Biodegradation of synthetic and naturally occurring mixtures of mono-cyclic aromatic compounds present in olive mill wastewaters by two aerobic bacteria

Two bacterial strains, Ralstonia sp. LD35 and Pseudomonas putida DSM 1868, were assayed for their ability to degrade the monocyclic aromatic compounds commonly found in olive mill wastewaters (OMWs). The goal was to study the possibility of employing the two strains in the removal of these recalcitrant and toxic compounds from the effluents of anaerobic treatment plants fed with OMWs. At first, the two strains were separately assayed for their ability to degrade a synthetic mixture of nine aromatic acids present in OMWs, both in growing- and resting-cell conditions. Then, due to the complementary activity exhibited by the two strains, a co-culture of the two bacteria was tested under growing-cell conditions for degradation of the same synthetic mixture. Finally, the degradation activity of the co-culture on two fractions was studied. Both fractions one deriving from natural OMWs through reverse osmosis treatment and containing low-molecular weight organic molecules, and the other obtained from an anaerobic lab-scale treatment plant fed with OMWs, were rich in monocyclic aromatic compounds. The co-culture of the two strains was able to biodegrade seven of the nine components of the tested synthetic mix (2, 6-dihydroxybenzoic acid and 3, 4, 5-trimethoxybenzoic acid were the two undegraded compounds). In addition, an efficient biodegrading activity towards several aromatic molecules present in the two natural fractions was demonstrated.     

An aerobic fixed-phase biofilm reactor system for the degradation of the low-molecular weight aromatic compounds occurring in the effluents of anaerobic digestors treating olive mill wastewaters

An aerobic co-culture, prepared by combining Ralstonia sp. LD35 and Pseudomonas putida DSM1868, was recently found to be capable of extensively degrading many of the hydroxylated and/or methoxylated benzoic, phenylacetic and 3-phenyl-2-propenoic acids occurring in the olive mill wastewaters (OMWs). In the perspective of developing a biotechnological process for the degradation of low-molecular weight (MW) aromatic compounds occurring in the effluents of anaerobic digestors treating OMWs, the capability of this bacterial co-culture of biodegrading a synthetic mix of the above mentioned compounds and the aromatic compounds of an anaerobic OMW-treatment plant effluent in the physiological state of immobilised cells was investigated. Two aerobic fixed-bed biofilm reactors were developed by immobilising the co-culture cells on Manville silica beads and on polyurethane foam cubes. Both supports were found to give rise to a microbiologically stable and biologically active biofilm. The two biofilm reactors were found to be similarly capable of rapidly and completely biodegrading the components of a synthetic mix of nine monocyclic aromatic acids typically present in OMWs and the low-MW aromatic compounds occurring in the anaerobic effluent in batch conditions. However, in the same conditions, the silica bead-packed reactor was found to be more effective in the removal of high-MW phenolic compounds from the anaerobic effluent with respect to the polyurethane cube-packed reactor. These results are encouraging in the perspective of using the co-culture as immobilized cells for developing a continuous biotechnological process for the post-treatment of effluents with low-MW aromatic compounds produced by anaerobic digestors treating OMWs.     

Anaerobic and aerobic metabolism of diverse aromatic compounds by the photosynthetic bacterium Rhodopseudomonas palustris.

The purple nonsulfur photosynthetic bacterium Rhodopseudomonas palustris used diverse aromatic compounds for growth under anaerobic and aerobic conditions. Many phenolic, dihydroxylated, and methoxylated aromatic acids, as well as aromatic aldehydes and hydroaromatic acids, supported growth of strain CGA001 in both the presence and absence of oxygen. Some compounds were metabolized under only aerobic or under only anaerobic conditions. Two other strains, CGC023 and CGD052, had similar anaerobic substrate utilization patterns, but CGD052 was able to use a slightly larger number of compounds for growth. These results show that R. palustris is far more versatile in terms of aromatic degradation than had been previously demonstrated. A mutant (CGA033) blocked in aerobic aromatic metabolism remained wild type with respect to anaerobic degradative abilities, indicating that separate metabolic pathways mediate aerobic and anaerobic breakdown of diverse aromatics. Another mutant (CGA047) was unable to grow anaerobically on either benzoate or 4-hydroxybenzoate, and these compounds accumulated in growth media when cells were grown on more complex aromatic compounds. This indicates that R. palustris has two major anaerobic routes for aromatic ring fission, one that passes through benzoate and one that passes through 4-hydroxybenzoate.     

石油降解细菌的表型特性和系统发育分析

从3种不同土壤中分离和纯化得到10个石油降解细菌菌株,分离菌株均为好氧菌、革兰氏阴性菌、不形成芽孢的杆菌,10个菌株均能利用中等链长的烷烃、柴油和原油作为碳源,而不能以单环芳烃和多环芳烃为碳源。根据其生理生化性状和16SrDNA序列分析结果表明,分离菌株EVA5,EVA6,EVA7,EVA8和EVA9为假单胞菌（Pseudomonas spp.）,EVA10、EVA11、EVA12、EVA13和EVA14为不动杆菌（Acinetobacter spp.）,均属于Proteobac-teria的γ亚群。     

Characterization of Ligninolytic Bacteria and Analysis of Alkali-Lignin Biodegradation Products

Ligninolytic bacteria degrading lignin were isolates and identified, and their biodegradation mechanism of alkaline-lignin was investigated. Four strains with lignin degradation capability were screened and identified from the soil, straw, and silage based on their decolorizing capacity of aniline blue and colony size on alkaline-lignin medium. The degradation ratio of Bacillus aryabhattai BY5, Acinetobacter johnsonii LN2, Acinetobacter lwoffii LN4, and Micrococcus yunnanensis CL32 have been assayed using alkaline-lignin as the unique carbon source. Further, the Lip (lignin peroxidase) and Mnp (manganese peroxidase) activities of strains were investigated. Lip activity of A. lwoffii LN4 was highest after 72 h of incubation and reached 7151.7 U · l^–1. Mnp activity of M. yunnanensis CL32 was highest after 48 h and reached 12533 U · l^–1. The analysis of alkaline-lignin degradation products by GC-MS revealed that the strains screened could utilize aromatic esters compounds such as dibutyl phthalate (DBP), and decomposite monocyclic aromatic compounds through the DBP aerobic metabolic pathway. The results indicate that B. aryabhattai BY5, A. johnsonii LN2, A. lwoffii LN4, and M. yunnanensis CL32 have high potential to degrade alkaline-lignin, and might utilize aromatic compounds by DBP aerobic metabolic pathway in the process of lignin degradation.Key words: isolation, bacteria, alkali-lignin, biodegradation products     

Methylopila helvetica sp. nov. and Methylobacterium dichloromethanicum sp. nov.--novel aerobic facultatively methylotrophic bacteria utilizing dichloromethane

Eight strains of Gram-negative, aerobic, asporogenous, neutrophilic, mesophilic, facultatively methylotrophic bacteria are taxonomically described. These icl- serine pathway methylobacteria utilize dichloromethane, methanol and methylamine as well as a variety of polycarbon compounds as the carbon and energy source. The major cellular fatty acids of the non-pigmented strains DM1, DM3, and DM5 to DM9 are C18:1, C16:0, C18:0, Ccy19:0 and that of the pink-pigmented strain DM4 is C18:1. The main quinone of all the strains is Q-10. The non-pigmented strains have similar phenotypic properties and a high level of DNA-DNA relatedness (81-98%) as determined by hybridization. All strains belong to the alpha-subgroup of the alpha-Proteobacteria. 16S rDNA sequence analysis led to the classification of these dichloromethane-utilizers in the genus Methylopila as a new species - Methylopila helvetica sp.nov. with the type strain DM9 (=VKM B-2189). The pink-pigmented strain DM4 belongs to the genus Methylobacterium but differs from the known members of this genus by some phenotypic properties, DNA-DNA relatedness (14-57%) and 16S rDNA sequence. Strain DM4 is named Methylobacterium dichloromethanicum sp. nov. (VKM B-2191 = DSMZ 6343).     

Laccase from Pleurotus sajor-caju on functionalised SBA-15 mesoporous silica: Immobilisation and use for the oxidation of phenolic compounds

Laccase from Pleurotus sajor-caju was immobilised on functionalised SBA-15 mesoporous silica. The immobilisation process reached the equilibrium after about 100 min. In order to study the effect of loading (L) on activity of the immobilised laccase, the adsorption isotherm was built and the activity of the corresponding immobilised biocatalysts was determined. The activity of the immobilised preparations reached a maximum at L = 217 kU g_SBA-15⁻¹, whereas higher loadings gave rise to a less-efficient biocatalyst. The immobilised laccase was used for the oxidation of a mixture of four phenolic compounds (protocatechuic acid, ferulic acid, sinapic acid and caffeic acid) chosen among those present in olive mill wastewaters (OMWs). These compounds determine the phytotoxicity of OMWs. Different reaction rates were observed for the oxidation of the examined phenolic compounds. The biocatalyst was recycled and a conversion of 84 mol% at the 10th reuse and of about 60 mol% after the 14th reuse was obtained. In conclusion, the laccase immobilised on SBA-15 is a potential biocatalyst for bioremediation of OMWs, which is an important environmental problem in the regions around the Mediterranean Sea.     

Characterization of beta-ketoadipate pathway from multi-drug resistance bacterium, Acinetobacter baumannii DU202 by proteomic approach

In this study, the biodegradative activities of monocyclic aromatic compounds were determined from the multi-drug resistant (MDR) Acinetobacter baumannii, which were studied in the form of clinical isolates from a hospital in Korea. These bacteria were capable of biodegrading monocyclic aromatic compounds, such as benzoate and p-hydroxybenzoate. In order to determine which pathways are available for biodegradation in these stains, we conducted proteome analyses of benzoate and p-hydroxybenzoate-cultured A. baumannii DU202, using 2-DE/MS analysis. As genome DB of A. baumannii was not yet available, MS/MS analysis or de novo sequencing methods were employed in the identification of induced proteins. Benzoate branch enzymes [catechol 1,2-dioxygenase (CatA) and benzoate dioxygenase alpha subunit (BenA)] of the beta-ketoadipate pathway were identified under benzoate culture condition and p-hydroxybenzoate branch enzymes [protocatechuate 3,4-dioxygenase alpha subunit (PcaG) and 3-carboxy-cis,cis-muconate cycloisomerase (PcaB)] of the beta-ketoadipate pathway were identified under p-hydroxybenzoate culture condition, respectively, thereby suggesting that strain DU202 utilized the beta-ketoadipate pathway for the biodegradation of monocyclic aromatic compounds. The sequence analysis of two purified dioxygenases (CatA and PcaGH) indicated that CatA is closely associated with the CatA of Acinetobacter radiresistance, but PcaGH is only moderately associated with the PcaGH of Acinetobacter sp. ADP1. Interestingly, the fused form of PcaD and PcaC, carboxymuconolactone decarboxylase (PcaCD), was detected on benzoate-cultured A. baumannii DU202. These results indicate that A. baumannii DU202 exploits a different beta-ketoadipate pathway from other Acinetobacter species.     

Trichosporon porosum comb. nov., an anamorphic basidiomycetous yeast inhabiting soil,related to the loubieri/laibachii group of species that assimilate hemicelluloses and phenolic compounds

Several isolates representing the genus Trichosporon were collected over a 6-year period from soils in The Netherlands. Based on classical growth tests with carbon and nitrogen compounds these were identical. Three of these (CBS 8396, CBS 8397 and CBS 8522) were subjected to molecular analysis of the D1/D2 region of the large subunit of rDNA. This confirmed that the three strains were identical, yet distinct from other members of the genus. Conspecificity was demonstrated with the type strain (CBS 2040) of Apiotrichum porosum Stautz (1931), with the exception that A. porosum, which had been isolated from exudate of a yew tree, differed morphologically from the soil strains. Based on the identity of DNA base sequences, morphology was not considered to be an adequate parameter to separate otherwise identical strains into two genera. Therefore, the new combination Trichosporon porosum is presented. Based on molecular sequence analysis, T. porosum may be related to T. sporotrichoides, within a weakly related clade that includes species such as Trichosporon laibachii and Trichosporon loubieri. The strains of T. porosum degrade phenolic compounds and hemicelluloses, which are characteristics with potential ecological importance in soil habitats. Characters distinguishing the nine species of the laibachii/loubieri group of species were listed. These include traditionally used tests as well as assimilation patterns of some aliphatic and phenolic compounds. Based on these tests, species such as Trichosporon multisporum and T. laibachii could be separated.     

Novel halophilic aerobic anoxygenic phototrophs from a Canadian hypersaline spring system

The first enumeration of cultivable obligately aerobic phototrophic bacteria from a terrestrial saline spring was accomplished in the East German Creek system (salinity approximately 6%), near Lake Winnipegosis, Manitoba, Canada. Occurring at densities up to 3.3 x 10(7) CFU/ml of sample, aerobic phototrophs comprised 15-36% of the total cultivable bacterial population in the diatom- and chlorophyte-dominated aerobic microbial mats. Many of the representative strains isolated for phenotypic characterization and phylogenetic analysis possessed <96% 16S rDNA sequence overlap with published species, including an obligately aerobic phototrophic gammaproteobacterium displaying only 92.9% 16S rDNA sequence similarity to Congregibacter litoralis. The springs yielded the most highly halotolerant aerobic anoxygenic phototroph yet recorded, strain EG11, which grew with 26% NaCl.     

Three New Phenolic Amides from the Roots of Eggplant (Solanum melongena L.)

The isolation of four phenolic amides, four phenolic compounds and an aromatic amine from the roots of eggplant is described. The phenolic amides were identified as N-trans-feruloyl tyramine (V), N-trans-p-coumaroyl tyramine (VII), N-trans-feruloyl octopamine (VIII) and N-trans-p-coymaroyl octopamine (IX). The three amides V, VIII and IX are new compounds. Furthermore, four phenolic compounds were identified as vanillin (I), isoscopoletin (II), ethyl caffeate (IV) and ferulic acid (VI). The aromatic amine was identified as p-aminobenzal-dehyde (III).     

Horizontal transfer of genetic determinants for degradation of phenol between the bacteria living in plant and its rhizosphere

Phenol and other monocyclic aromatic compounds (MACs) are highly water-soluble and volatile pollutants that plants are unable to completely degrade. Endophytic bacteria with MAC-degrading ability will facilitate phytoremediation, beneficial to plant survival in contaminated soil. Endophytic bacteria, strains FX1-FX3, and rhizosphere bacteria, strains FX0, FX4, and FX5, were isolated from the root tissue of a corn plant (Zea mays) and the corn rhizosphere near a chemical plant, respectively. The strains FX1-FX5 were able to grow on phenol and reduce phenol concentration, but the strain FX0 was unable to. The strains FX1, FX3, and FX4 were classified as Pseudomonas fluorescens and FX0, FX2, and FX5 as Burkholderia cepacia. The plasmids isolated from the strains FX1-FX5 were found to possess similar traits and to be loaded with a gene encoding the catechol 2, 3-dioxygenase (C23O), a key enzyme in the phenol degradation pathway. Alignment and phylogenetic analysis inferred that in situ horizontal transfer of the C23O gene might have occurred. The horizontal transfer of the C23O gene between endophytic and rhizosphere bacteria was proved by using conjugal matings experiment, in which the transconjugants were found to acquire the plasmid with the C23O gene, able to grow on phenol and degrade phenol.     

The genome sequence of an anaerobic aromatic-degrading denitrifying bacterium,strain EbN1

Recent research on microbial degradation of aromatic and other refractory compounds in anoxic waters and soils has revealed that nitrate-reducing bacteria belonging to the Betaproteobacteria contribute substantially to this process. Here we present the first complete genome of a metabolically versatile representative, strain EbN1, which metabolizes various aromatic compounds, including hydrocarbons. A circular chromosome (4.3 Mb) and two plasmids (0.21 and 0.22 Mb) encode 4603 predicted proteins. Ten anaerobic and four aerobic aromatic degradation pathways were recognized, with the encoding genes mostly forming clusters. The presence of paralogous gene clusters (e.g., for anaerobic phenylacetate oxidation), high sequence similarities to orthologs from other strains (e.g., for anaerobic phenol metabolism) and frequent mobile genetic elements (e.g., more than 200 genes for transposases) suggest high genome plasticity and extensive lateral gene transfer during metabolic evolution of strain EbN1. Metabolic versatility is also reflected by the presence of multiple respiratory complexes. A large number of regulators, including more than 30 two-component and several FNR-type regulators, indicate a finely tuned regulatory network able to respond to the fluctuating availability of organic substrates and electron acceptors in the environment. The absence of genes required for nitrogen fixation and specific interaction with plants separates strain EbN1 ecophysiologically from the closely related nitrogen-fixing plant symbionts of the Azoarcus cluster. Supplementary material on sequence and annotation are provided at the Web page .Electronic Supplementary Material Supplementary material is available for this article at Dedicated to Prof. Dr. h.c. Gerhard Gottschalk on the occasion of his 70th birthday.     

Aromatic-degrading Sphingomonas isolates from the deep subsurface.

An obligately aerobic chemoheterotrophic bacterium (strain F199) previously isolated from Southeast Coastal Plain subsurface sediments and shown to degrade toluene, naphthalene, and other aromatic compounds (J. K. Fredrickson, F. J. Brockman, D. J. Workman, S. W. Li, and T. O. Stevens, Appl. Environ. Microbiol. 57:796-803, 1991) was characterized by analysis of its 16S rRNA nucleotide base sequence and cellular lipid composition. Strain F199 contained 2-OH14:0 and 18:1 omega 7c as the predominant cellular fatty acids and sphingolipids that are characteristic of the genus Sphingomonas. Phylogenetic analysis of its 16S rRNA sequence indicated that F199 was most closely related to Sphingomonas capsulata among the bacteria currently in the Ribosomal Database. Five additional isolates from deep Southeast Coastal Plain sediments were determined by 16S rRNA sequence analysis to be closely related to F199. These strains also contained characteristic sphingolipids. Four of these five strains could also grow on a broad range of aromatic compounds and could mineralize [14C]toluene and [14C]naphthalene. S. capsulata (ATCC 14666), Sphingomonas paucimobilis (ATCC 29837), and one of the subsurface isolates were unable to grow on any of the aromatic compounds or mineralize toluene or naphthalene. These results indicate that bacteria within the genus Sphingomonas are present in Southeast Coastal Plain subsurface sediments and that the capacity for degrading a broad range of substituted aromatic compounds appears to be common among Sphingomonas species from this environment.     

Anaerobic oxidation of 2-chloroethanol under denitrifying conditions by<Emphasis Type="Italic"> Pseudomonas stutzeri</Emphasis> strain JJ

A bacterium that uses 2-chloroethanol as sole energy and carbon source coupled to denitrification was isolated from 1,2-dichloroethane-contaminated soil. Its 16 S rDNA sequence showed 98% similarity with the type strain of Pseudomonas stutzeri (DSM 5190) and the isolate was tentatively identified as Pseudomonas stutzeri strain JJ. Strain JJ oxidized 2-chloroethanol completely to CO(2) with NO(3)(- )or O(2) as electron acceptor, with a preference for O(2) if supplied in combination. Optimum growth on 2-chloroethanol with nitrate occurred at 30 degrees C with a mu(max) of 0.14 h(-1) and a yield of 4.4 g protein per mol 2-chloroethanol metabolized. Under aerobic conditions, the mu(max) was 0.31 h(-1). NO(2)(-) also served as electron acceptor, but reduction of Fe(OH)(3), MnO(2), SO(4)(2-), fumarate or ClO(3)(-) was not observed. Another chlorinated compound used as sole energy and carbon source under aerobic and denitrifying conditions was chloroacetate. Various different bacterial strains, including some closely related Pseudomonas stutzeri strains, were tested for their ability to grow on 2-chloroethanol as sole energy and carbon source under aerobic and denitrifying conditions, respectively. Only three strains, Pseudomonas stutzeri strain LMD 76.42, Pseudomonas putida US2 and Xanthobacter autotrophicus GJ10, grew aerobically on 2-chloroethanol. This is the first report of oxidation of 2-chloroethanol under denitrifying conditions by a pure bacterial culture.     

Leifsonia gen. nov., a genus for 2,4-diaminobutyric acid-containing actinomycetes to accommodate "Corynebacterium aquaticum" Leifson 1962 and Clavibacter xyli subsp. cynodontis Davis et al. 1984

"Corynebacterium aquaticum" was first proposed by Leifson in 1962 but not included in the approved lists of bacterial names in 1980. This species has been left from reclassification of the genus Corynebacterium because of the unusual chemotaxonomic characteristics such as 2,4-diaminobutyric acid (DAB) in the peptidoglycan and menaquinones of MK-10 and MK-11. A close relationship of "C. aquaticum" to the genera Agromyces and Rathayibacter has been pointed out from the viewpoint of chemotaxonomic profiles and phylogeny based on the 16S rDNA sequences. An analysis of DAB isomers of the peptidoglycan distinguished "C. aquaticum" clearly from these genera by possessing both L-DAB and D-DAB. We also found that the type strain of Clavibacter xyli subsp. cynodontis and two strains of amine-decomposing bacteria showed the similar chemotaxonomic features and formed a cluster with "C. aquaticum" in the phylogenetic tree based on 16S rDNA sequences in the family Microbacteriaceae. Considering these results, we propose a new genus Leifsonia to accommodate the four strains. The four species, Leifsonia aquatica sp. nov., nom. rev., comb. nov. (type species, type strain=JCM 1368), Leifsonia shinshuensis sp. nov. (type strain=DB102=JCM 10591), Leifsonia naganoensis sp. nov. (type strain=DB103=JCM 10592), and Leifsonia cynodontis comb. nov. (type strain=JCM 9733=ICMP 8790), were proposed here for the strains.     

Construction of a Corynebacterium glutamicum platform strain for the production of stilbenes and (2S)-flavanones

Corynebacterium glutamicum is an important organism in industrial biotechnology for the microbial production of bulk chemicals, in particular amino acids. However, until now activity of a complex catabolic network for the degradation of aromatic compounds averted application of C. glutamicum as production host for aromatic compounds of pharmaceutical or biotechnological interest. In the course of the construction of a suitable C. glutamicum platform strain for plant polyphenol production, four gene clusters comprising 21 genes involved in the catabolism of aromatic compounds were deleted. Expression of plant-derived and codon-optimized genes coding for a chalcone synthase (CHS) and a chalcone isomerase (CHI) in this strain background enabled formation of 35 mg/L naringenin and 37 mg/L eriodictyol from the supplemented phenylpropanoids p-coumaric acid and caffeic acid, respectively. Furthermore, expression of genes coding for a 4-coumarate: CoA-ligase (4CL) and a stilbene synthase (STS) led to the production of the stilbenes pinosylvin, resveratrol and piceatannol starting from supplemented phenylpropanoids cinnamic acid, p-coumaric acid and caffeic acid, respectively. Stilbene concentrations of up to 158 mg/L could be achieved. Additional engineering of the amino acid metabolism for an optimal connection to the synthetic plant polyphenol pathways enabled resveratrol production directly from glucose. The construction of these C. glutamicum platform strains for the synthesis of plant polyphenols opens the door towards the microbial production of high-value aromatic compounds from cheap carbon sources with this microorganism.     

Functional Gene-Guided Discovery of Type II Polyketides from Culturable Actinomycetes Associated with Soft Coral Scleronephthya sp

Compared with the actinomycetes in stone corals, the phylogenetic diversity of soft coral-associated culturable actinomycetes is essentially unexplored. Meanwhile, the knowledge of the natural products from coral-associated actinomycetes is very limited. In this study, thirty-two strains were isolated from the tissue of the soft coral Scleronephthya sp. in the East China Sea, which were grouped into eight genera by 16S rDNA phylogenetic analysis: Micromonospora, Gordonia, Mycobacterium, Nocardioides, Streptomyces, Cellulomonas, Dietzia and Rhodococcus. 6 Micromonospora strains and 4 Streptomyces strains were found to be with the potential for producing aromatic polyketides based on the analysis of KS(α) (ketoacyl-synthase) gene in the PKS II (type II polyketides synthase) gene cluster. Among the 6 Micromonospora strains, angucycline cyclase gene was amplified in 2 strains (A5-1 and A6-2), suggesting their potential in synthesizing angucyclines e.g. jadomycin. Under the guidance of functional gene prediction, one jadomycin B analogue (7b, 13-dihydro-7-O-methyl jadomycin B) was detected in the fermentation broth of Micromonospora sp. strain A5-1. This study highlights the phylogenetically diverse culturable actinomycetes associated with the tissue of soft coral Scleronephthya sp. and the potential of coral-derived actinomycetes especially Micromonospora in producing aromatic polyketides.     

Effect of purine derivatives, papaverine hydrochloride, and imidazole on enterotoxin formation by Clostridium perfringens type A

The percentage sporulation and enterotoxin specific activity were improved for all of five Clostridium perfringens strains, and numbers of heat-resistant spores were improved for four of five strains by replacing proteose peptone with peptone in Duncan-Strong (DS) medium. When raffinose replaced starch in DS, peptone was superior to proteose peptone in increasing percentage sporulation, numbers of heat-resistant spores, and enterotoxin formation for four of five strains. Enterotoxin levels for a strain varied when different lots of the same peptone were used. Additional experiments were conducted with three C. perfringens strains grown in DS medium with peptone. Enterotoxin specific activity was increased for three strains by adding papaverine (hydrochloride crystalline), for two strains by adding each of caffeine and 3-isobutyl-l-methylxanthine, for one strain by adding each of theophylline, 6-mercaptopurine, and 2-amino-6-mercaptopurine, and for none of the strains by adding imidazole. When enterotoxin formation was improved for a strain by one of the compounds, percentage sporulation increased, but growth decreased. Effective compounds also increased numbers of heat-resistant spores for strains H6 and R42, but slightly or not at all for strain E13. The action of these compounds was concentration dependent, with the optimal concentration differing between compounds and between strains grown in the presence of the same compound.