Chemostat enrichment and isolation of Hyphomicrobium EG

A stable mixed bacterial culture was obtained by chemostat enrichment using dimethyl-sulphoxide as a carbon and energy source. This culture could not only rapidly oxidize dimethyl-sulphoxide but also dimethyl-sulphide. Enzyme determinations indicated that an important part of it consisted of methylotrophs, which assimilated carbon via the serine pathway. Indeed plate counts revealed the majority of the community to be a Hyphomicrobium species. This organism, designated Hyphomicrobium EG, is an obligate methylotroph which can only grow aerobically on several different C₁-compounds. Its performance on dimethyl-sulphoxide was compared with that of the community and of another recently isolated strain, Hyphomicrobium S. The mixed culture, Hyphomicrobium EG and Hyphomicrobium S had a _max of 0.08, 0.08 and 0.014 h^-1 respectively. The K_S for dimethyl-sulphoxide was the same for all three cultures (3–6 M), whereas that for dimethyl-sulphide of Hyphomicrobium EG after growth on dimethyl-sulphoxide was 3-fold higher than that of the other two cultures (48 and 16 M respectively). After growth on dimethyl-sulphide it improved to 3 M. Dimethyl-sulphide respiration was maximal at a concentration of 100 M; higher concentrations were inhibitory. One of the accompanying organisms, a pink methylotroph, was able to derive energy from the oxidation of thiosulphate. Available cultures of Thiobacillus MS1 that were reported to be able to utilize dimethyl-sulphide could no longer metabolize this compound.     

Cryptic growth within a binary microbial culture

Summary The ability of viable cells of the species Pseudomonas putida and Hyphomicrobium sp. to metabolize the particulate and soluble cellular organic constituents of both species was studied in a series of batch experiments. Both P. putida and Hyphomicrobium sp. were grown in individual batch reactors on either the ¹⁴C-labelled soluble or the particulate debris of sonicated cells of each species derived from steady-state chemostat cultures. Cell generation times (t _g)observed for P. putida cultivated on soluble organic material originating from either sonicated P. putida or Hyphomicrobium sp. cells, were t _g= 2.0 h and t _g= 6.3 h, respectively. Corresponding t _gvalues of Hyphomicrobium sp. on soluble organic material originating from sonicated P. putida and Hyphomicrobium so. were, respectively, 11.6 h and 4.3 h. While particulate debris originating from either species was solubilized by both P. putida and Hyphomicrobium sp., no increases in cell numbers were observed for either species. The data indicate that bacteria are capable of scavenging soluble material released upon cell lysis at near maximal rates; solubilization of debris also occurred but at much slower overall rates with no observable cell replication. The results reaffirm that cryptic growth and turnover of cellular biomass can be significant under situations of low substrate flux or starvation conditions. Offprint requests to: J. D. Bryers     

Identification of a periplasmic dimethylsulphoxide reductase in Hyphomicrobium EG grown under chemolithoheterotrophic conditions with dimethylsulphoxide as carbon source

Hyphomicrobium EG can grow with dimethylsulphoxide as sole carbon and energy source with oxygen as electron acceptor. In the present work we have found that the dimethylsulphoxide reductase of this bacterium could be assayed with dithionite-reduced methylviologen as reductant but not with NADH. Sub-cellular fractionation of Hyphomicrobium EG showed that the dimethylsulphoxide reductase was a periplasmic enzyme. An antibody to the dimethylsulphoxide reductase of Rhodobacter capsulatus cross-reacted with a polypeptide in the periplasmic fraction from Hyphomicrobium EG which had the same M _r as the dimethylsulphoxide reductase of Rhodobacter capsulatus. It is suggested that the reduction of dimethylsulphoxide in Hyphomicrobium involves respiratory electron transfer.Abbreviations DMSO dimethylsulphoxide - DMS dimethylsulphide     

Dichloromethane as the sole carbon source forHyphomicrobium sp. strain DM2 under denitrification conditions

Hyphomicrobium sp. strain DM2 was found to grow anaerobically in the presence of nitrate with methanol, formaldehyde, formate or dichloromethane. The estimated growth rate constants with methanol and dichloromethane under denitrification conditions were 0.04 h^–1 and 0.015 h^–1, respectively, which is twofold and fourfold lower than the rates of aerobic growth with these substrates. Slight accumulation of nitrite was observed in all cultures grown anaerobically with nitrate. Dichloromethane dehalogenase, the key enzyme in the utilization of this carbon source, was induced under denitrification conditions to the same specific activity level as under aerobic conditions. In a fed batch culture under denitrification conditionsHyphomicrobium sp. DM2 cumulatively degraded 35 mM dichloromethane within 24 days. This corresponds to a volumetric degradation rate of 5 mg dichloromethane/l·h and demonstrates that denitrificative degradation offers an attractive possibility for the development of anaerobic treatment systems to remove dichloromethane from contaminated groundwater.     

Degradation of methamidophos by <Emphasis Type="Italic">Hyphomicrobium</Emphasis> species MAP-1 and the biochemical degradation pathway

Methamidophos is one of the most widely used organophosphorus insecticides usually detectable in the environment. A facultative methylotroph, Hyphomicrobium sp. MAP-1, capable of high efficiently degrading methamidophos, was isolated from methamidophos-contaminated soil in China. It was found that the addition of methanol significantly promoted the growth of strain MAP-1 and enhanced its degradation of methamidophos. Further, this strain could utilize methamidophos as its sole carbon, nitrogen and phosphorus source for growth and could completely degrade 3,000 mg l⁻¹ methamidophos in 84 h under optimal conditions (pH 7.0, 30°C). The enzyme responsible for methamidophos degradation was mainly located on the cell inner membrane (90.4%). During methamidophos degradation, three metabolites were detected and identified based on tandem mass spectrometry (MS/MS) and gas chromatography-mass spectrometry (GC–MS) analysis. Using this information, a biochemical degradation pathway of methamidophos by Hyphomicrobium sp. MAP-1 was proposed for the first time. Methamidophos is first cleaved at the P–N bond to form O,S-dimethyl hydrogen thiophosphate and NH₃. Subsequently, O,S-dimethyl hydrogen thiophosphate is hydrolyzed at the P–O bond to release –OCH₃ and form S-methyl dihydrogen thiophosphate. O,S-dimethyl hydrogen thiophosphate can also be hydrolyzed at the P–S bond to release –SCH₃ and form methyl dihydrogen phosphate. Finally, S-methyl dihydrogen thiophosphate and methyl dihydrogen phosphate are likely transformed into phosphoric acid.     

Genetic manipulation of the restricted facultative methylotroph Hyphomicrobium X by the R-plasmid-mediated introduction of the Escherichia coli pdh genes

The inability of Hyphomicrobium X to grow on compounds such as pyruvate and succinate is most likely due to the absence of a functional pyruvate dehydrogenase (PDH) complex. Further support for this was sought by studying the effect of the introduction of the Escherichia coli pdh genes in Hyphomicrobium X on the pattern of substrate utilization by the latter organism. These genes were cloned by in vivo techniques using the broad-host range conjugative plasmid RP4: :Mucts. Plasmid RP4 derivatives containing pdh genes were selected by their ability to complement a pyruvate dehydrogenase deletion mutant of E. coli, strain JRG746 recA (ace-1pd) 18. The plasmids thus obtained could be transferred through an intermediary host (C600 recA), selecting only for an antibiotic resistance coded for by RP4 and back into JRG746 or other E. coli pdh mutants, upon which they still conferred the wild type phenotype. Enzyme assays showed that the latter strains, when carrying plasmid RP4 pdh1 also possessed PDH complex activity. Conjugation between the auxotrophic E. coli JRG746 (RP4 pdh1) strain and Hyphomicrobium X on pyruvate minimal agar gave rise to progeny which, on the basis of its morphology (stalked bacteria), their ability to grow on C₁-compounds and to denitrify (now also with pyruvate) were identified as hyphomicrobia. This Hyphomicrobium X transconjugant was also able to grow in minimal medium with succinate, but no other novel growth substrates have been identified so far. An analysis of protein extracts with 2-dimensional gel electrophoresis indicated that Hyphomicrobium X and JRG746 only synthesized all three components of the PDH complex when carrying plasmid RP4 pdh1. These results are compatible with the suggested significance of the lack of a functional PDH complex in wild type Hyphomicrobium X.Abbreviations PDH pyruvate dehydrogenase - TCA tricarboxylic acid Dedicated to Prof. H. G. Schlegel on the occasion of his 60th birthday     

Pleomorphy of a budding bacterium on various carbon sources

Pleomorphy inHyphomicrobium T37 was manipulated by the use of different carbon (C₁) sources. Growth on methanol medium is characterized by the classical morphology as found inH. vulgare cells, accompanied by some cellular pleomorphy in the form of dichotomous lobing. Extensive cellular and colonial pleomorphy was observed on media containing methylamine.We thank Mrs. P. M. Scarborough and Mrs. J. Hardy for technical assistance.     

Quantification of Hyphomicrobium Populations in Activated Sludge from an Industrial Wastewater Treatment System as Determined by 16S rRNA Analysis

The bacterial community structure of the activated sludge from a 25 million-gal-per-day industrial wastewater treatment plant was investigated using rRNA analysis. 16S ribosomal DNA (rDNA) libraries were created from three sludge samples taken on different dates. Partial rRNA gene sequences were obtained for 46 rDNA clones, and nearly complete 16S rRNA sequences were obtained for 18 clones. Seventeen of these clones were members of the beta subdivision, and their sequences showed high homology to sequences of known bacterial species as well as published 16S rDNA sequences from other activated sludge sources. Sixteen clones belonged to the alpha subdivision, 7 of which showed similarity to Hyphomicrobium species. This cluster was chosen for further studies due to earlier work on Hyphomicrobium sp. strain M3 isolated from this treatment plant. A nearly full-length 16S rDNA sequence was obtained from Hyphomicrobium sp. strain M3. Phylogenetic analysis revealed that Hyphomicrobium sp. strain M3 was 99% similar to Hyphomicrobium denitrificans DSM 1869^T in Hyphomicrobium cluster II. Three of the cloned sequences from the activated sludge samples also grouped with those of Hyphomicrobium cluster II, with a 96% sequence similarity to that of Hyphomicrobium sp. strain M3. The other four cloned sequences from the activated sludge sample were more closely related to those of the Hyphomicrobium cluster I organisms (95 to 97% similarity). Whole-cell fluorescence hybridization of microorganisms in the activated sludge with genus-specific Hyphomicrobium probe S-G-Hypho-1241-a-A-19 enhanced the visualization of Hyphomicrobium and revealed that Hyphomicrobium appears to be abundant both on the outside of flocs and within the floc structure. Dot blot hybridization of activated sludge samples from 1995 with probes designed for Hyphomicrobium cluster I and Hyphomicrobium cluster II indicated that Hyphomicrobium cluster II-positive 16S rRNA dominated over Hyphomicrobium cluster I-positive 16S rRNA by 3- to 12-fold. Hyphomicrobium 16S rRNA comprised approximately 5% of the 16S rRNA in the activated sludge.     

Interactions in a mixed bacterial population growing on methane in continuous culture

The growth of a mixed methane-utilizing culture in a continuous flow fermenter has been studied under both methane and oxygen limitation. Small additions of methanol have been shown to inhibit the methane-utilizing moiety in the culture and it has been shown that the Hyphomicrobium sp. in the mixed culture removes any inhibitory methanol. The interaction between the methane-utilizing Pseudomonas sp., and the Hyphomicrobium sp. has been explained and a model of the continuous mixed culture under oxygen limitation has been formulated. Qualitative predictions of transient phenomena by the model have been verified experimentally.     

The use of various substrates and substrate concentrations by a Hyphomicrobium sp. isolated from soil: Effect on growth rate and growth yield

The content of Hyphomicrobium sp. was estimated from a clay loam soil using the most probable number technique with methanol as the sole carbon source. The method enumerated Hyphomicrobia as 0.2% of the total bacteria determined by acridine orange direct counts. Hyphomicrobium sp. was not able to use C-C compounds such as glucose or acetate for growth. Maximal growth yield and growth rate were obtained when the concentration of methanol was in the range of 0.5–5 mg C/liter. Substrate affinity measurements revealed K_s values of 0.8 m and 5.8 m when the methanol concentration was 0.5–2.5 m and 5–200 m, respectively. Hyphomicrobium sp. had the ability to assimilate volatile organic compounds from air for growth. A growth yield of 0.7 mg/liter cell carbon was obtained in a mineral medium that contained no additions of organic compounds but had been stored for 4 weeks in flasks, allowing volatile compounds from the air to dissolve in the medium. When air was pumped into the culture during cultivating, the growth yield was proportional to the flow rate of air into the culture. Correspondence to: Kari Aa     

Denitrification of nitrate and nitric acid with methanol as carbon source

Summary A methanol/nitrate-medium and anaerobic conditions yielded an enrichment culture which consisted ofHyphomicrobium andParacoccus. This mixed culture proved to be very effective in denitrification of solutions containing high concentrations of nitrate and free nitric acid when grown in a chemostat (D=0.04 h^-1). With 0.1 mol/l nitric acid solution as feed medium the pH in the culture vessel adjusted itself to 5.8. For the reduction of 1 g NO₃–N 2.6 g methanol were consumed and 0.56 g cells were produced.     

Chemolithotrophic potential of a Hyphomicrobium species,capable of growth on methylated sulphur compounds

The yield of Hyphomicrobium EG on dimethyl sulphoxide, dimethyl sulphide and methylamine, considering the metabolic pathways of these compounds, suggested that the organism gained energy from the oxidation of the sulphur moiety of the former compounds. Indeed, a comparison of chemostat cultures of Hyphomicrobium EG grown on methylamine in the presence and absence of sulphide or thiosulphate proved this obligate methylotroph to be a chemolithoheterotroph. The apparent Y_sulphide and Y_thiosulphate were comparable, being 8–10 g dry weight/mol. In batch cultures thiosulphate concentrations up to 10 mM had a stimulatory effect on the growth rate of Hyphomicrobium EG, whereas higher concentrations increased the organisms doubling time.Enzyme- and respiration data showed that the organism had constitutive enzymes for the breakdown of dimethyl sulphoxide although they were clearly regulated to need. Addition of sulphide or thiosulphate to methylamine-limited chemostat cultures of Hyphomicrobium EG not only resulted in the induction of enzymes necessary for their breakdown, but also caused the enzymes for dimethyl sulphoxide metabolism, especially methyl mercaptan oxidase, to be induced. The formation of H₂O₂, a product of the latter enzyme, was reflected in the relatively high catalase activities during growth on dimethyl sulphoxide and in the organisms inability to grow on this compound in the presence of a catalase inhibitor.Abbreviations DMSO dimethyl sulphoxide - DMS dimethyl sulphide - MM methyl mercaptan - TMAO trimethylamine N-oxide - D dilution rate - GSH redticed glutathione - DCPIP 2,6-dichlorophenolindophenol - PMS phenazine methosulphate - PES phenazine ethosulphate - RubPCase ribulose 1,5-bisphosphate carboxylase - PEPCase phosphoenol pyruvate carboxylase - Wurster's blue (TMPD) N,N,N,N-tetramethyl-p-phenylenediamine     

Oxygen-dependent desulphation of monomethyl sulphate by Agrobacterium sp. M3C

Agrobacterium sp. M3C, previously isolated from canal-water for its ability to grow on monomethyl sulphate, degraded this ester with stoichiometric liberation of inorganic sulphate. In contrast with the biodegradation of monomethyl sulphate in Hyphomicrobium sp., and of other longer-chain alkyl sulphates in Pseudomonas spp., the pathway in Agrobacterium appeared not to involve a sulphatase enzyme capable of catalysing ester-bond hydrolysis. No such sulphatase was detectable under a range of conditions of bacterial culture, or using various methods for preparing cell-extracts, or different assay conditions. There was no incorporation of ¹⁸O-label from H₂ ¹⁸O into the liberated inorganic sulphate. No methanol was detectable during biodegradation, and the organism was incapable of growth on methanol, and did not produce methanol dehydrogenase activity when grown on monomethyl sulphate. Tracer studies using mono[¹⁴C]-methyl sulphate indicated that formate serine and glycine were produced during the biodegradation. The presence of these amino acids, together with high activity of hydroxypyruvate reductase, indicated the operation of the serine pathway common in methylotrophs. Use of an oxygen electrode in conjunction with monomethyl[³⁵S]sulphate showed that release of ³⁵SO₄ ^2- was dependent on availability of O₂, and that there was equimolar stoichiometry among monomethyl sulphate degraded, O₂ consumed and ³⁵SO₄ ^2- released. A proposed pathway for the degradation involved an initial mono-oxygenation to methanediol monosulphate with subsequent elimination of SO₄ ^2- and concomitant formation of formaldehyde. The pathway was compared with degradation mechanisms for other C₁ compounds and for other sulphate esters.     

The influence of NaCl on the degradation rate of dichloromethane byHyphomicrobium sp.

The degradation of dichloromethane by the pure strainHyphomicrobium GJ21 and by an enrichment culture, isolated from a continuously operating biological trickling filter system, as well as the corresponding growth rates of these organisms were investigated in several batch experiments. By fitting the experimental data to generally accepted theoretical expressions for microbial growth, the maximum growth rates were determined. The effect of NaCl was investigated at salt concentrations varying from 0 to 1000 mM. Furthermore the dichloromethane degradation was investigated separately in experiments in which a high initial biomass concentration was applied. The results show that microbial growth is strongly inhibited by increased NaCl concentrations (50% reduction of _max at 200–250 mM NaCl), while a certain degree of adaptation has taken place within an operational system eliminating dichloromethane. A critical NaCl concentration for growth of 600 mM was found for the microbial culture isolated from an operational trickling filter, while a value of 375 mM was found for the pure cultureHyphomicrobium GJ21. The substrate degradation appears to be much less susceptible to inhibition by NaCl. Even at 800 mM NaCl relatively high substrate degradation rates are still observed, although this process is again dependent on the NaCl concentration. Here the substrate elimination is due to the maintenance requirements of the microorganisms. The inhibition of the dichloromethane elimination was also investigated in a laboratory scale trickling filter. The results of these experiments confirmed those obtained in the batch experiments. At NaCl concentrations exceeding 600 mM a considerable elimination of dichloromethane was still observed for during several months of operation. These observations indicate that the inhibition of microbial growth offers a significant control parameter against excessive biomass growth in biological trickling filters for waste gas treatment.     

Isolation of a dimethylsulfide-utilizingHyphomicrobium species and its application in biofiltration of polluted air

The methylotrophic bacteriumHyphomicrobium VS was enriched and isolated, using activated sewage sludge as inoculum in mineral medium containing dimethylsulfide (DMS) at a low concentration to prevent toxicity. DMS concentrations above 1 mM proved to be growth inhibiting.Hyphomicrobium VS could use DMS, dimethylsulfoxide (DMSO), methanol, formaldehyde, formate, and methylated amines as carbon and energy source. Carbon was assimilated via the serine pathway. DMS-grown cells respired sulfide, thiosulfate, methanethiol, dimethyldisulfide and dimethyltrisulfide.To testHyphomicrobium VS for application in biofiltration of air polluted with volatile sulfur compounds two laboratory scale trickling biofilters with polyurethane and lava stone as carrier material were started up by inoculation with this bacterium. Both methanol- and DMS-grown cells could be used. Only a short adaptation period was needed. Short term experiments showed that high concentrations of DMS (1–2 µmol 1^–1) were removed very efficiently by the biofilters at space velocities up to 100 h^–1.Abbreviations VSC volatile sulfur compounds - DMS dimethylsulfide - DMDS dimethyldisulfide - DMTS dimethyltrisulfide - MT methanethiol - DMSO dimethylsulfoxide     

SILICON UPTAKE BY ALGAE WITH NO KNOWN Si REQUIREMENT. I. TRUE CELLULAR UPTAKE AND pH-INDUCED PRECIPITATION BY PHAEODACTYLUM TRICORNUTUM (BACILLARIOPHYCEAE) AND PLATYMONAS SP. (PRASINOPHYCEAE)1

Phaeodactylum tricornutum Bohlin, the one diatom known to lack a silicon requirement for growth, and the prasinophyte Platymonas sp. are two representatives of a taxonomically diverse group of planktonic algae that have been reported to take up Si without a demonstrable requirement for the element. For both species, removal of Si from solution during growth in batch culture has at least two components; true biological uptake throughout the growth of the culture, and spontaneous inorganic precipitation of a solid silicate phase–probably Mg₂Si₃O₈ (sepiolite)–under the elevated pH conditions that prevail late in batch growth. It is not clear to what extent previous observations of Si uptake by algae without siliceous frustules may be influenced by inorganic, non-cellular precipitation. The kinetics of true cellular uptake of Si are similar in Phaeodalylum and Platymonas, and different from those reported for the Si-requiring diatoms. Uptake follows hyperbolic saturation kinetics in both species, with half-saturation concentrations of 97.4 μM in Phaeodactylum and 80.9 μM in Platymonas, as compared to ca. 1–6 μM in diatoms that form siliceous frustules. Uptake by Phaeodactylum and Platymonas is not substrate-saturated until the dissolved Si concentration of the medium exceeds 200 μM. Concentrations this high do not occur in the surface layer of the ocean, and the kinetics suggest that both species deposit much less silica in nature than they can be induced to deposit in culture.     

Isolation and Characterization of a Hyphomicrobium Species and its Polysaccharide Formation from Methanol

A microorganism which could utilize methanol as the sole source of carbon and excreted a new polysaccharide was isolated from soil. This isolate was a stalked bacterium which reproduced by a budding process, and could grow on only methanol, formaldehyde or methylamines as the carbon source. The most suitable nitrogen source for growth was the ammonium ion. The optimum pH and temperature for growth were about 7.0 and 30°C, respectively. The cell growth was inhibited by blue light irradiation. Amino acid composition and fatty acid composition of the cells and electrophoretic behavior of methanol dehydrogenase were also studied. On the basis of these properties as well as taxonomical studies, the isolate (strain JTS-811) was identified as belonging to the genus Hyphomicrobium. This strain had different characteristics as compared to those described for other Hyphomicrobium isolates. At present, it is difficult to give a specific name to this strain, because classification of hyphomicrobia is not clear.     

Growth limitation of submerged aquatic macrophytes by inorganic carbon

1. This study determined the effects of CO₂ and HCO₃- enrichment on in situ growth of two submerged macrophytes, Elodea canadensis and Callitriche cophocarpa, in two Danish lakes: Lake Hampen and Lake Væng. Lake Hampen is an oligotrophic low-alkaline lake (0.4 meq ^?1) and Lake Væng is mesotrophic with an alkalinity of 1.1 meq 1-^?1. In Lake Hampen experiments were carried out throughout the growth season, whereas experiments in Lake Væng were restricted to late summer. The CO₂ and HCO₃-enrichment procedures used increased the concentration of free-CO₂ by 500–1000 μM and the concentration of HCO₃- by about 80 μM. 2. The concentration of free-CO₂ in Lake Hampen was about five times atmospheric equilibrium concentration (55 μM) in early summer declining to virtually zero at the end of summer. 3. Under ambient conditions Callitriche, which is restricted to CO₂ use, was unable to grow and survive in both lakes. In contrast, Elodea, which has the potential to use HCO₃- in photosynthesis, grew at rates varying from 0.046 to 0.080 day^?1 over the season. 4. Under CO₂ enrichment the growth rate of Callitriche varied from 0.089 to 0.124 day^?1 and for Elodea from 0.076 to 0.117 day^?1 over the season. Enrichment with HCO₃-affected Elodea only and only to a limited extent. This may be a result of insufficient increase in [HCO₃-] upon enrichment or to a limited capacity of the plants to take up HCO₃-. 5. The substantial stimulation of in situ growth of Elodea and Callitriche by enhanced concentrations of free-CO₂ shows that inorganic carbon is an important determinant of growth of submerged macrophytes and that inorganic carbon limitation of in situ growth may be a common phenomenon in nature, even in lakes with an alkalinity as high a 1 meq 1-^?1. Inorganic carbon, however, is only one of many parameters important for growth, and the growth rates of Elodea at both ambient and high free-CO₂ were closely coupled to day length and photon irradiance, indicating that light had an ultimate control on growth.     

Metabolism of allantoin in Hyphomicrobium species

Hyphomicrobium species are able to use allantoin as a nitrogen source for growth. Allantoin is broken down to glyoxylate and ammonia by the consecutive action of allantoinase, allantoicase, ureidoglycolase and urease.     

Improved growth conditions for hyphomicrobium sp. B-522 and two additional strains

Growth yields and rates of 3 hyphomicrobia were improved by varying components of or adding compounds to medium 337. Methanol (0.5% v/v), and similarly methylamine·HCl (3.38g/l), were optimal among 22 C-sources tested; increasing the methylamine·HCl concentration to 5.07g/l gave higher Hyphomicrobium B-522 yields but also prolonged lag periods. Ten C-sources (organic acids, alcohols) stimulated growth slightly but significantly, even in subcultures. Sugar compounds were not utilized. Strains B-522 and ZV-580 were stimulated by l-lysine and gluconate, while NQ-521 gr was stimulated by aspartate.N-Sources tested were inorganic (3), organic (3), or complex (3). (NH₄)₂SO₄ (0.5g/l) was optimal for strains ZV-580 and NQ-521 gr, but Hyphomicrobium B-522 grew best with urea-N. With NH ₄ ⁺ , strain B-522 grew as homogeneous suspension, all other N-sources caused clumping and pellicle formation. Inorganic requirements (PO ₄ ^3- , Mg, Ca, Fe, Mn, Mo) of strains B-522 and ZV-580 were optimized. Addition of Ni, Co, or Zn had no effect; metals 44 or Cu, resulted in growth inhibition.Vitamin B₁₂ stimulated Hyphomicrobium B-522; 2.5g/l B₁₂ decreased the doubling time from 9.3–10.8h to 5.4–5.8h. All combined single improvements resulted in a protein increase of 557% (B-522), 141% (NQ-521 gr), or 109% (ZV-580), respectively.