius. 2002. Microbial cellulose utilization: fundamentals and biotechnology. Microbiol. Mol. Biol. Rev. 66:506-577). It is desirable to obtain faster growing strains in order to decrease production costs. 
       SUMMARY 
       [0009]    The present instrumentalities advance the art and overcome the problems outlined above by providing methods for selection of microbes with growth rates higher than previously reported. The expectation of modest ability to increase the growth rate of cellulolytic microbes, for example, is based on the observation that cultures of  C. thermocellum  saturate (completely occupy) the substrate with cellulase. Efforts to increase the specific activity of cellulase enzymes systems via protein engineering have been unsuccessful. With the insoluble cellulose fully loaded with cellulase and with extensive efforts having failed to achieve meaningful increases in cellulase activity, the large increases disclosed herein are entirely unexpected. By realizing higher growth rates, it is anticipated that better bioprocessing efficiency can be achieved resulting in increased economy. 
         [0010]    There are a number of industrial processes for the conversion of lignocellulosic materials for which the present invention would be suitable. These include, but are not limited to consolidated bioprocessing (CBP), simultaneous saccharification and fermentation (SSF) and simultaneous saccharification and co-fermentation (SSCF), involving both hexose and pentose-utilizing organisms. 
         [0011]    In an embodiment, a method of selecting a bacterium comprises: culturing a bacterium on a solid medium until colonies are formed; selecting a bacterial colony that has one or more morphological characteristics associated with a subpopulation of  C. thermocellum  with a high growth rate; isolating the bacterial colony; and growing the bacterial colony in liquid broth to produce a bacterium with a high growth rate. In a more advanced embodiment, continuous culture in a pH auxostat—or similar continuous culture configuration that selects for microorganisms with high growth rates—is used to obtain cultures that exhibit a growth rate yet higher than both the original culture and strains obtained by isolating large colonies. 
         [0012]    In one embobiment the bacterium used for selection can, for example be any one of the genus of  Clostridium  such as, but not limited to:  Clostridium thermosulfurogenes, Clostridium cellulolyticum, Clostridium thermocellum, Clostridium thermohydrosulfuricum, Clostridium thermoaceticum, Clostridium thermosaccharolyticum, Clostridium tartarivorum, Clostridium thermocellulaseum, Clostridium thermolacticum, Clostridium hungatei; Clostridium phytofermentans; Clostridium cellulolyticum; Clostridium aldrichii; Clostridium termitididis.    
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0013]      FIG. 1A  shows colonies of  C. thermocellum  on an agar-cellobiose MTC medium. 
           [0014]      FIG. 1B  shows a close-up view of the  C. thermocellum  of  FIG. 1   a.    
           [0015]      FIG. 2  illustrates comparative growth rates of auxostat-selected, colony-selected and non-selected  C. thermocellum.    
           [0016]      FIG. 3  illustrates the development of faster-growing strains in a pH auxostat maintained at various pH values. 
       
    
    
     DETAILED DESCRIPTION 
       [0017]    There will now be shown and described a method for selecting cellulolytic microbes with high growth rates. As used herein, a “high growth rate” is a growth rate higher than 0.17 h −1  for  C. thermocellum  and all other described cellulolytic microbes that grow optimally at 60 degrees C. or less or a growth rate higher than 0.4 h −1  for  Anaerocellum thermophilum .) In one aspect the growth rate for  C. thermocellum  is typically between 0.17 h −1  and 0.50 h −1  and more typically between 0.40 h −1  and 0.50 h −1 . 
         [0018]    Selection of large colonies, and in particular auxostat cultivation, was shown to be an effective means of obtaining cultures that exhibited growth rates on cellulose that were substantially higher than previously observed in  C. thermocellum . These approaches may be generally utilized for cellulolytic microbes, such as but not limited to bacteria, genetically modified yeast, anaerobic and/or thermophilic bacteria, and  Clostridium  species. 
         [0019]    It has been observed that there is considerable heterogeneity in cultures of  C. thermocellum  with respect to colony size, with some colonies exhibiting larger size than others. Colonies with sizes ranging from about 1 mm to about 3 mm were identified as those capable of producing the observed high growth rates. Cultures of  C. thermocellum  grown from the such colonies exhibited growth rates approximately substantially higher than four times faster than the control culture. A further substantial, nearly 2-fold, increase in growth rate was obtained by selection in auxostat culture. The ability to obtain growth rate increases of this magnitude was entirely unexpected and has no antecedent in the literature on cellulolytic microbes. 
         [0020]    The observed growth rates of cellulolytic microbes, the presence of subpopulations with growth rates higher than previously described, and demonstration of methods to obtain microbes with high growth rates on cellulose are important in an applied context for the reasons outlined above. 
         [0021]    As used herein the units of “growth rate” is the specific growth rate, μ, defined as (rate of cell formation)/(cell concentration). Typical units of the numerator in are g cells/L/hr. Typical units for the denominator are g cells/L. Hence, μ has units of 1/hr. 
         [0022]    In an exponentially-growing batch culture, the cell concentration X is given by: 
         [0000]        X=X   o   e   μt , 
         [0000]    where X o  is the initial cell concentration, and t is time.
 
By way of example, in order to determine the time necessary for doubling, td, we let X/X o =2:
 
         [0000]        td =ln 2/μ.
 
       Example 1 
     Selection of a High Growth-Rate Strain 
     Materials and Methods 
       [0023]    The strain  Clostridium thermocellum  ( C. thermocellum ) ATCC 27405 was used in the following examples.  C. thermocellum  is an anaerobic, thermophilic bacterium possessing cellulolytic and ethanogenic abilities that make it capable of directly converting a cellulosic substrate into ethanol. 
         [0024]      C. thermocellum  was maintained either in MTC medium with 3% Avicel, 3% cellobiose (Ozkan, Desai et al. 2001), or in a chemically-defined media (Johnson, Madia et al. 1981) modified as follows: cellobiose or cellulose, 10 g/L; KH 2 PO 4 , 4.25 g/L; (NH 4 ) 2 SO 4 , 2.1 g/L; MgCl 2 .6H 2 O, 1.0 g/L; CaCl 2 .2H 2 O, 0.15 g/L; FeSO 4 .7H 2 O, 0.002 g/L; Na-citrate, 3.0 g/L; L-cysteine, 1.0 g/L; rasazurin, 0.002 g/L; trace elements and vitamins. Medium was prepared in an anaerobic chamber with an atmosphere of CO 2 /N 2 /H 2  (10%/85%/5%). 
         [0025]    Isolation of  C. thermocellum  was carried out by reactivating  C. thermocellum  from a frozen state in batch culture on MTC-cellobiose and then anaerobically plating on agar-cellobiose MTC. After about one week of incubation at 55° C. the colonies developed within the agar layer were examined and single spatially-separated colonies were transferred to fresh MTC medium. After isolation of individual colonies, the obtained clones were maintained without freezing at low positive temperatures (from 2 to 6° C.) on Avicel-containing MTC medium. 
         [0026]    Batch and continuous fermentations on cellobiose (10 g/liter) and microcrystalline cellulose (Avicel PH 105, FMC, Philadelphia) (10 g/liter) were carried out in 2.5-liter round-bottom reactors (Sartorius A+) with agitation at 100 rpm and flow of ultra-pure N 2  (100 ml/min). The cultures were at constant pH 6.8 and 60° C. All cultivation parameters, including 1 N KOH titration rate, were logged to a computer. Every hour, 6.7 ml of culture were automatically pumped to a fraction collector (Waters) with simultaneous acidification to pH 1.5 with H 2 SO 4  to stop metabolic activity. 
         [0027]    The solids (residual cellulose and cells) were collected by centrifugation, washed, dried at 70° C., and then weighed. The outflow gases were analyzed by a CO 2  infrared gas analyzer (LiCor 800, Lincoln, Nebr.) and by mass-spectrometry using a RGA100 (Stanford Research System, CA). Three gas constituents —CO 2 , H 2 , and CH 4 — were monitored and logged to a computer every 15 seconds. The liquid phase was analyzed by HPLC as described elsewhere (Zang and Lynd, 2005). 
         [0028]    Continuous cultivation was performed in two regimes: chemostat (fixed dilution rate) and pH-auxostat. Having fixed pH of the fed medium (pH 8.5) the constant acidity of cultural liquid (pH 6.8) was electronically maintained by addition of fresh medium. Direct logging of the titration rate allowed for recordation of the instant specific growth as the algebraic sum of dilution rate and the apparent rate of biomass concentration. 
         [0000]    Selection of High Growth-Rate  C. thermocellum    
         [0029]    As described above, frozen  C. thermocellum  was reactivated, plated, incubated, separated, and transferred to fresh MTC medium. The colonies were heterogeneous in size, morphology and color. One set was large, round and bright-yellow. The sizes of the large colonies ranged from about 1 mm to about 5 mm. Another set was slim, oblong and pale-yellow ( FIGS. 1A and 1B ). The large bright-yellow colonies were cultured in MTC medium with 3% Avicel, 3% cellobiose, or the chemically-defined medium of Johnson (vide supra). 
         [0030]    A  C. thermocellum  culture obtained from a large-colony isolate was maintained for two weeks in a pH-auxostat during which the growth rate was observed to continuously increase. Whereas the original culture exhibited a growth rate of 0.11 hr −1 , the large-colony isolate exhibited a growth rate of 0.28 hr −1 , and the auxostat-selected isolated exhibited a growth rate of 0.48 hr −1 . The growth rates were measured from the rate of CO 2  formation, which is proportional to biomass under the absence of a growth limitation.  FIG. 2  shows the curves are a best-fit exponential regression, x=x 0 exp(μt), where x 0  is initial cell mass, t is time, and μ is specific growth rate. The growth rates for the pH-auxostat pre-cultivated large colony-selected  C. thermocellum  (A), primary large colony-selected  C. thermocellum  (B) and non-selected  C. thermocellum  (C) were 0.48 h −1 , 0.28 h −1 , and 0.11 h −1  respectively. In short, the pH-auxostat selected large colony-isolate of  C. thermocellum  had a growth rate ˜4.4 time greater than that of the non-selected  C. thermocellum  control in this experiment, the primary large colony  C. thermocellum  isolate had a growth rate ˜2.3 times greater than the non-selected  C. thermocellum , and the auxostat-selected culture had a growth rate 1.7 times greater than the large colony isolate. The magnitude of these differences was entirely unexpected. 
         [0031]    The effect of pH on growth rate was assessed on large colony  C. thermocellum  isolate maintained in an auxostat over a period of about 20 days. As shown in  FIG. 3 , cells initially grew at the rate of about 0.2 h −1 . However, as the culture evolved at constant pH between 6.7 and 6.8, the growth rate increased to 0.50 h −1 . At a lower pH of 6.4, the growth rate remained high between around 0.25 h −1  and 0.30 h −1 . The culture was interrupted on day 3 and day 8 in order to test the ability of the fast strain to recover after perturbations. 
         [0032]    The disclosed microbes may be utilized in a saccharification process, including a Simultaneous Saccharification and Fermentation (SSF) process as well as a consolidated bioprocessing (CBP) process with no added enzymes. Methods of utilizing cellulolytic microbes for the conversion of cellulosic material into ethanol are known. Cellulosic materials that may be converted by the presently described microbes include any feedstock that contains cellulose, such as wood, corn, corn stover, sawdust, bark, leaves, agricultural and forestry residues, grasses such as switchgrass or miscanthus or mixed prairie grasses, ruminant digestion products, municipal wastes, paper mill effluent, newspaper, cardboard or combinations thereof. 
         [0033]    The description of the specific embodiments reveals general concepts that others can modify and/or adapt for various applications or uses that do not depart from the general concepts. Therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not limitation. 
         [0034]    All references mentioned in this application are incorporated by reference to the same extent as though fully replicated herein.