Cellulomonas Carbonis Sp. Nov., Isolated from Coal Mine Soil

%paper no. ije034934 charlesworth ref: ije034934& New Taxa - Actinobacteria

International Journal of Systematic and Evolutionary Microbiology (2012), 62, 000–000 DOI 10.1099/ijs.0.034934-0

Cellulomonas carbonis sp. nov., isolated from coal mine soil

Zunji Shi, Guosheng Luo and Gejiao Wang

Correspondence State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Gejiao Wang Huazhong Agricultural University, Wuhan, Hubei 430070, PR China [email protected] or [email protected] A Gram-positive, aerobic, motile, rod-shaped bacterium, designated strain T26T, was isolated from subsurface soil of Tianjin coal mine, China. Colonies were yellow–white, convex, circular, smooth and non-transparent on R2A agar. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain T26T was closely related to members of the genus Cellulomonas and a member of the genus Actinotalea with 96.8–94.7 % and 96.7 % gene sequence similarities, T respectively. The peptidoglycan type of strain T26 was A4b, containing L-ornithine–D-glutamic acid as the interpeptide bridge. The cell-wall sugars were rhamnose, galactose, xylose and

inositol. The major fatty acids (.10 %) were anteiso-C15 : 0 (33.6 %), anteiso-C15 : 1 A (22.1 %),

C16 : 0 (14.4 %) and C14 : 0 (12.1 %). The predominant respiratory quinone was MK-9(H4) and the genomic DNA G+C content was 74.4 mol%. The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol-mannosides and phosphatidylinositol. Comparison of phenotypic and phylogenetic characteristics between strain T26T and related organisms revealed that the new isolate represented a novel species of the genus Cellulomonas, for which the name Cellulomonas carbonis sp. nov. is proposed. The type strain is T26T (5CGMCC 1.10786T5KCTC 19824T5CCTCC AB2010450T).

The family Cellulomonadaceae was originally defined by 2008), blood (Brown et al., 2005), cerebrospinal fluid Stackebrandt & Prauser (1991) and included the genera (Funke et al., 1995), a decayed elm tree (Rivas et al., 2004), Cellulomonas (18 species), Oerskovia (4 species) and sediment (Jones et al., 2005), air (Lee et al., 2008) and reed Actinotalea (1 species). The genus Cellulomonas was first periphyton (Rusznya´k et al., 2011), respectively. The described by Bergey et al. (1923) and later emended by typical characteristics of members of the genus Clark (1952) and Stackebrandt et al. (2006). At the time of Cellulomonas are Gram-positive, slender irregular rods writing, the 18 recognized species of the genus with cellulolytic activity, containing menaquinone MK- Cellulomonas were Cellulomonas flavigena (type species), 9(H4) as the predominant respiratory quinone, having C. biazotea, C. cellasea, C. fimi, C. gelida, C. uda (Bergey et anteiso-C15 : 0 and C16 : 0 as the major fatty acids al., 1923), C. hominis (Funke et al., 1995), C. humilata (Stackebrandt et al., 2006) and with a high DNA G+C (Collins & Pascual, 2000), C. iranensis, C. persica (Elberson content (68.5–76.0 mol%). Other diagnostic characteristics et al., 2000), C. xylanilytica (Rivas et al., 2004), C. terrae for the genus Cellulomonas include rhamnose (Rha) as the (An et al., 2005), C. denverensis (Brown et al., 2005), C. major cell-wall sugar in most of the strains, except for C. bogoriensis (Jones et al., 2005), C. composti (Kang et al., gelida, C. uda, C. composti (Kang et al., 2007) and C. 2007), C. aerilata (Lee et al., 2008), C. chitinilytica (Yoon et aerilata (Lee et al., 2008) (data are not available for C. al., 2008) and C. phragmiteti (Rusznya´k et al., 2011). These hominis, C. bogoriensis and C. phragmiteti). The diagnostic species were mostly isolated from soil (Collins & Pascual, diamino acid in position 3 of the peptide subunit of the 2000; Elberson et al., 2000; An et al., 2005). Others were peptidoglycan is ornithine (Orn) with the interpeptide isolated from compost (Kang et al., 2007; Yoon et al., bridge containing either D-aspartic acid (D-Asp) (for C. flavigena, C. persica, C. bogoriensis, C. iranensis and C. phragmiteti)orD-glutamic acid (D-Glu) (for all the other Abbreviations: DPG, diphosphatidylglycerol; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PI, 13 species) (Stackebrandt et al., 2006). The polar lipids phosphatidylinositol; PIM, phosphatidylinositol mannosides; PL, have been reported for six species of the genus phospholipid; PGL, phosphoglycolipid. Cellulomonas and included diphosphatidylglycerol (DPG) The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene and phosphatidylglycerol (PG) (for C. aerilata and C. sequence of strain T26T is HQ702749. chitinilytica), PG (for C. bogoriensis), DPG, phosphatidy- Four supplementary figures are available with the online version of this linositol (PI) and phosphatidylethanolamine (PE) (for C. paper. composti), DPG and PI (for C. flavigena) and DPG, PG, PI,

034934 G 2012 IUMS Printed in Great Britain 1

%paper no. ije034934 charlesworth ref: ije034934&

Cellulomonas carbonis sp. nov.

Cells of strain T26T were Gram-staining-positive, aerobic, in this study and showed very similar polar lipids to strain motile and rod-shaped. Colonies were yellow–white, T26T (Fig. S4). The polar lipid data and 16S rRNA gene convex, circular, smooth and non-transparent after 3 days sequence phylogenies of both strain T26T and C. bogoriensis incubation on R2A agar at 28 uC. The strain grew on R2A DSM 16987T were consistent with their assignment to the agar, nutrient agar, full-strength TSA and LB agar, but did genus Cellulomonas. The DNA G+C content of strain not grow on MacConkey agar. Detailed results of the T26T was 74.4 mol%. The cell-wall peptidoglycan type was morphological, physiological and biochemical character- A4b, containing L-Orn–D-Glu. The cell-wall sugars were istics are given in the species description. A scanning rhamnose (80.9 %), galactose (3.3 %), xylose (5.3 %) and electron and transmission electron micrographs showing inositol (6.3 %). All of these results for strain T26T were the general morphology of cells of strain T26T are available very similar to those of the type species of the genus as Fig. S1a and b, respectively. The main phenotypic Cellulomonas, C. flavigena DSM 20109T, and to other differences between strain T26T and closely related type species of the genus. Strain T26T could be differentiated strains are shown in Table 1. from members of the genera Actinotalea and Oerskovia on T the basis of differences in the respiratory quinone [MK- The 1442 bp 16S rRNA gene sequence of strain T26 was ] analysed to determine the phylogenetic position of the 9(H4) vs MK-10(H4) and peptidoglycan type (L-Orn–D- novel strain. Strain T26T shared gene sequence similarities Glu vs L-Lys–L-ThrrD-Asp or L-Lys–L-ThrrD-Glu), of 96.8–94.7 % with other species of the genus respectively. Cellulomonas and a member of the genus Actinotalea. On the basis of the close relationship and distinctive T Strain T26 showed the highest 16S rRNA gene sequence phenotypic and phylogenetic differences between strain T similarities with C. cellasea DSM 20118 (96.8 %), A. T26T and other closely related species, it is proposed that T T fermentans DSM 3133 (96.7 %), C. hominis DMMZ CE40 strain T26T represents a novel species of Cellulomonas, with T (96.6 %), C. denverensis W6929 (96.5 %) and C. the name Cellulomonas carbonis sp. nov. bogoriensis 69B4T (96.4 %). A phylogenetic tree constructed using the neighbour-joining algorithm showed that strain Description of Cellulomonas carbonis sp. nov. T26T was always very closely associated with C. bogoriensis 69B4T; other species of the genus Cellulomonas fell into Cellulomonas carbonis (car.bo9nis. L. gen. n. carbonis of other nearby clusters, but all in a large cluster (Fig. 1). This coal, of charcoal). cluster also included four strains of the genus Oerskovia T T Cells are Gram-positive-staining, aerobic, motile and rod- and Paraoerskovia marina CTT-37 , however, strain T26 shaped (0.5–0.862.0–2.4 mm). Colonies are yellow–white, was distinct from members of the genus Oerskovia based convex, circular, smooth and non-transparent after on the peptidoglycan structure (see below). In addition, P. u T T incubation on R2A agar at 28 C for 3 days. Grows on marina CTT-37 could be distinguised from strain T26 on R2A agar, nutrient agar, full-strength TSA and LB agar, but the basis of the cellular fatty acid content and peptidogly- does not grow on MacConkey agar. Temperature range for can structure (Khan et al., 2009). The maximum- growth is 4–45 uC, and optimal temperature for growth is parsimony and the maximum-likelihood trees (Fig. S2a, 28 uC. Growth occurs with NaCl concentrations in the b) also supported the phylogenetic position obtained with range 0–7 % and with pH 6–10 (optimum, pH 7.0). the neighbour-joining algorithm. Catalase-positive and oxidase-negative. Positive in tests Strain T26T gave a negative result in tests for urease activity for b-galactosidase activity but negative result for arginine but a positive result for the hydrolysis of carboxymethyl dihydrolase and urease. Hydrolyses carboxymethyl cel- cellulose and aesculin. The major cellular fatty acids lulose, starch, gelatin, aesculin, but not DNA, casein, (.10 %) were anteiso-C15 : 0 (33.6 %), anteiso-C15 : 1 A tyrosine or Tween 80. Does not produce indole from (22.1 %), C16 : 0 (14.4 %) and C14 : 0 (12.1 %) on R2A tryptophan, but H2S is produced from cysteine and NH3 is medium (details are shown in Table 2). The fatty acid produced from peptone. Positive in tests for nitrate profile of the novel strain was very similar to that of the reduction but negative result in methyl-red and Voges– type species of the genus Cellulomonas, C. flavigena DSM Proskauer tests. Assimilates D-glucose, L-arabinose, man- T 20109 . The major respiratory quinone was MK-9(H4) nose, N-acetylglucosamine, maltose, gluconate, sucrose, (Fig. S3), which was the same as that of members of the glycogen, salicin, melibiose, D-sorbitol, xylose, lactose, D- genus Cellulomonas and was different from A. fermentans galactose, D-fructose and raffinose. Does not assimilate L- which contained MK-10(H4) as the major respiratory rhamnose, D-ribose, inositol, mannitol, L-fucose, L-serine, quinone (Yi et al., 2007). Strain T26T exhibited similar L-proline, L-alanine, L-histidine, capric acid, adipic acid, major polar lipids, DPG and PG, when compared with malic acid, citric acid, phenylacetic acid, trisodium citrate, other type strains of the genus Cellulomonas. In addition, itaconic acid, suberate, sodium malonate, sodium acetate, strain T26T also contained phosphatidylinositol manno- lactate, propionate, caprate, valerate, potassium 5-ketoglu- sides (PIM), phosphatidylinositol (PI), an unknown conate, 3-hydroxybenzoate, potassium 2-ketogluconate, 3- phospholipid (PL) and an unknown phosphoglycolipid hydroxybutyrate or 4-hydroxybenzoate (API 20NE and (PGL) (Fig. S4). C. cellasea DSM 20118T, C. bogoriensis API ID 32 GN). In API ZYM tests, positive result for DSM 16987T and A. fermentans DSM 3133T were analysed esterase (C4), acid phosphatase, a-galactosidase, b-galacto- http://ijs.sgmjournals.org 3

%paper no. ije034934 charlesworth ref: ije034934&

Cellulomonas carbonis sp. nov.

74 Cellulomonas bogoriensis DSM 69B4T (X92152) Cellulomonas carbonis T26T (HQ702749) Cellulomonas gelida DSM 20111T (X83800) Cellulomonas uda DSM 20107T (X83801) Cellulomonas iranensis OT (AF064702) 95 Cellulomonas composti TR7-06T (AB166887) Cellulomonas phragmiteti KB23T (AM902253) IT (AF064701) 70 Cellulomonas persica Cellulomonas flavigena DSM 20109T (X83799) Cellulomonas aerilata 5420S-23T (EU560979) Cellulomonas cellasea DSM 20118T (X83804) 85 Cellulomonas chitinilytica X.bu-bT (AB268586) Cellulomonas biazotea DSM 20112T (X83802) 100 Cellulomonas fimi DSM 20113T (X79460) Cellulomonas terrae DB5T (AY884570) Cellulomonas humilata ATCC 25174T (X82449) 100 Cellulomonas xylanilytica XIL11T (AY303668) 99 Cellulomonas hominis DMMZ CE40T (X82598) Cellulomonas denverensis W6929T (AY501362) 75 Oerskovia jenensis DSM 46000T (AJ314848) 95 Oerskovia paurometabola DSM 14281T (AJ314851) 100 Oerskovia turbata DSM 20577T (X83806) Oerskovia enterophila DSM 43852T (X83807) Paraoerskovia marina CTT-37T (AB445007) Actinotalea fermentans DSM 3133T (X83805) Pseudoclavibacter soli KP02T (AB329630)

T 99 Pseudoclavibacter chungangensis CAU59 (FJ514934) 100 Pseudoclavibacter helvolus DSM 20419T (X77440) 100 Demequina salsinemoris KV-810T (AB471559) Demequina globuliformis YM24-125T (AB522643)

100 Demequina aestuarii JC2054T (DQ010160) SV45T (EF451745) 0.01 Demequina lutea Demequina oxidasica YM05-1041T (AB522640) 100 Demequina aurantiaca YM12-102T (AB522641)

Fig. 1. A neighbour-joining tree based on 16S rRNA gene sequences showing the phylogenetic position of strain T26T. Bootstrap values (.70 %) based on 1000 replications are shown at branch nodes. Bar, 10 substitutions per 1000 nt. sidase, a-glucosidase, b-glucosidase, naphthol-AS-BI-phos- (30 mg), cefoxitin (30 mg), kanamycin (30 mg), streptomy- phohydrolase, N-acetyl-b-glucosaminidase and leucine cin (10 mg), tobramycin (10 mg), vancomycin (30 mg), arylamidase activities, but negative result for alkaline ofloxacin (5 mg), norfloxacin (10 mg), erythromycin phosphatase, esterase lipase (C8), lipase (C14), trypsin, (15 mg), minocin (30 mg), tetracycline (30 mg), neomycin valine arylamidase, a-chymotrypsin, cystine arylamidase, (30 mg), amoxicillin (30 mg) and nitrofurantoin (300 mg). b-fucosidase, b-glucuronidase and a-mannosidase. Acid is The peptidoglycan type is A4b, containing L-ornithine–D- produced from D-glucose, cellobiose, D-fructose, D-man- glutamic acid. Cell-wall sugars are rhamnose, galactose, nose, D-galactose, maltose, sucrose, D-xylose, trehalose, xylose and inositol. Major fatty acids are anteiso-C15 : 0, lactose, raffinose, turanose, L-arabinose, D-sorbitol, meli- C16 : 0,C14 : 0 and anteiso-C15 : 1 A. The major respiratory biose and N-acetylglucosamine, but not from D-mannitol, quinone is MK-9(H4). Major polar lipids are DPG, PG, D-ribose, D-arabinose, inositol, L-sorbose, melezitose, L- PIM and PI. rhamnose, ribitol or propylene glycol. Sensitive to poly- The type strain, T26T (5CGMCC 1.10786T5KCTC myxin B (25 mg), novobiocin (5 mg), teicoplanin (30 mg), 19824T5CCTCC AB2010450T), was isolated from coal cefalotin (30 mg), chloramphenicol (30 mg), rifampicin mine soil from Tianjin, China. The G+C content of the (5 mg), ampicillin (10 mg), carbenicillin (100 mg), lincomy- genomic DNA of the type strain is 74.4 mol%. cin (2 mg), cephalosporin V (30 mg), cephalosporin IV http://ijs.sgmjournals.org 5 %paper no. ije034934 charlesworth ref: ije034934&

Z. Shi, G. Luo and G. Wang

Table 2. Cellular fatty acid contents (%) of strain T26T and some type strains of the genera Cellulomonas and Actinotalea

Taxa: 1, strain T26T;2,A. fermentans DSM 3133T;3,C. cellasea DSM 20118T;4,C. bogoriensis DSM 16987T;5,C. flavigena DSM 20109T;6,C. hominis DSM 9581T;7,C. gelida DSM 20111T;8,C. uda DSM 20107T;9,C. biazotea DSM 20112T; 10, C. fimi DSM 20113T; 11, C. terrae KCTC 19081T; 12, C. humilata ATCC 25174T; 13, C. xylanilytica LMG 21723T; 14, C. chitinilytica DSM 17922T; 15, C. composti KCTC 19030T; 16, C. aerilata DSM 18649T; 17, C. phragmiteti DSM 22512T. Data for strains 1–5 are from this study. Data for taxa 6–17 are from Funke et al. (1995), Kang et al. (2007), Yoon et al. (2008), Lee et al. (2008) and Rusznya´k et al. (2011). Data are not available for C. iranensis ATCC 700643T, C. persica ATCC 700642T or C. denverensis ATCC BAA-788T.–,,1 % or not detected.

Fatty acid 1 2 3 4567891011121314151617

C13 : 0 – – – – 1.8 1.0 2.0 1.0 – – – – – – – – – anteiso-C13 : 0 1.22.51.11.0––––1.3– – – – –––– C14 : 0 12.1 20.4 4.1 9.6 12.1 8.0 4.0 4.0 4.0 7.3 3.6–3.8 1.0–1.8 4.6–4.8 – 9.0 3.9 6.8 iso-C14 : 0 5.3 7.9 10.8 – 3.4 1.0 2.0 2.0 8.9 4.6 5.1–5.9 7.8–8.9 7.7–9.6 3.0 – 5.1 1.6 anteiso-C15 : 1 22.1 – 9.3 4.1 21.5 – – – 2.2 2.1 – 2.4–3.3 1.9–2.6 – – 9.2 19.0 A

C15 : 0 1.6 – 1.4 1.7 6.8 8.0 8.0 8.0 – – – – – – – – 2.0 iso-C15 : 0 1.2 – 3.8 2.0 2.6 2.0 4.0 4.0 7.4 – 15.2–18.5 10.9–12.2 18.6–22.1 3.0 – – 2.5 anteiso-C15 : 0 33.6 35.1 51.9 42.5 34 42.0 43.0 43.0 41.2 44.9 44.5–45.6 32.8–44.9 40.1–45.5 61.3 44.0 49.7 37.8 C16 : 0 14.4 27.6 5.2 30.5 12.6 16.0 17.0 17.0 9.6 12.7 8.0–9.5 11.2–12.8 9.5–10.6 2.9 14.6 20.0 23.3 iso-C16 : 0 6.1 6.2 9.6 2.1 1.5 3.0 3.0 3.0 8.8 7.6 – 1.7–7.0 1.8–2.3 1 – 6.7 1.6 C17 : 0 –––––4.02.02.03.7–1.8–2.3 1.2–2.1 1.8–2.7 2.4 – – – iso-C17 : 0 ––––––1.01.01.2– – 0.8–1.6 – 5.4 – – – anteiso-C17 : 0 1.4 – 1.9 3.9 1.1 7.0 7.0 7.0 8.4 12.9 7.4–7.6 11.6–13.7 5.5–6.6 15.9 – 4.3 3.4 C18 : 0 –––––3.01.01.01.95.47.1–8.2 0.8–1.8 10.7–13.0 3 7.9 – –

Acknowledgements Cowan, S. T. & Steel, K. J. (1965). Manual for the Identification of Medical Bacteria. UK: Cambridge University Press. We are grateful to the DSMZ for providing the type strains C. cellasea DSM 20118T, C. bogoriensis DSM 16987T, C. flavigena DSM 20109T Dussault, H. P. (1955). An improved technique for staining red and A. fermentans DSM 3133T and to Prof. Jean Euze´by (E´ cole halophilic bacteria. J Bacteriol 70, 484–485. National Ve´te´rinaire, France) for the etymology of the species name. Elberson, M. A., Malekzadeh, F., Yazdi, M. T., Kameranpour, N., This work was supported by a Major International Collaborative Noori-Daloii, M. R., Matte, M. H., Shahamat, M., Colwell, R. R. & Project of National Natural Science Foundation of China Sowers, K. R. (2000). Cellulomonas persica sp. nov. and Cellulomonas (31010103903). iranensis sp. nov., mesophilic cellulose-degrading bacteria isolated from forest soils. Int J Syst Evol Microbiol 50, 993–996. Fan, H., Su, C., Wang, Y., Yao, J., Zhao, K., Wang, Y. & Wang, G. References (2008). Sedimentary arsenite-oxidizing and arsenate-reducing bacteria associated with high arsenic groundwater from Shanyin, An, D.-S., Im, W.-T., Yang, H.-C., Kang, M. S., Kim, K. K., Jin, L., Kim, Northwestern China. J Appl Microbiol 105, 529–539. M. K. & Lee, S.-T. (2005). Cellulomonas terrae sp. nov., a cellulolytic and xylanolytic bacterium isolated from soil. Int J Syst Evol Microbiol Felsenstein, J. (1981). Evolutionary trees from DNA sequences: a 55, 1705–1709. maximum likelihood approach. J Mol Evol 17, 368–376. Bagnara, C., Toci, R., Gaudin, C. & Belaich, J. P. (1985). Isolation and Fitch, W. M. (1971). Toward defining the course of evolution: characterization of a cellulolytic microorganism, Cellulomonas minimum change for a specific tree topology. Syst Zool 20, 406–416. fermentans sp. nov. Int J Syst Bacteriol 35, 502–507. Funke, G., Ramos, C. P. & Collins, M. D. (1995). Identification of Bergey, D. H., Harrison, F. C., Breed, R. S., Hammer, B. W. & some clinical strains of CDC coryneform group A-3 and A-4 bacteria Huntoon, F. M. (editors) (1923). Bergey’s Manual of Determinative as Cellulomonas species and proposal of Cellulomonas hominis sp. nov. Bacteriology. Baltimore, MD: Williams and Wilkins. for some group A-3 strains. J Clin Microbiol 33, 2091–2097. Brown, J. M., Frazier, R. P., Morey, R. E., Steigerwalt, A. G., Pellegrini, Guindon, S., Lethiec, F., Duroux, P. & Gascuel, O. (2005). PHYML G. J., Daneshvar, M. I., Hollis, D. G. & McNeil, M. M. (2005). Online–a web server for fast maximum likelihood-based phylogenetic Phenotypic and genetic characterization of clinical isolates of CDC inference. Nucleic Acids Res 33 (Web Server issue, ), W557–W559. coryneform group A-3: proposal of a new species of Cellulomonas, Hinz, K. H., Ryll, M. & Ko¨ hler, B. (1998). Detection of acid production Cellulomonas denverensis sp. nov. J Clin Microbiol 43, 1732–1737. from carbohydrates by Riemerella anatipestifer and related organisms Cappuccino, J. G. & Sherman, N. (2002). Microbiology: a Laboratory using the buffered single substrate test. Vet Microbiol 60, 277–284. Manual, 6th edn. Menlo Park, CA: Benjamin/Cummings. Jones, B. E., Grant, W. D., Duckworth, A. W., Schumann, P., Weiss, N. Clark, F. E. (1952). The generic classification of the soil corynebac- & Stackebrandt, E. (2005). Cellulomonas bogoriensis sp. nov., an teria. Int Bull Bacteriol NomTax 2, 45–56. alkaliphilic cellulomonad. Int J Syst Evol Microbiol 55, 1711–1714. Collins, M. D. & Pascual, C. (2000). Reclassification of Actinomyces Kang, M. S., Im, W. T., Jung, H. M., Kim, M. K., Goodfellow, M., Kim, K. humiferus (Gledhill and Casida) as Cellulomonas humilata nom. K., Yang, H. C., An, D. S. & Lee, S. T. (2007). Cellulomonas composti sp. corrig., comb. nov. Int J Syst Evol Microbiol 50, 661–663.

6 International Journal of Systematic and Evolutionary Microbiology 62

Dear Authors,

Please find enclosed a proof of your article for checking.

When reading through your proof, please check carefully authors’ names, scientific data, data in tables, any mathematics and the accuracy of references. Please do not make any unnecessary changes at this stage. All necessary corrections should be marked on the proof at the place where the correction is to be made; please write the correction clearly in the margin (if in the text they may be overlooked).

Any queries that have arisen during preparation of your paper for publication are listed below and indicated on the proof. Please provide your answers when returning your proof.

Please return your proof by email ([email protected]) or Fax [+44 (0)118 988 1834] within 2 days of receipt.

Query no. Query

1 Author: I have changed ‘Na-lactate’ to the chemical formula for sodium lactate so that this list is consistent. Please can you check the formula that I have added?

2 The in-text citation "Weeger et al., 1999" was missing from your reference list. I have added a reference that I think is correct. Please can you check this carefully?

3 Author: I have added this reference as it was missing from your reference list. Please can you check that this is the correct reference and amend if necessary?

4 Author. I could not find any data that had this symbol in your table. Please can you check the data carefully and add this symbol where needed or delete this footnote to the table. SGM reprint ordering system

As a result of declining reprint orders and feedback from many authors who tell us they have no use for their free reprints, SGM has decided to phase out our practice of sending 25 free reprints to all corresponding authors. Authors of articles published in 2013 onwards will no longer receive free reprints.

When the final version of this article has been authorized for printing, you will receive an email containing a link to the SGM Reprint Service. You can forward this email to your co-authors if you wish, so that they can order their own reprints directly, or to your finance or purchasing department, if orders are placed centrally.

When you click on the link in the email, you will be taken to an order page to place your reprint order. Like most online ordering sites, it will be necessary to set up an account and provide a delivery address while placing your order, if you do not already have an account. Once an account and delivery address have been set up, these details will be stored by the system for use with future orders. Payments can be made by credit card, PayPal or purchase order.

For articles that are published in 2012, authors will be provided with a discount code that will allow them to order 25 free reprints, as well as any additional reprints they wish to purchase. This code will be valid for 90 days, and applies only to the paper for which it was issued. As reprint orders are despatched by courier, there is a charge for postage and packing, even on orders that consist only of free reprints.

SUMMARY N You can create or update your reprint account at any time at http://sgm-reprints.charlesworth.com/ N You will be sent an email when the reprints of this paper are ready for ordering N You cannot order reprints of this paper before this email has been sent, as your paper will not be in the system N You will also receive a discount code that will allow you to order 25 free reprints (postage & packing applies) N Reprints can be ordered at any time after publication, although the discount code is only valid for 90 days

The ordering details and discount code will be emailed to the author listed as the corresponding author on the journal’s manuscript submission system. If your paper has been published (the final version, not the publish-ahead-of-print version) but you have not received your discount code, email [email protected] quoting the journal, paper number and publication details.

Corresponding authors will also receive an email including a link to download the published PDF of their paper. You can forward this link to co-authors or others, and they can also use it to download the published PDF. The link can be used up to 25 times. This email will be sent out at around the time your rticle is published online.

If you have any questions or comments about the new reprint-ordering system or about the link to your published paper, email [email protected]