Facultative Anaerobe Caldibacillus debilis GB1: Characterization and Use in a Designed Aerotolerant, Cellulose-Degrading Coculture with Clostridium thermocellum

Abstract

Development of a designed coculture that can achieve aerotolerant ethanogenic biofuel production from cellulose can reduce the costs of maintaining anaerobic conditions during industrial consolidated bioprocessing (CBP). To this end, a strain of Caldibacillus debilis isolated from an air-tolerant cellulolytic consortium which included a Clostridium thermocellum strain was characterized and compared with the C. debilis type strain. Characterization of isolate C. debilis GB1 and comparisons with the type strain of C. debilis revealed significant physiological differences, including (i) the absence of anaerobic metabolism in the type strain and (ii) different end product synthesis profiles under the experimental conditions used. The designed cocultures displayed unique responses to oxidative conditions, including an increase in lactate production. We show here that when the two species were cultured together, the noncellulolytic facultative anaerobe C. debilis GB1 provided respiratory protection for C. thermocellum, allowing the synergistic utilization of cellulose even under an aerobic atmosphere.

INTRODUCTION

Isolation and characterization of organisms with the ability to degrade and ferment cellulose are essential steps toward enhancing biofuel production via consolidated bioprocessing (CBP) (1). Designing a culture that could achieve both cellulose degradation and ethanol production under nonreduced conditions would reduce the costs and complexity of culturing strictly anaerobic bacteria, as this would eliminate the need for maintaining a reduced environment. Clostridium thermocellum is perhaps the best-studied model organism capable of CBP under anaerobic conditions (2, 3). Coculturing a cellulolytic organism with a noncellulolytic partner capable of producing the desired end products at high rates and yields has been shown to improve the overall biofuel production capability via CBP (4) and increased cellulose hydrolysis rates (4, 5). Many biofuel-producing cellulolytic bacteria, including C. thermocellum, lack aerotolerance and have not been shown to be able to tolerate greater than 2% O₂ in the atmosphere (6, 7). While members of the genus Clostridium are typically described to be strict anaerobes, the genomes of some members, including Clostridium straminisolvens, Clostridium acetobutylicum, and Clostridium intestinalis, encode genes required for aerotolerance, and these organisms have been shown to grow in microaerobic environments (6, 8, 9). Moreover, genetic modification of C. acetobutylicum resulted in a significant increase in aerotolerance, such that the recombinant strain was able to grow in the presence of atmospheric concentrations of oxygen (10).

Previously, it was reported that cocultures containing a Clostridium species paired with facultative aerobes can lead to oxygen removal in a sealed vessel followed by fermentation (11–13) or achieve aerotolerance via respiratory protection under microaerobic conditions (14). The importance of biofilms and the use of mixed cultures to help lignocellulosic degradation have also recently been highlighted (15). Ronan et al. (12) highlighted the potential of stable, apparently aerotolerant, cellulolytic communities to ferment cellulose to ethanol. Wushke et al. (16) also described a stable enrichment culture, B4-1, which included Caldibacillus debilis-like and C. thermocellum-like bacteria, that was capable of cellulolytic growth and fermentation in the presence of oxygen. The C. debilis strain was subsequently isolated and named GB1.

The aims of the present paper were to (i) characterize C. debilis GB1 and compare it with the type strain, C. debilis Tf DSM 16016 (here referred to as C. debilis DSM 16016), and (ii) demonstrate that the aerotolerant phenotype observed in enrichment culture B4-1 can be achieved using a coculture of C. debilis GB1 and C. thermocellum DSM 1237. To this end, we compared the aerobic and anaerobic growth and end product formation of C. debilis GB1, isolated from the cellulolytic aerotolerant ethanol-producing enrichment B4-1, with those of the type strain, C. debilis Tf DSM 16016, isolated by Banat et al. (17). Furthermore, enrichment culture B4-1, primarily comprised of a C. thermocellum-like strain and C. debilis GB1, was compared with a coculture containing C. thermocellum DSM 1237 and C. debilis GB1 under both aerobic and anaerobic conditions.

MATERIALS AND METHODS

Culturing method.

Cultures of C. thermocellum DSM 1237 and C. debilis Tf^T DSM 16016 were obtained from the DSMZ culture collection. Enrichment culture B4-1 and C. debilis GB1, isolated from B4-1, were derived from previous work (16). Modified 1191 medium (18), in which a concentration of yeast extract (0.76 g/liter) lower that used previously was used and whose pH was adjusted to 7.2, was used for all experiments. Various sugars were used as carbon and energy sources (at 2 g/liter), including xylose, glucose, ribose, sucrose, arabinose, rhamnose, mannose, maltose, cellobiose, sorbitol, xylan, and trehalose, as specified in the methods used for the different experiments (see below).

Sealed Balch tubes (26 ml) and 110-ml serum bottles, supplied from Bellco Glass Inc. (Vineland, NJ) and Fisher Scientific (Toronto, ON, Canada), respectively, were used to carry out the experiments. Balch tubes were used for all substrate utilization experiments (see Table 1) to compare C. debilis GB1 with C. debilis DSM 16016. Anaerobic Balch tubes and serum bottles were prepared using the methodology described by Islam et al. (18), while aerobic tubes and serum bottles were prepared using an atmosphere in the headspace and no addition of the reductant sodium sulfide. Experiments designed to compare aerobic and anaerobic conditions (see Table 3) utilized serum bottles with 10 ml of medium and a 100-ml headspace.

TABLE 1.

Comparison of substrates of C. debilis strain DSM 16016 and GB1 grown under static conditions in sealed Balch tubes on modified 1191 medium

Growth condition and substrate	DSM 16016						GB1
	Final growth pHa	Protein concnb (μg/ml)	End product concnc (mM)				Final growth pHa	Protein concnb (μg/ml)	End product concnc (mM)
			Lactate	Acetate	Formate	Ethanol			Lactate	Acetate	Formate	Ethanol
Aerobicd
Cellobiose	5.5	118	0.6	14.4	6.3	—e	5.5	106	—	8.9	9.6	9.0
Xylose	5.4	165	0.8	14.7	4.7	—	5.8	72	0.1	7.2	8.4	—
Anaerobic
Cellobiose	7.2	—	—	—	—	—	5.4	143	—	3.7	9.3	6.6
Xylose	6.7	—	—	—	—	—	5.3	125	—	3.9	8.2	5.4

^aStandard deviation, <±0.1.

^bStandard deviation, <±6 μg/ml.

^cStandard deviation, <±15%.

^dEnd products produced by the medium control were subtracted. DSM 16016 produced 0.1 mM lactate, 1.5 mM acetate, and 0.9 mM formate, and GB1 produced 0.1 mM lactate, 1.5 mM acetate, and 0.5 mM formate. Strains DSM 16016 and GB1 also grew on xylose, ribose, glucose, mannose, cellobiose, sucrose, maltose, and trehalose under aerobic conditions.

^e—, concentrations were less than or equal to those for the yeast extract controls.

TABLE 3.

End products after 72 and 168 h of incubation at 60°C on modified 1191 medium^a

Growth condition and microorganism or consortium	Amt of end product (mmol/liter of cell culture) at 72/168 h						Amt of O2 consumed (mmol/liter of cell culture) at 72/168 hd	Redox balance at 72/168 h
	Lactateb	Acetateb	Formateb	Ethanolb,c	CO2b	H2b
Anaerobice
C. debilis GB1f	0.1	5.5	10.1	6.5	BD	BDg		0.77
C. thermocellum	1.5	5.1	2.0	4.2	8.7	15.9		0.80
C. thermocellum + C. debilis GB1	0.5	3.8	5.0	6.3	4.5	6.7		0.73
B4-1	2.6	7.2	10.7	11.0	5.6	4.1		0.84
Aerobic
C. debilis GB1	BD/BD	2.5/4.0	BD/BD	BD/BD	7.5/41.2	BD/BD	6.1/35.4	1.22/1.16
C. thermocellum + C. debilis GB1	6.6/1.3	8.7/BD	4.4/6.1	5.5/BD	13.2/86.2	12.4/9.3	8.0/97.0	0.46/90.5
B4-1	2.4/4.2	12.4/17.3	4.5/5.2	3.8/3.8	48.2/41.2	9.2/11.5	32.0/34.0	1.31/1.01

^aThe cultures were shaken at 75 rpm. End products produced by the yeast extract control were subtracted for each condition (for GB1 grown aerobically, CO₂ was present at 0.8 mmol/liter of cell culture and O₂ was present at 15 mmol/liter of cell culture; for GB1 grown anaerobically, 0.1 mM lactate, 1.5 mM acetate, and 0.4 mM formate were present). C. thermocellum did not produce any appreciable end products from the medium control.

^bStandard deviation, ≤±10% for monocultures and ≤±20% for cocultures.

^cWelch's t test found a statically significant (P ≤ 0.05) difference in ethanol production between C. thermocellum alone and C. thermocellum in the coculture under anaerobic conditions (21).

^dAt time zero, 100.7 mmol of O₂/liter of cell culture was available. Standard deviation, ≤±25%.

^eOnly data for 72 h are shown for samples grown anaerobically, as no change was observed at 168 h.

^fAll cultures were grown with excess cellulose (5 g/liter), with the exception of the pure culture of C. debilis GB1, where cellobiose (5 g/liter) was used.

^gBD, below the detection (quantification) limit (0.1 mM/liter of cell culture).

Cell growth.

Prior to each experiment, cells were passaged once under the relevant condition to allow adaptation to substrate and/or, in the case of cocultures, to allow adaptation to growth together. All experiments were carried out in triplicate using a 10% inoculum. All cultures were grown at 60°C unless otherwise stated. Cultures grown for end product sampling with sampling points at 48 h postinoculation (hpi) (see Table 1) were grown in sealed aerobic Balch tubes containing 10 ml of medium and a 17-ml headspace. For the assays whose results are presented in Tables 3 and 4, cultures were grown in serum bottles using 10 ml of medium with a 100-ml headspace to ensure oxygen excess throughout growth. Shaking (75 rpm) was used to equilibrate the medium with the gas phase. For analysis of end products, samples were taken at 72 hpi and 168 hpi. All cultures for which the results are presented in Table 3 were grown under conditions of carbon excess (5 g/liter) with either cellulose or cellobiose. All cultures grown aerobically on cellulose were transferred with a cut off pipette tip, as an intact biofilm appeared to enhance the inoculation efficiency. During cell growth under relevant conditions, aerobiosis was also visually assessed via detection of the pink color of resazurin dye in the medium.

TABLE 4.

Aerobic end product consumption/production by C. debilis GB1 at 0, 72, and 168 h of incubation at 60°C on modified 1191 medium^a

Substrate	Amt of end product (mmol/liter of cell culture) at 0/72/168 h					Amt of O2 consumedb (mmol/liter of cell culture) at 0/72/168 h
	Lactatec	Acetatec	Formatec	Ethanolc	CO2d
Lactatee	21.7/21.8/22.2	BD/BD/BDf	BD/BD/BD	BD/BD/BD	BD/BD/BD	BD/0.8/BD
Formatee	BD/BD/BD	BD/BD/BD	23.1/23.0/23.0	BD/BD/BD	BD/BD/BD	BD/0.5/0.5
Acetatee	BD/BD/BD	21.7/21.0/15.7	BD/BD/BD	BD/BD/BD	BD/0.7/1.3	BD/6.3/11.0
Ethanole	BD/BD/BD	BD/9.8/13.0	BD/BD/BD	21.3/11.7/6.4	BD/0.9/2.2	BD/3.4/8.9
Mixed end productsg	2.7/2.9/2.9	2.5/1.4/BD	2.9/3.2/3.0	2.9/0.4/0.3	BD/1.1/2.8	BD/11.3/19.1
Cellobiose with mixed end productsg	2.7/2.9/3.0	2.7/4.3/BD	2.9/3.2/1.6	2.7/0.5/0.2	BD/1.3/3.0	BD/13.7/18.5
Uninoculated mixed end product controlg,h	2.4	2.1	2.6	2.7	BD	BD

^aThe cultures were shaken at 75 rpm. The end products produced and oxygen consumed from the yeast extract control were subtracted for each condition (for GB1 grown aerobically, CO₂ was present at 0.8 mmol/liter of cell culture and O₂ was present at 15 mmol/liter of cell culture; for GB1 grown anaerobically, 0.1 mM lactate, 1.5 mM acetate, and 0.4 mM formate were present). C. thermocellum did not produce any appreciable end products from the medium control.

^bAt time zero, 100.7 mmol of O₂/liter of cell culture was available. Standard deviation, ≤±10%.

^cStandard deviation, <±10%.

^dStandard deviation, <±15%.

^eAdded at ∼20.0 mM.

^fBD, below the detection limit.

^gWhere applicable, ∼2.5 mM each end product and 1.1 mM cellobiose were added.

^hData are for 168 h only.

Physiological characterization.

The optical density at 600 nm (OD₆₀₀) was used to monitor cell growth in liquid medium with soluble substrates. When the cells were grown on cellulose, substrate degradation, pH, end product synthesis data, and protein concentrations were used to verify growth. Protein concentrations were estimated by the Bradford assay (19), using a NanoDrop 1000 spectrometer.

Sugar and end product analysis.

Culture samples (1 ml) were collected after 48 hpi and stored at −20°C until analyzed. The concentrations of cellobiose, glucose, ribose, trehalose, sorbitol, rhamnose, arabinose, maltose, mannose, sucrose, lactate, formate, acetate, and ethanol were measured by high-pressure liquid chromatography (HPLC) using a Dionex ICS 3000 system equipped with a Bio-Rad Aminex-87H column and run at 30°C and 0.75 ml/min with 0.02 mM sulfuric acid. A Shodex 101 refractive index detector was used on all compounds being analyzed.

CO₂, H₂, and O₂ concentrations were determined using a multiple gas analyzer number 1 gas chromatograph (GC) system (model 8610-0070; SRI Instruments, Torrance, CA) equipped with a thermal conductivity detector (TCD) and argon as the carrier gas. The columns and methods used were those previously described by Rydzak et al. (20).

Welch's t test was used to determine if the levels of ethanol production presented in Table 3 were significantly changed (P < 0.05) (21). The glucose equivalents consumed were estimated from the end products produced using the following formula: C₆ = (C₁/6) + (C₂/3) + (C₃/2), where C₆ is the concentration (mM) of glucose equivalents consumed, C₁ is the concentration (mM) of formate and CO₂, C₂ is the concentration (mM) of acetate and ethanol, and C₃ is the concentration (mM) of lactate (22).

Bioinformatic analyses.

The C. thermocellum DSM 1237 (NC_009012) and C. debilis DSM 16016 (NZ_ARVR01000000) genomes were viewed, and a search for genes was performed using the IMG/er program (23).

Culture purity confirmation.

Monoculture purity was determined via 16S rRNA gene PCR amplification and sequencing using primers 8F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1541R (5′-AAGGAGGTGATCCAGCCGCA-3′), and the consistency of the end product production phenotype was determined from the literature and previous experiments (16, 20). The purity of the cocultures (i.e., confirmation that they contained only C. thermocellum DSM 1237 and C. debilis DSM 16016) was confirmed by PCR using the 16S rRNA interspacer primers ITS-F (5′-GTCGTAACAAGGTAGCCGTA-3′) and ITS-Reub (5′-GCCAAGGCATCCACC-3′). The resulting amplicons were compared with the interspacer profiles of the pure cultures using agarose gel electrophoresis (24).

Nucleotide sequence accession numbers.

GB1 sequences for the 16S rRNA and cpn-60 genes were submitted to NCBI under the accession numbers KF652080 and KP259875, respectively.

RESULTS

Caldibacillus strain comparison.

C. debilis GB1 was deposited at DSMZ and designated DSM 29516. Comparison of the C. debilis DSM 16016 and C. debilis GB1 16S rRNA and cpn-60 gene sequences (DSM 16016 locus tags, A3EQDRAFT_10000 and A3EQDRAFT_00206, respectively; GB1 GenBank accession numbers, KF652080 and KP259875, respectively) revealed high sequence identities (99%) for both. The growth of both strains on cellobiose at 60°C revealed that strain GB1 grew similarly to DSM 16016, with both strains reaching similar cell densities, as determined by measurement of the OD₆₀₀ (0.13), and producing similar total protein concentrations (Table 1). The growth of both strains on cellobiose was also measured and found to be similar at different temperatures (50, 55, 60, 65, and 70°C), as seen in Table 2. The growth rates for strain GB1 and DSM 16016 were found to be optimal at between 55 and 60°C (Table 2). Due to the low OD₆₀₀ and the large amount of cell lysis after C. debilis reaches stationary phase, which are typical of C. debilis, the total amount of protein per milliliter was used as an additional indicator of growth (Table 1).

TABLE 2.

Doubling times of C. debilis GB1 and DSM 16016 at temperatures of between 50 and 70°C

Temp (°C)	Doubling time (h)a
	GB1	DSM 16016
50	6.25	6.00
55	1.78	2.18
60	1.71	3.00
65	3.29	2.96
70	5.33	3.00

^aStandard deviation, ≤3%.

A comparison of sugar utilization and end product synthesis by strains GB1 and DSM 16016 under aerobic and anaerobic conditions is summarized in Table 1. An increase in total protein concentrations, a decrease in pH, and an increase in end product synthesis (compared to the results for the no-substrate control) revealed that both strain DSM 16016 and strain GB1 can grow aerobically on cellobiose, xylose, glucose, ribose, sucrose, mannose, maltose, and trehalose but not on sorbitol, arabinose, rhamnose, or xylan.

As presently described, members of the Caldibacillus genus are obligate aerobes (25). While C. debilis DSM 16016 was indeed confirmed to be a strict aerobe under the conditions tested, the novel isolate, strain GB1, was capable of both aerobic and anaerobic growth on the carbon sources tested: xylose and cellobiose (Table 1). Different end product synthesis patterns were observed under aerobic and anaerobic growth conditions for strain GB1. Under aerobic growth conditions (defined in Table 1), C. debilis GB1 produced acetate at a higher concentration, while ethanol and formate formation remained at comparable levels (the exceptions were xylose and ribose, with which the GB1 cells did not produce any ethanol). Note that H₂ and CO₂ were not measured in the experiments whose results are presented in Table 1.

A comparison of GB1 growth under anaerobic versus aerobic conditions with shaking (Table 3) revealed that formate as an end product was completely eliminated and only acetate and CO₂ production was observed under aerobic conditions.

Caldibacillus debilis plus Clostridium thermocellum coculture.

Interspacer analysis (24) at the time points at which samples were taken in the coculture experiments (Table 3) showed that each coculture was comprised of its component organisms, and no contaminating organisms were detected.

Table 3 also compares the end product synthesis patterns, obtained under aerobic and anaerobic conditions, of the designed coculture of C. debilis plus C. thermocellum and monocultures of the two bacteria used in the designed coculture with those of the initial B4-1 enrichment culture. The designed coculture produced the same aerotolerant cellulolytic phenotype as the environmental enrichment culture B4-1 growing under aerobic conditions, with both cocultures producing the end products formate, acetate, lactate, ethanol, CO₂, and H₂ at various concentrations at 72 hpi. The maintenance of aerobic conditions using shaking (Table 3) was confirmed visually (by detection of the pink color of the resazurin dye) throughout growth and at the sampling time points via detection of excess oxygen.

The enrichment and designed cocultures showed a similar response to oxygen for certain end products. Both cocultures displayed a decrease in formate and ethanol concentrations and an increase in CO₂, acetate, and H₂ concentrations. Under aerobic conditions, both the designed coculture and B4-1 show a significant shift towards lactate production, an aerotolerant fermentative pathway, compared to the monoculture.

Further incubation under an aerobic atmosphere (168 hpi) resulted in further metabolism of both the substrate and the fermentation end products by the designed coculture mostly to the respirative product CO₂. The reduction in the concentrations of several end products at between 72 and 168 hpi in the aerobic coculture is consistent with respiration of the fermentative end products after the consumption of the sugars. Due to this apparent ability to consume end products in coculture, the ability to oxidize fermentation end products was verified in pure culture using C. debilis (Table 4). Acetate and ethanol were oxidized, consistent with the reduction in the amount of O₂, while formate was metabolized at significant levels only in the presence of cellobiose.

DISCUSSION

Caldibacillus strain comparison.

First described as Geobacillus debilis in 2004 (17), the type strain, DSM 16016, was characterized as a Gram-variable, rod-shaped, spore-forming, obligatory aerobic organism with a growth temperature range of 50 to 70°C. Upon reevaluation of its taxonomic classification, it was renamed Caldibacillus debilis, becoming the type strain for the new genus (25). Previous characterization of C. debilis strains showed the ability to grow aerobically on several substrates, including formate (17), in one strain, but none had been found to grow in the absence of oxygen. Our observed end product profiles revealed mixed fermentative and respiratory metabolism under an aerobic atmosphere, even though the type strain, DSM 16016, was indeed an obligate aerobe (Table 1). While carbon sources were similar for both strains, aqueous end product concentrations between strains were different (Table 1). When grown in static tubes in the presence of air, the redox indicator resazurin stayed oxidized throughout the experiment and both strains consumed oxygen but displayed incomplete respiration by the synthesis of acetate and formate. Strain DSM 16016 (but not GB1) also synthesized lactate as a major end product. GB1 produced ethanol under aerobic conditions (resazurin oxidized to pink) or anaerobic conditions, making it more attractive than DSM 16016 as an aerobic partner for CBP.

The decrease and subsequent elimination of formate production by GB1 in response to exposure to higher soluble O₂ (Table 3) concentrations likely stem from the fact that pyruvate:formate lyase (PFL) is inactivated in the presence of O₂ (26). End product data (Table 3) further suggest that PFL is used under anaerobic conditions, whereas pyruvate dehydrogenase (PDH) is most likely used under aerobic conditions, consistent with the enzymes used under aerobic conditions by members of the closely related genus Geobacillus (27). Analysis of the C. debilis DSM 16016 genome revealed copies of genes encoding both of these enzymes. A copy of AdhE, an enzyme linked to high levels of ethanol production (28), was also found in C. debilis DSM 16016, but AdhE did not seem to be active under the growth conditions tested.

Typically, under aerobic conditions, organisms produce CO₂ and H₂O. However, C. debilis produces acetate as well. Aerobic acetate production is commonly used to generate ATP and deal with the excess acetyl coenzyme A generated by the high carbon flux through glycolysis (29). A unique characteristic that makes C. debilis GB1 desirable for use for biofuel production via CBP is its low level of lactate production under all conditions tested, which is rare in wild-type thermophilic bacilli, such as Geobacillus species (27, 30, 31). While both C. debilis GB1 and strain DSM 16016 can grow aerobically using respirofermentative metabolism (32), only GB1 maintains a low-lactate phenotype.

Representatives of the thermophilic bacillus genera Ureibacillus, Aneurinibacillus, Brevibacillus, Geobacillus, and Alicyclobacillus (33) have variable phenotypic characteristics regarding anaerobic metabolism, with both strictly aerobic and facultatively anaerobic members being represented. Here we describe a facultatively anaerobic strain within the genus Caldibacillus. Anaerobic fermentative metabolism makes C. debilis GB1, which produces only low levels of lactate, an interesting candidate for designed coculture development to process lignocellulosic substrates under either aerobic or anaerobic conditions via CBP.

Caldibacillus debilis plus Clostridium thermocellum coculture.

The enrichment culture B4-1 displayed an aerotolerant cellulolytic phenotype that is of interest for CBP (16). Subsequent isolation and characterization of C. debilis GB1 from this enrichment demonstrated that C. debilis GB1 is most likely the organism lending functional aerotolerance to the enrichment. A coculture of C. debilis GB1 and C. thermocellum DSM 1237 was tested for cellulose degradation and ethanol production under nonreduced conditions. We compared the designed coculture with the original B4-1 enrichment culture, containing C. debilis GB1 and an uncharacterized C. thermocellum strain as major components, for their capacity to degrade cellulose. While both a pink color from the resazurin dye and excess oxygen were observed throughout, C. debilis may reduce local concentrations of oxygen via respiration and/or via oxygen-scavenging enzymes, creating a reduced microenvironment for C. thermocellum. One possible requirement for creation of this anaerobic microenvironment may be a solid substrate for biofilm creation (15). Indeed, under aerobic conditions, several products generated from the cocultures require enzymes that are sensitive to oxygen, including H₂ and formate (20).

Figure 1 shows the total amount of ethanol, the amount of glucose equivalents consumed, and the ethanol yield per glucose equivalent. Previous studies of C. thermocellum growing on 1191 medium on cellulose have shown that 6.1 mM glucose equivalents is converted to end products (12, 34) in batch cultures, and our experiments showed that 5.2 mM glucose equivalents was consumed. Under our conditions, C. thermocellum produces 4.2 mM ethanol, similar to the findings of other work performed under similar conditions (34, 35). The enrichment and defined cocultures appear to act synergistically under aerobic conditions to allow the production of end products in amounts larger than the amounts produced by either of the C. debilis or C. thermocellum monocultures. Synergies have been observed before in cocultures containing C. thermocellum (36).

FIG 1.

Comparison of the total amount of ethanol produced, the total amount of glucose equivalents consumed, and the efficiency of ethanol production per glucose equivalent consumed at 72 h.

Typically, lignocellulosic ethanol production is rate limited due to the cellulose degradation rate (1, 2).The increase in the total amount of carbon consumed under coculture conditions may be due to the ability of C. debilis to continue to metabolize the cellobiose released through the action of the cellulosomes even after C. thermocellum has stopped growing due to a low pH (under anaerobic conditions, the final pH is ∼5.3; data not shown), the alleviation of nutrient deficiencies, and/or the consumption of end products for utilization in respiration leading to a pH that does not limit the growth of C. thermocellum (under aerobic conditions, the final pH is ∼6.2; data not shown). Increasing the total amount of carbon consumed in CBP systems is a key element, as an increase in the total amount of ethanol produced may enhance the economic viability of this process (2).

Islam et al. (18) showed high levels of lactate production by C. thermocellum DSM 1237 with high substrate loading and during stationary phase. The shift to lactate production in C. thermocellum may be an indicator of cellular stress or of metabolic activity with limited growth (20). Cellular and/or oxidative stress may be important factors leading to high lactate levels in the designed and enriched cocultures at 72 hpi. The genome of C. thermocellum DSM 1237 contains several annotated genes that may be involved in oxygen tolerance, including copies of genes for alkyl hydroperoxidase, superoxide dismutase, and Mn-catalase. These genes may be necessary to deal with reactive oxygen species and to reduce low levels of oxidative stress. It is possible that these can be expressed only while the organism is growing under microaerobic conditions, which are provided by the conditions of coculture with C. debilis.

C. thermocellum has previously been shown to consume lactate (20). The ability of the coculture to consume the fermentative products (lactate, acetate, ethanol) at 168 hpi appears to be an additive effect of the coculture, since C. debilis GB1 appears to be capable of both acetate and ethanol oxidation (Table 4). While the backconsumption of ethanol is not ideal for CBP, the novelty of the use of (micro)aerobic cellulosic degradation to produce ethanol makes it an interesting subject of study.

While the aerotolerant phenotype was conserved in both the B4-1 enrichment culture and the designed cocultures, there were some differences between the end product synthesis patterns of the enrichment culture and those of the designed coculture. Comparison of the enrichment and designed cultures should be done with caution, as there could be other undetected organisms within the enrichment culture (16). Respiratory protection of an obligate anaerobe under aerobic conditions has previously been observed with Clostridium phytofermentans and Saccharomyces cerevisiae cdt-1 (14).

Conclusion.

Previously described models for culturing of an obligate anaerobe and facultative anaerobe on a solid substrate include oxygen depletion as the first step, followed by anaerobic fermentation (11, 13). In the current research, we observed simultaneous anaerobic fermentation and respiration. It is probable that the presence of C. debilis created an anaerobic microenvironment to allow C. thermocellum to ferment, even though the macroenvironment remained aerobic (Table 3).

This study demonstrates that cocultures containing only C. debilis and C. thermocellum possess both aerotolerance and cellulolytic capabilities, even though C. thermocellum and the cellulosome are known to be aerosensitive (M. Resch, J. O. Baker, Q. I. Xu, W. S. Adney, S. R. Decker, M. E. Himmel, and B. Donohoe, U.S. patent application US 13/953,220). This ability demonstrates potential CBP functionality under aerobic, anaerobic, or aerotransient conditions.