J. Gen. App!. Microbic!. Vol. 5. No. 3 1959 " TAXONOMIC STUDIES ON THE GENUS PEDIOCOCCUS " ATSUSHI NAKAGAWA* and KAKUO KITAHARA Division of Zymomycology Receivedfor publicationApril 6. 1959 Since BALCKE(1) first named the tetrads-forming cocci in spoiled beer as Pediococcus ceyevisiae, various specific names (2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12) have been applied by many investigators to those strains of pedio- cocci associated with brewery products. In recent years, PEDERSON(13, 14) recognized the importance of pe- diococci in fermenting vegetables such as sauerkraut and pickles, and attempted the classification of many pediococci. According to the opinion of PEDERSONappearing in BERGEY'S Manual (15), the genus Pediococcus is included in the family Lactobacillaceae and is summarized into two species : Pediococcus ceyevisiae BALCKEand Pedio- coccus acidilactici LINDNER. The former species which is capable of growing in beer and hopped wort includes pediococci distributing in beer as well as in fermenting vegetables. But this opinion is still a debatable problem and is not yet settled today. More recently, two curious species were described by Japanese workers. One is mevalonic acid requiring strain, Pediococcus mevalovorus, which has been isolated by KITAHARA& NAKAGAWA(16) from beer, and the other is strongly halophilic Pediococcus soyae isolated from soy-mashes by SAKAGUCHI (17) and YAMAZATO(18) independently at almost the same time, which can- not be included into any of the above mentioned species. We have therefore attempted taxonomic studies on the pediococci iso- lated from mash, yeasts and beer including our unpublished strains and also on the authentic strains supplied by many investigators. As a conclusion, we have found that the pediococci growing in beer are quite different from those of fermenting vegetables in many taxonomic properties, and we wish to propose that the genus Pediococcus should at least be classified into five species, i.e., P. ceyevisiae, P. acidilactici, P. pentosaceus, P. halophilus and P. urinae-equi. The taxonomic properties of these species and the reason for classifying them into five species are described in herein as follows. EXPERIMENTAL 1. Strains Taxonomic studies have been made on the strains isolated by the * Asahi Breweries Co. Ltd., Tokyo. 95 96 A. NAKAGAWAand K. KITAHARA present authors, and those supplied by many investigators. These strains are described as follows. Strain No. Source § Acid sensitive group (Pediococcus urinae-equi & P. halophilus) *P . halophilus MEES (1934) .... P. hal..... anchovy (Tetracoccus No. 1 ORLA-JENSEN1919) pickles *P . urinae-equi MEES (1934) .... P. u.e..... horse-urine Tetrakokken Stamm von S. STRESE & S. WINDIscH (1955) (19) .... beer, brewing .... C, F, T, Sp-2 & Sp-4 -water, horse-urine °A -38 , A-40, N-21 & A-28 isolated by A. NAKAGAWA & K. KITAHARA(1956) .... yeasts *P . soyae K. SAKAGUCHI(1958) .... d-2, d-8 & dl-2.... soy-mash P. soyae K. YAMAZATO(1957) (18) .... soy-mash § Intermediate group (P. pentosaceus & P. acidilactici) *P . pentosaceus MEES (1934) .... P, pt..... yeast (Tetracoccus No. 2 ORLA-JENSEN1919) *P . damnosus Delft ....904.... beer ? *P . damnosus var. perniciosus Delft ....908.... beer ? *P . cerevisiae PEDERSON(1949) .... fermenting .... B-168, E-66, F-166, K-64, N-82, N-88 & P-611 vegetables *Streptococcus citrovorus Hammer (1920).... cit..... dairy products *Leuconostoc citrovorum ATCC .... 8081 .... dairy products P. lindneri Henneberg isolated by K. KITAHARA(1935) .... P. lin .... sake mash *Leuconostoc mesenteroides ATCC 8042 .... P-60.... °S-20 by A . NAKAGAWA& K. KITAHARA(1956) .... mash § Alkali sensitive group (P, cerevisiae) *P . damnosus MEES (1934) .... M-1.... beer *P . damnosus var. perniciosus MEES (1934) .. M-3.... beer *P3 damnosus var , diastaticus ANDREWS& GILLILAND(195 2) .... P. di..... beer P. mevalovorus KITAHARA& NAKAGAWA (1958) .... A-41 .... beer °8 -8 ,11-2,14-5,19-2.... forty strains isolated by A. NAKAGAWA & K. KITAHARA(1957) .... yeasts & beer ° .Unpublished strain(s), * .... Received strain(s). Remarks : Two strains, "904" & "908", were obtained through the courtesy of Dr. PEDERSON. 2. Method of isolation Many techniques intended for th e isolationIso a ion oto microorganisms inm pure culture have been reported. Whe n only one kind of organism predominates comparativelymicroflora,its pure isolation is in easy although such a case rarely occurs. 1959 "Taxonomic Studies on the Genus Pediococcus " 97 In most cases owing to the existence of various undesired organisms, a pure culture is directly employed upon consideration of the best condition for the desired organism, or after enrichment cultures. In order to isolate pediococci directly in pure culture, the following method has been generally adopted. Each sample is inoculated in the most favourable natural media (pH 5.0 to 7.0) at 25, 30° or higher, employing aerobic or anaerobic (C02-atmos- phere) plate culture or a high-layered agar-culture. In case of the aerobic plate culture (generally with the addition of calcium carbonate), the majority of the organisms are capable of develop- ing into colonies, while those of P. cerevisiae can hardly or very slowly develop into visible colonies. In order to inhibit the growth of other mixed organisms, chemical substances are often added to the media. Sodium-acetate •3H2O (2.0%), used as a buffer, inhibits the growth of catalase-positive bacteria such as Gram-negative Enterobacteriaceae and Gram-positive Tetracoccus (34). Antibiotics, for instance actidion (5 p.p.m.), fermicidin (10 p.p.m.) and eurocidin (30 p.p.m.) (17), are also employed to avoid the growth of yeasts and molds. Anaerobic and a high-layered cultures suppress the growth of aerobic organisms such as Bacillus, Acetobacter and molds. When a desired organism, such as P. cerevisiae, propagates more slowly than other mixed organisms in enrichment culture, its pure isolation becomes a very difficult matter. Table 1. Methods employed in pure culture for each species of Pediococcus. 98 A. NAKAGAWAand K. KITAHARA VOL. 5 We have almost succeeded in obtaining a pure culture for the slowly propagating cocci by inoculating them into the following media, and cultur- ing anaerobically or in a Burn-tube. Media: End-fermented beer with the addition of sodium acetate and D- mannose*, pH 5.0. Table 1 presents just an example of the conditions in enrichment or direct pure culture for each species of Pediococcus, although this is not always the best condition for propagation. 3. General characteristics As the genus Pediococcus is included in the family Lactobacillaceae (15), it possesses the same general properties as true lactic acid bacteria and the general characteristics of each strain are described as pediococci in this section. Cultural characters The propagating velocity of pediococci slows down in the order of P. pentosaceus, P. acidilactici, P. urinae-equi, P, halophilus and P. cerevisiae under both (micro) aerobic and anaerobic atmosphere, however, the growth of P. cerevisiae under anaerobic conditions (C02-atmosphere) is much mark- able than aerobic. Liquid culture : No strain can grow on liquid surface. Color of the cell sediments is usually white but occasionally yellowish, and the sediments are granular to viscous in the order of P. cerevisiae, P. acidilactici, P. pentosaceus, P. urinae-equi and P. halophilus. Among the five species, acid-sensitive strains belonging to P. halo- philus and P. urinae-equi tend to produce plenty of cell-sediments in the media. The growth media of the strains belonging to P, cerevisiae generally remain clear except P. perniciosus (5, 9), but others often make the media turbid especially at the time when cultures are young. Solid culture: Stab culture: all strains grow uniformly along the stab-canal in fili- f orm or papillate. Strains belonging to P. cerevisiae and P. soyae do not show any surface growth while other strains reveal either raised or leafy surface growth. Streak culture: strains show poor sweaty growth and their colonies are semi-transparent. When strains of P. cerevisiae are cultured in a high-layer, round or lens shaped colonies often develop from the bottom of the test tube. * Fermentable-sugar of the cocci and non fermentable-sugar of hop-tolerant Lacto- bacillus. 1959 '' Taxonomic Studies on the Genus Pediococcus '' 99 Growth in milk: Unlike the strains of Streptococcus, the majority of the strains do not show any change in milk (pH 5.6) or in Litmus-milk. But some strains belonging to P. urinae-equi (19), those in fermenting vege- tables (14) and P. citrovorum (15) ferment milk slowly with slightly producing an acid. Growth in sugar-free media: Strains generally show good growth in cabohydrate-free natural media except those of P. cerevisiae. Morphology and Physiology All strains are spheres, 0.6-0.8, or 0.8-1.0 ,u in diameter and never show any ovoid forms. Most of them appear as tetrad or diploid forms but never occur in chained forms. Capsuls are sometimes or often observed. Like other lactic acid bacteria, they are Gram-positive, non-motile, non- spore-forming, and do not reduce nitrates or liquefy gelatin. Indol formation also is never detected. Catalase reaction : Hitherto, all true lactic acid bacteria are considered to be catalase negative. There is only one exception on some strains belonging to Pediococcus pentosaceus as reported by FELTON, EVANS & NIVEN, (20). But even such strains give only weak positive reactions and their activity to the enzyme is not so distinct in contrast to the so-called Tetracoccus (34) which morphologically resembles to Pediococcus. Like P. pentosaceus, some strains of P. urinae-equi exhibited a some- what positive catalase reaction when they were cultured in the media con- taining a trace of sugar like Y.T.G. media (20). + + .... A-38, A-40 (P. urinae-equi), P. pt. B-168, E-66, K-64, N-82, N-88 (P. pentosaceus). + .... P-611, cit., 8081 (P. pentosaceus). -- .... P.u.e., A-28, N-21 (P, urinae-equi P.