Production of polyhydroxyalkanoic acids by Ralstonia eutropha and Pseudomonas oleovorans from an oil remaining from biotechnological rhamnose production

Screening experiments identified several bacteria which were able to use residual oil from biotechnological rhamnose production as a carbon source for growth. Ralstonia eutropha H16 and Pseudomonas oleovorans were able to use this waste material as the sole carbon source for growth and for the accumulation of polyhydroxyalkanoic acids (PHA). R. eutropha and P. oleovorans accumulated PHA amounting to 41.3% and 38.9%, respectively, of the cell dry mass, when these strains were cultivated in mineral salt medium with the oil from the rhamnose production as the sole carbon source. The accumulated PHA isolated from R. eutropha consisted of only 3-hydroxybutyric acid, whereas the PHA isolated from P. oleovorans consisted of 3-hydroxyhexanoic acid, 3-hydroxyoctanoic acid, 3-hydroxy decanoic acid, and 3-hydroxydodecanoic acid. The composition was confirmed by gas chromatography of the isolated polyesters. Batch and fed-batch cultivations in stirred-tank reactors were done. Received: 15 June 1999 / Received revision: 10 August 1999 / Accepted: 13 August 1999     

Accumulation and mobilization of storage lipids by Rhodococcus opacus PD630 and Rhodococcus ruber NCIMB 40126

The time course of the accumulation of triacylglycerols (TAGs) in Rhodococcus opacus PD630 or of TAGs plus polyhydroxyalkanoates (PHA) in Rhodococcus ruber NCIMB 40126 with gluconate or glucose as carbon source, respectively, was studied. In addition, we examined the mobilization of these storage compounds in the absence of a carbon source. R. opacus accumulated TAGs only after the exhaustion of ammonium in the medium, and, with a fixed concentration of the carbon source, the amounts of TAGs in the cells increased with decreasing concentrations of ammonium in the medium. When these cells were incubated in the absence of an additional carbon source, about 90% of these TAGs were mobilized and used as endogenous carbon source, particularly if ammonium was available. R. ruber accumulated a copolyester consisting of 3-hydroxybutyrate and 3-hydroxyvalerate already during the early exponential growth phase, whereas TAGs were synthesized and accumulated mainly during the late exponential and stationary growth phases. In the stationary growth phase, synthesis of TAGs continued, whereas PHA was partially mobilized. In the absence of an additional carbon source but in the presence of ammonium, mobilization of TAGs started first and was then paralleled by the mobilization of PHA, resulting in an approximately 90% and 80% decrease of these storage compounds, respectively. During the accumulation phase, interesting shifts in the composition of the two storage compounds occurred, indicating that the substrates of the PHA synthase and the TAG synthesizing enzymes were provided to varying extents, depending on whether the cells were in the early or late exponential or in the stationary growth phase. Received: 12 January 2000 / Received revision: 22 February 2000 / Accepted: 25 February 2000     

Processes of liquefaction/solubilization of Spanish coals by microorganisms

Several fundamental aspects of microbial coal liquefaction/solubilization were studied. The liquefied/solubilized products from coal by microorganisms were analysed. The liquid products analysed by IR titration and UV/visible spectrometry showed some alterations with regard to the original coal. Humic acids extracted from the liquefied lignite showed a reduction in the average molecular weight and a increase in the condensation index, probably due to depolymerization caused by microorganisms. The mechanisms implicated in coal biosolubilization by two fungal strains, M2 (Trichoderma sp.) and M4 (Penicillium sp.) were also studied. Extracellular peroxidase, esterase and phenoloxidase enzymes appear to be involved in coal solubilization. Received: 15 June 1998 / Received revision: 23 November 1998 / Accepted: 29 November 1998     

Degradation of alicyclic molecules by Rhodococcus ruber CD4

The present work describes investigations on the bacterial degradation of the alicyclic molecule cyclododecane. It represents a structure where the initial degradative steps have to be similar to a “subterminal” attack as there is no “terminal” part of the molecule. We were able to show that the gram-positive bacterium Rhodococcus ruber CD4 DSM 44394 oxidizes cyclododecane to the corresponding alcohol and ketone, the latter being subject to ring fission by a Baeyer-Villiger oxygenase. This key enzyme is an NADPH- and O₂-dependent flavoprotein with a substrate specificity for bigger rings. The further metabolism of the resulting lactone gives rise to an ω-hydroxyalkanoic acid that is susceptible to common β-oxidation. Due to its alicyclic character and its ring size, cyclododecane is comparable to aliphatic bridge components that are an important element in the coal texture. They contribute to the three-dimensional coal structure and thus could serve as a valuable target for the oxidative abilities of R. ruber CD4 to reduce the molecular mass of coal. Received: 2 July 1998 / Received revision: 27 October 1998 / Accepted: 30 October 1998     

Biodegradation of polyhydroxyalkanoic acids

Stimulated by the commercial availability of bacteriologically produced polyesters such as poly[(R)-3-hydroxybutyric acid], and encouraged by the discovery of new constituents of polyhydroxyalkanoic acids (PHA), a considerable body of knowledge on the metabolism of PHA in microorganisms has accumulated. The objective of this essay is to give an overview on the biodegradation of PHA. The following topics are discussed: (i) general considerations of PHA degradation, (ii) methods for identification and isolation of PHA-degrading microorganisms, (iii) characterization of PHA-degrading microorganisms, (iv) biochemical properties of PHA depolymerases, (v) mechanisms of PHA hydrolysis, (vi) regulation of PHA depolymerase synthesis, (vii) molecular biology of PHA depolymerases, (viii) influence of the physicochemical properties of PHA on its biodegradability, (ix) degradation of polyesters related to PHA, (x) biotechnological aspects of PHA and PHA depolymerases. Received: 28 May 1996 / Received revision: 5 August 1996 / Accepted: 12 August 1996     

Lipid storage compounds in marine bacteria

Forty psychrophile or psychrotrophic crude-oil-utilizing marine bacteria were investigated for their ability to accumulate lipid storage compounds in the cytoplasm during cultivation under nitrogen-limiting conditions. Most of them (73%) were able to accumulate specialized lipids like polyhydroxyalkanoic acids (PHA) while other lipids such as wax esters occurred in two isolates. Accumulation of PHA occurred predominantly at low temperatures (4–20 °C) as demonstrated for three isolates. Electron microscopy revealed polyphosphate inclusions occurring in two isolates in addition to PHA. Cells of the isolate Acinetobacter sp. 211 were able to synthesize and accumulate lipid inclusions during growth on acetate, ethanol, olive oil, hexadecanol and heptadecane. The composition of the lipid inclusions depended on the compounds provided as carbon source. Wax esters and acylglycerols occurred mainly during the cultivation on olive oil; in contrast, wax esters and free alcohols occurred during cultivation on hexadecanol. Total fatty acids in cells of the Acinetobacter sp. 211 amounted to 25% of the cellular dry weight in olive-oil-grown cells. Palmitic acid was the main fatty acid in the lipids when the cells were cultivated on acetate or ethanol (44% and 32% of total fatty acids respectively). In contrast, fatty acids occurring in the lipids during cultivation on hexadecanol, heptadecane or olive oil were related to the carbon source. The fatty acids present in the accumulated lipids consisted predominantly of saturated and unsaturated straight-chain fatty acids with a chain length ranging from 12 to 18 carbon atoms. Analysis of the lipid-granule-associated proteins in cells of Acinetobacter sp. 211 revealed a protein of 39 kDa as the predominant protein species. Received: 2 July 1996 / Received revision: 3 September 1996 / Accepted: 28 September 1996     

A transketolase mutant of Corynebacterium glutamicum

A transketolase mutant was first isolated from Corynebacterium glutamicum, an organism of industrial importance. The mutant strain exhibited an absolute requirement for shikimic acid or the aromatic amino acids and vitamins for growth, and also failed to grow on ribose or gluconic acid as sole carbon source, even with the aromatic supplement. All of these defective properties were fully restored in spontaneous revertants, indicating the existence of a single transketolase in C. glutamicum that was indispensable both for aromatic biosynthesis and for utilization of these carbohydrates in vivo. The transketolase mutant accumulated ribulose extracellularly when cultivated in glucose medium with shikimic acid, but no ribose was detected. Received: 10 April 1998 / Received revision: 26 May 1998 / Accepted: 14 June 1998     

Intermediary sulfur compounds in pyrite oxidation: implications for bioleaching and biodepyritization of coal

Accumulation of elemental sulfur during pyrite oxidation lowers the efficiency of coal desulfurization and bioleaching. In the case of pyrite bioleaching by Leptospirillum ferrooxidans, an iron(II)-ion-oxidizing organism without sulfur-oxidizing capacity, from the pyritic sulfur moiety about 10% elemental sulfur, 2% pentathionate, and 1% tetrathionate accumulated by a recently described cyclic pyrite oxidation mechanism. In the case of pure cultures of Thiobacillus ferrooxidans and mixed cultures of L. ferrooxidans and T. thiooxidans, pyrite was nearly completely oxidized to sulfate because of the capacity of these cultures to oxidize both iron(II) ions and sulfur compounds. Pyrite oxidation in acidic solutions, mediated chemically by iron(III) ion, resulted in an accumulation of similar amounts of sulfur compounds as obtained with L. ferrooxidans. Changes of pH to values below 2 or in the iron ion concentration are not decisive for diverting the flux of sulfur compounds. The literature on pyrite bioleaching is in agreement with the findings indicating that the chemistry of direct and indirect pyrite leaching is identical. Received: 20 April 1998 / Received revision: 27 August 1998 / Accepted: 3 September 1998     

In vivo-decolorization of coal-derived humic acids by laccase-excreting fungus Trametes versicolor

Lignite (brown coal) can be liquefied/solubilized with several fungi by different mechanisms. When applied industrially, only catalytic mechanisms can compete with chemical methods. The well-known fungal ligninolytic peroxidases are at a disadvantage, in that the relatively expensive hydrogen peroxide must be used as a cofactor. Comparing several fungal strains, we observed that the fungus Trametes versicolor is able to decolorize coal-derived humic acids, producing a considerable amount of laccase in the process. During this reaction the amount of humic acids decreases whilst that of fulvic acids increases; this was verified by optical density measurement and GPC after the two substance classes had been separated. Received: 27 August 1998 / Received revision: 4 November 1998 / Accepted: 7 November 1998     

Chemically defined media for commercial fermentations

The use of chemically defined media is gaining popularity in some commercial fermentations, particularly for the preparation of biological products. Although these media are still not frequently developed for industrial processes, they do exhibit favorable characteristics at large scale that are not observed with traditional complex media. This review focuses on the application, development, and practical considerations, especially process economics, of fermentations in chemically defined media in an industrial environment. Received: 3 August 1998 / Received revision: 16 November 1998 / Accepted: 21 November 1998     

Mechanisms of coal solubilization by the deuteromycetes Trichoderma atroviride and Fusarium oxysporum

Three different mechanisms can be envisaged that are used by fungi to solubilize coal: the production of alkaline substances, the extrusion of chelators and, of special interest in the scope of biotechnology, the action of enzymes. Whether these mechanisms are operating separately or in various combinations has not yet been finally assessed. The two deuteromycetes Fusarium oxysporum and Trichoderma atroviride solubilize coal by synergistic effects of various different mechanisms depending on the cell metabolism. F. oxysporum seems to solubilize coal by increasing the pH of the mycelial surroundings and by the action of chelators induced during growth in glutamate-containing media (without involvement of enzymes). T. atroviride, on the other hand, appears to use, in addition to an alkaline pH and a high chelator activity, at least two classes of enzyme activity to attack coal: hydrolytic activity for coal solubilization and ligninolytic activity for degradation of humic acids. Received: 3 February 1998 / Received revision: 31 August 1998 / Accepted: 3 September 1998     

Evidence for and expression of a laccase gene in three basidiomycetes degrading humic acids

The majority of lignin-degrading basidiomycetes are able to depolymerize humic acids. In this presentation the relationship and possible similarities between enzymes involved in lignin degradation and humic acid depolymerization were examined on the genetic level. We have cloned fragments of the gene encoding the extracellular ligninolytic enzyme laccase from Clitocybula dusenii, Nematoloma frowardii and a fungal strain designated i63-2, and compared the three sequences with those of several other published laccase genes. The sequenced fragments displayed a high homology both on the DNA (97%–77%) and amino acid (100%–85%) level. Furthermore, the expression of this gene in the above-mentioned fungi was demonstrated by a nested polymerase chain reaction with cDNA as template. Received: 3 February 1998 / Received revision: 31 August 1998 / Accepted: 3 September 1998     

Efficient production of polyhydroxyalkanoates from plant oils by Alcaligenes eutrophus and its recombinant strain

The ability of Alcaligenes eutrophus to grow and produce polyhydroxyalkanoates (PHA) on plant oils was evaluated. When olive oil, corn oil, or palm oil was fed as a sole carbon source, the wild-type strain of A. eutrophus grew well and accumulated poly(3-hydroxybutyrate) homopolymer up to approximately 80% (w/w) of the cell dry weight during its stationary growth phase. In addition, a recombinant strain of A. eutrophus PHB⁻4 (a PHA-negative mutant), harboring a PHA synthase gene from Aeromonas caviae, was revealed to produce a random copolyester of 3-hydroxybutyrate and 3-hydroxyhexanoate from these plant oils with a high cellular content (approximately 80% w/w). The mole fraction of 3-hydroxyhexanoate units was 4–5 mol% whatever the structure of the triglycerides fed. The polyesters produced by the A. eutrophus strains from olive oil were 200–400 kDa (the number-average molecular mass). The results demonstrate that renewable and inexpensive plant oils are excellent carbon sources for efficient production of PHA using A. eutrophus strains. Received: 3 September 1997 / Received revision: 10 November 1997 / Accepted: 16 November 1997     

Transformation of macromolecules from a brown coal by lignin peroxidase

Indirect evidence has suggested that lignin peroxidase (LiP) of the white-rot fungus Phanerochaete chrysosporium catalyses oxidative decolourisation and depolymerisation of macromolecules from brown coal in vivo. In this study we show that LiP catalyses these transformations in vitro. Unmethylated (USC45 coal) and methylated (MWSC6 coal) fractions of solubilised macromolecules (M _r > 30 000) from a brown coal were treated with a semi-purified preparation of LiP isozymes from P. chrysosporium. Both coal fractions were decolourised, losing between 26% and 39% of their absorbance at both 280 nm and 400 nm, in reactions that had an absolute requirement for H₂O₂ and veratryl alcohol. Neither coal fraction was transformed when the enzyme was heat-inactivated or in the presence of the LiP inhibitor metavanadate. Gel-permeation chromatography showed that MWSC6 coal but not USC45 was depolymerised and yielded low-molecular-mass (M _r < 30 000) fragments. Nine monomeric products were identified by GC-MS. Received: 20 March 1998 / Received revision: 3 September 1998 / Accepted: 3 September 1998     

An amperometric enzyme-linked immunosensor for Nitrobacter

A new amperometric enzyme-linked immunoassay for specific enumeration of Nitrobacter has been developed. This assay uses an electrode made of glassy carbon, on which the immunological reaction is carried out. The method is based on a competitive immunoassay principle, utilising monoclonal primary antibody and alkaline-phosphatase-labelled secondary antibody. The enzyme substrate 5-bromo-4-chloro-3-indolyl phosphate generates an electroactive product which is amperometrically detected. The effects of different parameters on the performance of the sensor have been studied. Quantitative detection of Nitrobacter using the immunosensor has been compared to a previously developed enzyme-linked immunosorbent assay showing compatible results. In addition, the overall assay time can be shortened with this new sensor. A detection limit of approximately 3 × 10⁶ Nitrobacter cells/ml was obtained. Received: 27 May 1998 / Received revision: 28 August 1998 / Accepted: 28 August 1998     

The influence of carbon source on the level and composition of ceramides of the Candida lipolytica yeast

Candida lipolytica yeast was grown batchwise on two different carbon sources, glucose and n-hexadecane. Free ceramides were quantitatively isolated from sphingolipid fractions of total lipids by a combination of column chromatography and preparative thin-layer chromatography. Their composition, after acid methanolysis, was analysed by gas-liquid chromatography. The ceramide content accounted for 2.6% of the total cell lipids in hexadecane-grown cells, which was 1.5 times higher than in glucose-grown cells. The fatty acid composition of ceramides was characterized by the predominance of fatty acids shorter than 20 carbon atoms and by high concentrations of fatty acids with 16 carbon atoms after growth on both carbon sources. The dominant fatty acid was hydroxylated 16:0 in the glucose-grown cells and 16:0 in the hexadecane-grown cells. The striking finding was the low degree of fatty acid hydroxylation and relatively high proportion of odd-numbered fatty acids in ceramide of the n-hexadecane-grown cells. The ceramides contained an unusual long-chain base composition. In hexadecane-grown cells more than 60% of the long-chain bases were C₁₉ phytosphingosine. In glucose-grown cells more than one-half of the total long-chain bases were tetrahydroxy bases, 4,5-dihydroxysphinganine and 4,5-dihydroxyeicosasphinganine. Received: 20 April 1998 / Received revision: 10 July 1998 / Accepted: 29 July 1998     

Identification of 5-hydroxyhexanoic acid, 4-hydroxyheptanoic acid and 4-hydroxyoctanoic acid as new constituents of bacterial polyhydroxyalkanoic acids

 A recombinant strain of Pseudomonas putida GPp104 (pHP1014::E146), which expressed the polyhydroxyalkanoic acid (PHA) synthase of Thiocapsa pfennigii exhibiting an unusual substrate specificity at a high level was incubated in two-stage batch or fed-batch accumulation experiments with 5-hydroxyhexanoic acid (5HHx) as carbon source in the second cultivation phase, copolyesters of 3-hydroxybutyric acid (3HB) plus 5HHx, or of 3HB, 3-hydroxyhexanoic acid (3HHx) plus 5HHx were accumulated as revealed by gas-chromatographic and ¹³C-NMR spectroscopic analysis. When the recombinant P. putida GPp104 was incubated with 4-hydroxyheptanoic acid (4HHp) as carbon source in the second cultivation phase, a copolyester consisting of 3HB, 3-hydroxyvaleric acid and 3- and 4-hydroxyheptanoic acid accumulated. Providing 4-hydroxyoctanoic acid as carbon source in the second cultivation phase led to the accumulation of a polyester that contained 1–2 mol% 4-hydroxyoctanoic acid besides 3-hydroxyoctanoic acid, 3HHx, 3-hydroxyvaleric acid and 3HB. In addition to PHA containing these new constituents, PHA with 4-hydroxyvaleric acid was accumulated from laevulinic acid. Eleven strains from five genera have been also analysed for their ability to utilize different carbon sources for colony growth, which might serve as potential precursors for the biosynthesis of PHA with unusual constituents. Although most of the carbon sources were utilized by some strains for colony growth, accumulation experiments gave no evidence for the accumulation of new PHA by these wild-type strains. Received: 22 April/Received revision: 23 May 1996/Accepted: 2 June 1996     

Fungal biosolubilization of Rhenish brown coal monitored by Curie-point pyrolysis/gas chromatography/mass spectrometry using tetraethylammonium hydroxide

Residues and coal fractions that remained after the biosolubilization of Rhenish brown coal by strains of Lentinula edodes and Trametes versicolor have been studied by Curie-point pyrolysis/gas chromatography/mass spectrometry using tetraethylammonium hydroxide (NEt₄OH) at 610 °C. To differentiate methyl derivatives of esters and ethers from free or bound hydroxyl and carboxyl groups NEt₄OH was used in the thermochemolysis experiments instead the commonly used tetramethylammonium hydroxide. A comparison of humic acid fractions before and after fungal attack shows considerable alteration of the soluble macromolecules of coal. Depending on the coal fraction studied and the fungi used, the assortment of fatty acid esters released during the pyrolysis varies significantly. Furthermore, dicarbonic acid ethyl diesters as well as ethyl derivatives of aromatic ethers and acids yield information about humic acid structure and the biosolubilization of brown coal. Variations in the mixture produced are possibly caused by differences in the pattern of extracellular enzymes secreted that attack the macromolecular structural elements of brown coal. Therefore pyrolysis of native and microbiologically altered geomacromolecules using NEt₄OH allows one to differentiate between free hydroxyl groups as well as substances that are attached to humic substances via ester or ether bridges, and their methylated counterparts. Received: 13 July 1998 / Received revision: 12 October 1998 / Accepted: 16 October 1998     

Establishment of a gene transfer system for Rhodococcus opacus PD630 based on electroporation and its application for recombinant biosynthesis of poly(3-hydroxyalkanoic acids)

A gene transfer system for Rhodococcus opacus PD630 based on electroporation was established and optimized employing the Escherichia coli-Rhodococcus shuttle vectors pNC9501 and pNC9503 as well as the E. coli-Corynebacterium glutamicum shuttle vector pJC1 as suitable cloning vectors for R. opacus PD630, resulting in transformation efficiencies up to 1.5 × 10⁵ CFUs/μg plasmid DNA. Applying the optimized electroporation protocol to the pNC9501-derivatives pAK68 and pAK71 harboring the entire PHB synthesis operon from Ralstonia eutropha and the PHA synthase gene phaC1 from Pseudomonas aeruginosa, respectively, recombinant PHA biosynthesis was established in R. opacus PD630 and the TAG-negative mutant ROM34. Plasmid pAK68 enabled synthesis and accumulation of poly(3HB) in R. opacus PD630 and ROM34 during cultivation under storage conditions from 1% (w/v) gluconate, of poly(3HB-co-3HV) from 0.2% (w/v) propionate and of poly(3HV) from 0.1% (w/v) valerate. Under storage conditions, recombinant strains of PD630 and ROM34 harboring pAK71 were able to synthesize and accumulate PHA of the medium chain length hydroxyalkanoic acids 3HHx, 3HO, 3HD and 3HDD from 0.1% (w/v) hexadecane or octadecane and a copolyester composed of 3HHp, 3HN and 3HUD from 0.1% (w/v) pentadecane or heptadecane. In the recombinant strains of PD630 and ROM34, the thiostrepton-induced overexpression of a 20 kDa protein was observed with its N-terminus exhibiting a homology of 60% identical amino acids to TipA from Streptomyces lividans. Received: 13 March 1999 / Received revision: 18 May 1999 / Accepted: 21 May 1999     

Biotechnology and microbiology of coal degradation

For several years it has been known that fungi and bacteria can attack and even liquefy low rank coals. This review covers the progress in coal biotechnology and microbiology, mainly during the last decade, from describing the first effects to elucidating the mechanisms used by the microorganisms. More than one mechanism is responsible for microbial coal degradation/liquefaction: oxidative enzymes (peroxidases, laccases), hydrolytic enzymes (esterases), alkaline metabolites and natural chelators. Due to the heterogeneous structure of coal, which is described in one section, and for economic reasons the review focuses on the enzymatic depolymerization of brown coal. Approaches which seem not so promising are discussed (anaerobic, reductive pathways, chemical pretreatment). Finally the possible applications and products in this field are summarized, as lignite with a worldwide production of about 940 million tons a year will continue to play an important economic role in the future. Received: 19 October 1998 / Received revision: 16 December 1998 / Accepted: 21 December 1998