Biological pretreatment of corn stover with Phlebia brevispora NRRL-13108 for enhanced enzymatic hydrolysis and efficient ethanol production.

Biological pretreatment of lignocellulosic biomass by white-rot fungus can represent a low-cost and eco-friendly alternative to harsh physical, chemical, or physico-chemical pretreatment methods to facilitate enzymatic hydrolysis. In this work, solid-state cultivation of corn stover with Phlebia brevispora NRRL-13018 was optimized with respect to duration, moisture content and inoculum size. Changes in composition of pretreated corn stover and its susceptibility to enzymatic hydrolysis were analyzed. About 84% moisture and 42 days incubation at 28°C were found to be optimal for pretreatment with respect to enzymatic saccharification. Inoculum size had little effect compared to moisture level. Ergosterol data shows continued growth of the fungus studied up to 57 days. No furfural and hydroxymethyl furfural were produced. The total sugar yield was 442 ± 5 mg/g of pretreated corn stover. About 36 ± 0.6 g ethanol was produced from 150 g pretreated stover per L by fed-batch simultaneous saccharification and fermentation (SSF) using mixed sugar utilizing ethanologenic recombinant Eschericia coli FBR5 strain. The ethanol yields were 32.0 ± 0.2 and 38.0 ± 0.2 g from 200 g pretreated corn stover per L by fed-batch SSF using Saccharomyces cerevisiae D5A and xylose utilizing recombinant S. cerevisiae YRH400 strain, respectively. This research demonstrates that P. brevispora NRRL-13018 has potential to be used for biological pretreatment of lignocellulosic biomass. This is the first report on the production of ethanol from P. brevispora pretreated corn stover. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:365-374, 2017.

Sodium Tetraborate Decahydrate Treatment Reduces Hydrogen Sulfide and the Sulfate-Reducing Bacteria Population of Swine Manure.

Emission of odorous and toxic gases from stored livestock manure is well documented and poses a serious health risk to farmers and livestock. Hydrogen sulfide emissions have been sharply rising with increasingly intensive livestock production and are of particular concern because of the acute toxicity of this gas. Numerous strategies, technologies, and chemical treatments have been used to control hydrogen sulfide emissions, but none have worked particularly well because they are neither cost-effective nor environmentally sustainable, or they are too toxic for animals. The inhibitory effect of the sodium tetraborate decahydrate (i.e., borax) treatment to reduce hydrogen sulfide production using sulfate-reducing bacteria was examined in shallow manure pits in a starter-grower swine facility. Monitoring of air emissions and DNA analysis revealed that treatment of stored swine manure effectively reduced hydrogen sulfide production, and the reduction correlated to a decrease in the sulfate-reducing bacteria population in the stored swine manure.

Examination of the Aerobic Microflora of Swine Feces and Stored Swine Manure.

Understanding antibiotic resistance in agricultural ecosystems is critical for determining the effects of subtherapeutic and therapeutic uses of antibiotics for domestic animals. This study was conducted to ascertain the relative levels of antibiotic resistance in the aerobic bacterial population to tetracycline, tylosin, and erythromycin. Swine feces and manure samples were plated onto various agar media with and without antibiotics and incubated at 37°C. Colonies were counted daily. Randomly selected colonies were isolated and characterized by 16S rRNA sequence analyses and additional antibiotic resistance and biochemical analyses. Colonies were recovered at levels of 10 to 10 CFU mL for swine slurry and 10 to 10 CFU g swine feces, approximately 100-fold lower than numbers obtained under anaerobic conditions. Addition of antibiotics to the media resulted in counts that were 60 to 80% of those in control media without added antibiotics. Polymerase chain reaction analyses for antibiotic resistance genes demonstrated the presence of a number of different resistance genes from the isolates. The recoverable aerobic microflora of swine feces and manure contain high percentages of antibiotic-resistant bacteria, which include both known and novel genera and species, and a variety of antibiotic resistance genes. Further analyses of these and additional isolates should provide additional information on these organisms as potential reservoirs of antibiotic resistance genes in these ecosystems.

Miscanthus×giganteus xylooligosaccharides: Purification and fermentation.

A procedure was developed to recover xylooligosaccharides (XOS) from Miscanthus×giganteus (M×G) hydrolyzate. M×G hydrolyzate was prepared using autohydrolysis, and XOS rich fractions were acquired using activated carbon adsorption and stepwise ethanol elution. The combined XOS fractions were purified using a series of ion exchange resin treatments. The end product, M×G XOS, had 89.1% (w/w) total substituted oligosaccharides (TSOS) composed of arabinose, glucose, xylose and acetyl group. Bifidobacterium adolescentis and Bifidobacterium catenulatum (health promoting bacteria) were cultured in vitro on M×G XOS and a commercial XOS source, which was used as a comparison. B. adolescentis grew to a higher cell density than B. catenulatum in both XOS cultures. Total xylose consumption for B. adolescentis was 84.1 and 84.8%, respectively for M×G and commercial XOS cultures; and for B. catenulatum was 76.6 and 73.6%, respectively. The xylobiose (X2), xylotriose (X3) and xylotetraose (X4) were almost utilized for both strains. Acetic and lactic acids were the major fermentation products of the XOS cultures.

Comparative lipid production by oleaginous yeasts in hydrolyzates of lignocellulosic biomass and process strategy for high titers.

Oleaginous yeasts can convert sugars to lipids with fatty acid profiles similar to those of vegetable oils, making them attractive for production of biodiesel. Lignocellulosic biomass is an attractive source of sugars for yeast lipid production because it is abundant, potentially low cost, and renewable. However, lignocellulosic hydrolyzates are laden with byproducts which inhibit microbial growth and metabolism. With the goal of identifying oleaginous yeast strains able to convert plant biomass to lipids, we screened 32 strains from the ARS Culture Collection, Peoria, IL to identify four robust strains able to produce high lipid concentrations from both acid and base-pretreated biomass. The screening was arranged in two tiers using undetoxified enzyme hydrolyzates of ammonia fiber expansion (AFEX)-pretreated cornstover as the primary screening medium and acid-pretreated switch grass as the secondary screening medium applied to strains passing the primary screen. Hydrolyzates were prepared at ∼18-20% solids loading to provide ∼110 g/L sugars at ∼56:39:5 mass ratio glucose:xylose:arabinose. A two stage process boosting the molar C:N ratio from 60 to well above 400 in undetoxified switchgrass hydrolyzate was optimized with respect to nitrogen source, C:N, and carbon loading. Using this process three strains were able to consume acetic acid and nearly all available sugars to accumulate 50-65% of cell biomass as lipid (w/w), to produce 25-30 g/L lipid at 0.12-0.22 g/L/h and 0.13-0.15 g/g or 39-45% of the theoretical yield at pH 6 and 7, a performance unprecedented in lignocellulosic hydrolyzates. Three of the top strains have not previously been reported for the bioconversion of lignocellulose to lipids. The successful identification and development of top-performing lipid-producing yeast in lignocellulose hydrolyzates is expected to advance the economic feasibility of high quality biodiesel and jet fuels from renewable biomass, expanding the market potential for lignocellulose-derived fuels beyond ethanol for automobiles to the entire U.S. transportation market. Biotechnol. Bioeng. 2016;113: 1676-1690. © 2016 Wiley Periodicals, Inc.

Irradiation of Yarrowia lipolytica NRRL YB-567 creating novel strains with enhanced ammonia and oil production on protein and carbohydrate substrates.

Increased interest in sustainable production of renewable diesel and other valuable bioproducts is redoubling efforts to improve economic feasibility of microbial-based oil production. Yarrowia lipolytica is capable of employing a wide variety of substrates to produce oil and valuable co-products. We irradiated Y. lipolytica NRRL YB-567 with UV-C to enhance ammonia (for fertilizer) and lipid (for biodiesel) production on low-cost protein and carbohydrate substrates. The resulting strains were screened for ammonia and oil production using color intensity of indicators on plate assays. Seven mutant strains were selected (based on ammonia assay) and further evaluated for growth rate, ammonia and oil production, soluble protein content, and morphology when grown on liver infusion medium (without sugars), and for growth on various substrates. Strains were identified among these mutants that had a faster doubling time, produced higher maximum ammonia levels (enzyme assay) and more oil (Sudan Black assay), and had higher maximum soluble protein levels (Bradford assay) than wild type. When grown on plates with substrates of interest, all mutant strains showed similar results aerobically to wild-type strain. The mutant strain with the highest oil production and the fastest doubling time was evaluated on coffee waste medium. On this medium, the strain produced 0.12 g/L ammonia and 0.20 g/L 2-phenylethanol, a valuable fragrance/flavoring, in addition to acylglycerols (oil) containing predominantly C16 and C18 residues. These mutant strains will be investigated further for potential application in commercial biodiesel production.

Bioabatement with hemicellulase supplementation to reduce enzymatic hydrolysis inhibitors.

A stepwise removal of inhibitory compounds by bioabatement combined with hemicellulase supplementation was conducted to enhance cellulose hydrolysis of liquid hot water-pretreated corn stover. Results showed that the fungus Coniochaeta ligniaria NRRL30616 eliminated most of the enzyme and fermentation inhibitors from liquid hot water-pretreated corn stover hydrolysates. Moreover, addition of hemicellulases after bioabatement and before enzymatic hydrolysis of cellulose achieved 20% higher glucose yields compared to non-treated samples. This work presents the mechanisms by which supplementation of the fungus with hemicellulase enzymes enables maximal conversion, and confirms the inhibitory effect of xylo-oligosaccharides in corn stover hydrolysates once the dominant inhibitory effect of phenolic compounds is removed.

Enhancement of xylose utilization from corn stover by a recombinant Escherichia coli strain for ethanol production.

Effects of substrate-selective inoculum prepared by growing on glucose, xylose, arabinose, GXA (glucose, xylose, arabinose, 1:1:1) and corn stover hydrolyzate (dilute acid pretreated and enzymatically hydrolyzed, CSH) on ethanol production from CSH by a mixed sugar utilizing recombinant Escherichia coli (strain FBR5) were investigated. The initial ethanol productivity was faster for the seed grown on xylose followed by GXA, CSH, glucose and arabinose. Arabinose grown seed took the longest time to complete the fermentation. Delayed saccharifying enzyme addition in simultaneous saccharification and fermentation of dilute acid pretreated CS by the recombinant E. coli strain FBR5 allowed the fermentation to finish in a shorter time than adding the enzyme simultaneously with xylose grown inoculum. Use of substrate selective inoculum and fermenting pentose sugars first under glucose limited condition helped to alleviate the catabolite repression of the recombinant bacterium on ethanol production from lignocellulosic hydrolyzate.

Savagea faecisuis gen. nov., sp. nov., a tylosin- and tetracycline-resistant bacterium isolated from a swine-manure storage pit.

A polyphasic taxonomic study using morphological, biochemical, chemotaxonomic and molecular methods was performed on three strains of a Gram-stain positive, non-sporeforming, motile aerobic rod-shaped bacterium resistant to tylosin and tetracycline isolated from a swine-manure storage pit. On the basis of 16S rRNA gene sequence analyses, it was confirmed that these isolates are highly related to each other and form a hitherto unknown lineage within the Planococcaceae. In particular, pairwise analysis of the 16S rRNA gene sequence demonstrated that the novel organism is closely related to members of the genus Sporosarcina (92.8-94.5 %), Pyschrobacillus (93.5-93.9 %) and Paenisporosarcina (93.3-94.5 %). The predominant fatty acids were found to consist of iso-C15:0 and iso-C17:1 ω10c and the G+C mol% was determined to be 41.8. Based on biochemical, chemotaxonomic, and phylogenetic evidence, it is proposed that these novel strains be classified as a novel genus and species, Savagea faecisuis gen nov., sp. nov. The type strain is Con12(T) (=CCUG 63563(T) = NRRL B-59945(T) = NBRC 109956(T)).

Evolved strains of Scheffersomyces stipitis achieving high ethanol productivity on acid- and base-pretreated biomass hydrolyzate at high solids loading.

BACKGROUND: Lignocellulosic biomass is an abundant, renewable feedstock useful for the production of fuel-grade ethanol via the processing steps of pretreatment, enzyme hydrolysis, and microbial fermentation. Traditional industrial yeasts do not ferment xylose and are not able to grow, survive, or ferment in concentrated hydrolyzates that contain enough sugar to support economical ethanol recovery since they are laden with toxic byproducts generated during pretreatment. RESULTS: Repetitive culturing in two types of concentrated hydrolyzates was applied along with ethanol-challenged xylose-fed continuous culture to force targeted evolution of the native pentose fermenting yeast Scheffersomyces (Pichia) stipitis strain NRRL Y-7124 maintained in the ARS Culture Collection, Peoria, IL. Isolates collected from various enriched populations were screened and ranked based on relative xylose uptake rate and ethanol yield. Ranking on hydrolyzates with and without nutritional supplementation was used to identify those isolates with best performance across diverse conditions. CONCLUSIONS: Robust S. stipitis strains adapted to perform very well in enzyme hydrolyzates of high solids loading ammonia fiber expansion-pretreated corn stover (18% weight per volume solids) and dilute sulfuric acid-pretreated switchgrass (20% w/v solids) were obtained. Improved features include reduced initial lag phase preceding growth, significantly enhanced fermentation rates, improved ethanol tolerance and yield, reduced diauxic lag during glucose-xylose transition, and ability to accumulate >40 g/L ethanol in <167 h when fermenting hydrolyzate at low initial cell density of 0.5 absorbance units and pH 5 to 6.

Pilot scale conversion of wheat straw to ethanol via simultaneous saccharification and fermentation.

The production of ethanol from wheat straw (WS) by dilute acid pretreatment, bioabatement of fermentation inhibitors by a fungal strain, and simultaneous saccharification and fermentation (SSF) of the bio-abated WS to ethanol using an ethanologenic recombinant bacterium was studied at a pilot scale without sterilization. WS (124.2g/L) was pretreated with dilute H2SO4 in two parallel tube reactors at 160°C. The inhibitors were bio-abated by growing the fungus aerobically. The maximum ethanol produced by SSF of the bio-abated WS by the recombinant Escherichia coli FBR5 at pH 6.0 and 35°C was 36.0g/L in 83h with a productivity of 0.43gL(-1)h(-1). This value corresponds to an ethanol yield of 0.29g/g of WS which is 86% of the theoretical ethanol yield from WS. This is the first report on the production of ethanol by the recombinant bacterium from a lignocellulosic biomass at a pilot scale.

Draft Genome Sequences of Streptococcus bovis Strains ATCC 33317 and JB1.

We report the draft genome sequences of Streptococcus bovis strain ATCC 33317 (CVM42251) isolated from cow dung and strain JB1 (CVM42252) isolated from a cow rumen in 1977. The strains were sequenced using the Genome Sequencer FLX 454 system. The genome sizes are approximately 2 Mb and 2.2 Mb, respectively.

Peptoniphilus stercorisuis sp. nov., isolated from a swine manure storage tank and description of Peptoniphilaceae fam. nov.

A species of a previously unknown Gram-positive-staining, anaerobic, coccus-shaped bacterium recovered from a swine manure storage tank was characterized using phenotypic, chemotaxonomic, and molecular taxonomic methods. Comparative 16S rRNA gene sequencing studies and biochemical characteristics demonstrated that this organism is genotypically and phenotypically distinct, and represents a previously unknown sub-line within the order Clostridiales, within the phylum Firmicutes. Pairwise sequence analysis demonstrated that the novel organism clustered within the genus Peptoniphilus, most closely related to Peptoniphilus methioninivorax sharing a 16S rRNA gene sequence similarity of 95.5%. The major long-chain fatty acids were found to be C14:0 (22.4%), C16:0 (15.6%), C16:1ω7c (11.3%) and C16 : 0 ALDE (10.1%) and the DNA G +C content was 31.8 mol%. Based upon the phenotypic and phylogenetic findings presented, a novel species Peptoniphilus stercorisuis sp. nov. is proposed. The type strain is SF-S1(T) ( = DSM 27563(T) = NBRC 109839(T)). In addition, it is proposed to accommodate the genera Peptoniphilus, Anaerococcus, Anaerosphaera, Finegoldia, Gallicola, Helcococcus, Murdochiella and Parvimonas in a new family of the order Clostridiales, for which the name Peptoniphilaceae fam. nov. is proposed; the type genus of the family is Peptoniphilus.

Regulated expression of polysaccharide utilization and capsular biosynthesis loci in biofilm and planktonic Bacteroides thetaiotaomicron during growth in chemostats.

Bacteroides thetaiotaomicron is a prominent member of the human distal gut microbiota that specializes in breaking down diet and host-derived polysaccharides. While polysaccharide utilization has been well studied in B. thetaiotaomicron, other aspects of its behavior are less well characterized, including the factors that allow it to maintain itself in the gut. Biofilm formation may be a mechanism for bacterial retention in the gut. Therefore, we used custom GeneChips to compare the transcriptomes of biofilm and planktonic B. thetaiotaomicron during growth in mono-colonized chemostats. We identified 1,154 genes with a fold-change greater than 2, with confidence greater than or equal to 95%. Among the prominent changes observed in biofilm populations were: (i) greater expression of genes in polysaccharide utilization loci that are involved in foraging of O-glycans normally found in the gut mucosa; and (ii) regulated expression of capsular polysaccharide biosynthesis loci. Hierarchical clustering of the data with different datasets, which were obtained during growth under a range of conditions in minimal media and in intestinal tracts of gnotobiotic mice, revealed that within this group of differentially expressed genes, biofilm communities were more similar to the in vivo samples than to planktonic cells and exhibited features of substrate limitation. The current study also validates the use of chemostats as an in vitro "gnotobiotic" model to study gene expression of attached populations of this bacterium. This is important to gut microbiota research, because bacterial attachment and the consequences of disruptions in attachment are difficult to study in vivo.

Conversion of switchgrass to ethanol using dilute ammonium hydroxide pretreatment: influence of ecotype and harvest maturity.

Switchgrass (Panicum virgatum L.) is a perennial C4 grass that is being developed as a bioenergy crop because it has high production yields and suitable agronomic traits. Five switchgrass biomass samples from upland and lowland switchgrass ecotypes harvested at different stages or maturity were used in this study. Switchgrass samples contained 317.0-385.0 g glucans/kg switchgrass dry basis (db) and 579.3-660.2 g total structural carbohydrates/kg switchgrass, db. Carbohydrate contents were greater for the upland ecotype versus lowland ecotype and increased with harvest maturity. Pretreatment of switchgrass with dilute ammonium hydroxide (8% w/w ammonium loading) at 170 degrees C for 20 min was determined to be effective for preparing switchgrass for enzymatic conversion to monosaccharides; glucose recoveries were 66.9-90.5% and xylose recoveries 60.1-84.2% of maximum and decreased with increased maturity at harvest. Subsequently, pretreated switchgrass samples were converted to ethanol by simultaneous saccharification and fermentation using engineered xylose-fermenting Saccharomyces cerevisiae strain YRH400. Ethanol yields were 176.2-202.01/Mg of switchgrass (db) and followed a similar trend as observed for enzymatic sugar yields.

Growth and fermentation of D-xylose by Saccharomyces cerevisiae expressing a novel D-xylose isomerase originating from the bacterium Prevotella ruminicola TC2-24.

BACKGROUND: Saccharomyces cerevisiae strains expressing D-xylose isomerase (XI) produce some of the highest reported ethanol yields from D-xylose. Unfortunately, most bacterial XIs that have been expressed in S. cerevisiae are either not functional, require additional strain modification, or have low affinity for D-xylose. This study analyzed several XIs from rumen and intestinal microorganisms to identify enzymes with improved properties for engineering S. cerevisiae for D-xylose fermentation. RESULTS: Four XIs originating from rumen and intestinal bacteria were isolated and expressed in a S. cerevisiae CEN.PK2-1C parental strain primed for D-xylose metabolism by over expression of its native D-xylulokinase. Three of the XIs were functional in S. cerevisiae, based on the strain's ability to grow in D-xylose medium. The most promising strain, expressing the XI mined from Prevotella ruminicola TC2-24, was further adapted for aerobic and fermentative growth by serial transfers of D-xylose cultures under aerobic, and followed by microaerobic conditions. The evolved strain had a specific growth rate of 0.23 h-1 on D-xylose medium, which is comparable to the best reported results for analogous S. cerevisiae strains including those expressing the Piromyces sp. E2 XI. When used to ferment D-xylose, the adapted strain produced 13.6 g/L ethanol in 91 h with a metabolic yield of 83% of theoretical. From analysis of the P. ruminicola XI, it was determined the enzyme possessed a Vmax of 0.81 µmole/min/mg protein and a Km of 34 mM. CONCLUSION: This study identifies a new xylose isomerase from the rumen bacterium Prevotella ruminicola TC2-24 that has one of the highest affinities and specific activities compared to other bacterial and fungal D-xylose isomerases expressed in yeast. When expressed in S. cerevisiae and used to ferment D-xylose, very high ethanol yield was obtained. This new XI should be a promising resource for constructing other D-xylose fermenting strains, including industrial yeast genetic backgrounds.

Dilute sulfuric acid pretreatment of corn stover for enzymatic hydrolysis and efficient ethanol production by recombinant Escherichia coli FBR5 without detoxification.

A pretreatment strategy for dilute H2SO4 pretreatment of corn stover was developed for the purpose of reducing the generation of inhibitory substances during pretreatment so that a detoxification step is not required prior to fermentation while maximizing sugar yield. The optimal conditions for pretreatment of corn stover (10%, w/v) were: 0.75% H2SO4, 160°C, and 0-5 min holding time. The conditions were chosen based on maximum glucose release after enzymatic hydrolysis, minimum loss of pentose sugars and minimum formation of sugar degradation products such as furfural and hydroxymethyl furfural. The pretreated corn stover after enzymatic saccharification generated 63.2 ± 2.2 and 63.7 ± 2.3 g total sugars per L at 0 and 5 min holding time, respectively. Furfural production was 0.45 ± 0.1 and 0.87 ± 0.4 g/L, respectively. The recombinant Escherichia coli strain FBR5 efficiently fermented non-detoxified corn stover hydrolyzate if the furfural content is <0.5 g/L.

Erratum to: Two novel species Enterococcus lemanii sp. nov. and Enterococcus eurekensis sp. nov., isolated from a swine-manure storage pit.

A polyphasic taxonomic study using morphological, biochemical, chemotaxonomic and molecular genetic methods was performed on six strains of an unknown Gram-positive, nonspore-forming, facultative anaerobic coccus-shaped bacterium isolated from a swine-manure storage pit. On the basis of 16S rRNA, RNA polymerase-subunit (rpoA), and the 60-kilodalton chaperonin (cpn60) gene sequence analyses, it was shown that all the isolates were enterococci but formed two separate lines of descent. Pairwise 16S rRNA sequence comparisons demonstrated that the two novel organisms were most closely related to each other (97.9 %) and to Enterococcus aquimarinus (97.8 %). Both organisms contained major amounts of C16:0, C16:1 ω7c, and C18:1 ω7c/12t/9t as the major cellular fatty acids. Based on biochemical, chemotaxonomic, and phylogenetic evidence, the names Enterococcus lemanii sp. nov. (type strain PC32(T) = CCUG 61260(T) = NRRL B-59661(T)) and Enterococcus eurekensis sp. nov. (type strain PC4B(T) = CCUG 61259(T) = NRRL B-59662(T)) are proposed for the hitherto undescribed species.

Response surface optimization of corn stover pretreatment using dilute phosphoric acid for enzymatic hydrolysis and ethanol production.

Dilute H(3)PO(4) (0.0-2.0%, v/v) was used to pretreat corn stover (10%, w/w) for conversion to ethanol. Pretreatment conditions were optimized for temperature, acid loading, and time using central composite design. Optimal pretreatment conditions were chosen to promote sugar yields following enzymatic digestion while minimizing formation of furans, which are potent inhibitors of fermentation. The maximum glucose yield (85%) was obtained after enzymatic hydrolysis of corn stover pretreated with 0.5% (v/v) acid at 180°C for 15min while highest yield for xylose (91.4%) was observed from corn stover pretreated with 1% (v/v) acid at 160°C for 10min. About 26.4±0.1g ethanol was produced per L by recombinant Escherichia coli strain FBR5 from 55.1±1.0g sugars generated from enzymatically hydrolyzed corn stover (10%, w/w) pretreated under a balanced optimized condition (161.81°C, 0.78% acid, 9.78min) where only 0.4±0.0g furfural and 0.1±0.0 hydroxylmethyl furfural were produced.

Two novel species Enterococcus lemanii sp. nov. and Enterococcus eurekensis sp. nov., isolated from a swine-manure storage pit.

A polyphasic taxonomic study using morphological, biochemical, chemotaxonomic and molecular genetic methods was performed on six strains of unknown Gram-positive, nonspore-forming, facultative anaerobic coccus-shaped bacteria isolated from a swine-manure storage pit. On the basis of the 16S rRNA, RNA polymerase α-subunit (rpoA) and 60 kDa chaperonin (cpn60) gene sequence analyses, it was shown that all the isolates were enterococci but formed two separate lines of descent. Pairwise 16S rRNA gene sequence comparisons demonstrated that the two novel organisms were most closely related to each other (97.9 %) and to Enterococcus aquimarinus (97.8 %). Both organisms contained major amounts of C(16:0), C(16:1) ω7c, C(16:1) ω7c, and C(18:1) ω7c/12t/9t as the major cellular fatty acids. Based on biochemical, chemotaxonomic and phylogenetic evidence, the names Enterococcus lemanii sp. nov. (type strain PC32(T) = CCUG 61260(T) = NRRL B-59661(T)) PPC27A = CCUG 61369; PPC38 = CCUG 61261 [corrected] and Enterococcus eurekensis sp. nov. (type strain PC4B(T) = CCUG 61259(T) = NRRL B-59662(T)) PPC15 = CCUG 61368; PPC107 = CCUG 61372 [corrected] are proposed for these hitherto undescribed species.