Anaerobic sequencing batch reactors for wastewater treatment: a developing technology

This paper describes and discusses the main problems related to anaerobic batch and fed-batch processes for wastewater treatment. A critical analysis of the literature evaluated the industrial application viability and proposed alternatives to improve operation and control of this system. Two approaches were presented in order to make this anaerobic discontinuous process feasible for industrial application: (1) optimization of the operating procedures in reactors containing self-immobilized sludge as granules, and (2) design of bioreactors with inert support media for biomass immobilization. Received: 22 May 2000 / Received revision: 20 July 2000 / Accepted: 21 July 2000     

Biological phosphate removal processes

Biological phosphate removal has become a reliable and well-understood process for wastewater treatment. This review describes the historical development of the process and the most important microbiological and process-engineering aspects. From a microbiological point of view, the role of␣poly(hydroxyalkanoates) as storage material in a dynamic process and the use of polyphosphate as an energy reserve are the most important findings. From a process-engineering point of view, the study of biological phosphate removal has shown that highly complex biological processes can be designed and controlled, provided that the importance of the prevailing microbiological ecological processes is recognised. Received: 3 April 1997 / Received revision: 9 June 1997 / Accepted: 14 June 1997     

Growth yield coefficients of Sphingomonas sp. strain P5 on various chlorophenols in chemostat culture

A polychlorophenol-degrading bacterium, Sphingomonas sp. strain P5, was grown in 2,6-dichlo-rophenol(26-DCP)-limited, 2,3,6-trichlorophenol(236-TCP)-limited, 2,4,6-trichlorophenol(246-TCP)-limited, 2,3,4,6-tetrachlorophenol(2346-TeCP)-limited, and pentachlorophenol(PCP)-limited chemostat cultures at a dilution rate of 0.02 ± 0.002 h⁻¹. The cultures were analyzed for the yield coefficient for growth on chlorophenol during steady-state conditions. The average growth yields coefficients (as carbon conversion efficiencies) were 0.252, 0.230, 0.219, 0.157, and 0.121 mol C mol C⁻¹ for 26-DCP, 236-TCP, 246-TCP, 2346-TeCP, and PCP respectively. The differences in growth yield can be interpreted in terms of the energetics of chlorinated carbon metabolism; i.e. substitution of the phenol moiety reduces the available metabolic energy by one electron per chlorine. The growth yield coefficients on chlorinated phenols were lower than the yield coefficients of heterotrophic growth reported in the literature on non-chlorinated and aliphatic compounds. Metabolic origins for low growth yield coefficients on (chlorinated) aromatic compounds are postulated. Received: 7 April 1997 / Received revision: 7 July 1997 / Accepted: 12 July 1997     

Purification of the main manganese peroxidase isoenzyme MnP2 from the white-rot fungus Nematoloma frowardii b19

The main manganese peroxidase isoenzyme MnP2 of the South American white-rot fungus Nematoloma frowardii b19 was purified to homogeneity using anion-exchange chromatography (Mono Q) and preparative isoelectric focusing. The purified enzyme has a molecular mass of 44 kDa and a pI of 3.2. Received: 23 May 1997 / Received revision: 1 July 1997 / Accepted: 4 July 1997     

Optimal conditions for bioconversion of ferulic acid into vanillic acid by Pseudomonas fluorescens BF13 cells

Pseudomonas fluorescens BF13 is especially capable of promoting the formation of vanillic acid during ferulic acid degradation. We studied the possibility of enhancing the formation of this intermediary metabolite by using suspensions of cells at high density. The bioconversion of ferulic into vanillic acid was affected by several parameters, such as the concentration of the biomass, the amount of ferulic acid that was treated, the carbon source on which the biomass was grown. The optimal yield of vanillic acid was obtained with 6 mg/ml cells pre-grown on p-coumaric acid and 2 mg/ml ferulic acid. Under these conditions the bioconversion rate was 95% in 5 h. Therefore BF13 strain represents a valid biocatalyst for the preparative synthesis of vanillic acid. Received: 1 July 1997 / Received revision: 28 October 1997 / Accepted: 16 November 1997     

Anthracyclines: isolation of overproducing strains by the selection and genetic recombination of putative regulatory mutants of Streptomycespeucetius var. caesius

In Streptomyces peucetius var. caesius, the production of anthracyclines was suppressed either by 330 mM d-glucose or 25 mM phosphate. In addition, the anthracycline doxorubicin and the glucose analogue 2-deoxyglucose inhibited the growth of this microorganism at concentrations of 0.025 mM and 10 mM respectively. Spontaneous and induced mutants, resistant to the action of these compounds, were isolated, tested and chosen by their ability to overproduce anthracyclines. Genetic recombination between representative mutants was carried out by the protoplast fusion technique. Some recombinants carrying resistance to doxorubicin, phosphate and 2-deoxyglucose produced more than 40-fold greater levels of anthracyclines than those obtained with the parental strain. This improvement resulted in total antibiotic titres of more than 2 g/l culture medium at 6 days of fermentation. Received: 14 April 1997 / Received revision: 19 June 1997 / Accepted: 4 July 1997     

Changes in intrinsic fluorescence during the production of viable but nonculturable Escherichia coli

The potential of intrinsic fluorescence spectroscopy to detect and differentiate viable but nonculturable bacteria in the presence of culturable bacteria was explored. Escherichia coli cells, starved for 210 days in nutrient-free normal saline, show new fluorescence emissions near 400 and 440 nm, and reduced emission near 340 nm. Received 7 July 1997/ Accepted in revised form 26 November 1997     

Expression and secretion of functional miniantibodies McPC603scFvDhlx in cell-wall-less L-form strains of Proteus mirabilis and Escherichia coli : A comparison of the synthesis capacities of L-form strains with an E. coli producer strain

The paper describes the synthesis of the phosphorylcholine-binding miniantibody McPC603scFvDhl x in cell-wall-less L-form strains of Escherichia coli and Proteus mirabilis. Cells of these strains were transformed with the plasmid pACK02scKan, carrying the miniantibody (miniAb) coding sequence under the control of the lac promoter. L-form transformants of both species were able to synthesize the functional miniAb as an extracellular soluble product. The highest quantities were obtained by P. mirabilis L-form strains after induction with 5 mM isopropyl β-d-thiogalactopyranoside (IPTG). Yields of 45–75 mg/l total antibody protein and of 10–18 mg/l functional miniAb were estimated in the growth medium of shaking cultures 40–80 h after induction with IPTG. About 10% of the active miniAb remained cell-bound. The yields of functional miniAb could be optimized by lowering the growth temperature from 37 °C to 26–32 °C and by supplementation of the medium with 80 mM sodium fumarate. A comparison of the specific activities revealed that the P. mirabilis L-form strains have a similar synthesis capacity (2–4 mg functional miniAb/g cell dry weight) to that of the producer strain E. coli RV308. The results show that the processes of correct folding and assembling of the miniAb molecules are possible without the periplasmic compartment. Received: 14 April 1997 / Received revision: 17 July 1997 / Accepted: 25 August 1997     

A new arabinofuranohydrolase from Bifidobacterium adolescentis able to remove arabinosyl residues from double-substituted xylose units in arabinoxylan

An arabinofuranohydrolase (AXH-d3) was purified from a cell-free extract of Bifidobacterium adolescentis DSM 20083. The enzyme had a molecular mass of approximately 100 kDa as determined by gel filtration. It displayed maximum activity at pH 6 and 30 °C. Using an arabinoxylan-derived oligosaccharide containing double-substituted xylopyranosyl residues established that the enzyme specifically released terminal arabinofuranosyl residues linked to C-3 of double-substituted xylopyranosyl residues. In addition, this arabinofuranohydrolase released arabinosyl groups from wheat flour arabinoxylan polymer but showed no activity towards p-nitrophenyl α-l-arabinofuranoside or towards sugar-beet arabinan, soy arabinogalactan, arabino-oligosaccharides and arabinogalacto-oligosaccharides. Received: 15 July 1996 / Received revision: 18 October 1996 / Accepted: 18 October 1996     

Application of agro-industrial by-products for riboflavin production by Eremothecium ashbyii NRRL 1363

Riboflavin production is significantly determined by the type and initial concentration of the carbon and nitrogen sources and also by other flavinogenic stimulants. Using an optimum carbon and nitrogen concentration, an industrial fermentation medium has been designed with molasses as the carbon source and peanut seed cake as the nitrogen source. In addition the stimulatory effect of some of the low-cost agro-industrial by-products on riboflavin yield was investigated. Received: 10 March 1996 / Received revision: 25 June 1996 / Accepted: 14 July 1996     

Effects of phenolic compounds on the growth and the fatty acid composition of Lactobacillus plantarum

The effects of different phenolic compound concentrations on the fatty acid composition of Lactobacillus plantarum isolated from traditional home-made olive brines were determined. Increasing amounts of caffeic and ferulic acids induced a gradual increase in the amounts of myristic, palmitoleic, stearic and 9,10-methylenehexadecanoic (C17Δ, where Δ represents the cyclopropane group) acid with a concomitant decrease of lactobacillic acid (C₁₉Δ). On the other hand, the addition of tannins induced an increase in the C₁₉Δ level at the expense of vaccenic acid content. The presence of acidic phenols and tannins also affected bacterial growth, inducing the most obvious effect with tannin at 1 g l⁻¹. Received: 1 July 1997 / Received revision: 9 September 1997 / Accepted: 15 September 1997     

Microbial oxidative metabolism of diclofenac: production of 4′-hydroxydiclofenac using Epiccocum nigrum IMI354292

The 4′-hydroxylated metabolite of diclofenac was produced by biocatalysis for probing specific human drug-metabolising enzymes (CYP2C9). An initial screen of 11 microorganisms was carried out (50 ml scale) to identify the organism best suited to the regioselective conversion of diclofenac to its 4′-hydroxylated metabolite. From this screen, the fungus Epicoccum nigrum IMI354292 was selected as the most suitable microorganism. Scale-up was carried out in a 30-l fermenter to which 2 g diclofenac was added. After 48 h, 50% of the diclofenac had been converted to it 4′-hydroxylated metabolite. The broth was then extracted with ethyl acetate and purified by chromatography and crystallisation. This yielded 0.3 g 4′-hydroxydiclofenac with a purity of at least 99%. The 4′-hydroxydiclofenac produced by E. nigrum was characterised by HPLC, mass spectrometry and NMR. Received: 28 July 1997 / Received revision: 8 December 1997 / Accepted: 14 December 1997     

New insights and novel developments in clostridial acetone/butanol/isopropanol fermentation

Clostridial acetone/butanol fermentation used to rank second only to ethanol fermentation by yeast in its scale of production and thus is one of the largest biotechnological processes known. Its decline since about 1950 has been caused by increasing substrate costs and the availability of much cheaper feedstocks for chemical solvent synthesis by the petrochemical industry. The so-called oil crisis in 1973 led to renewed interest in novel fermentation and product recovery technologies as well as in the metabolism and genetics of the bacterial species involved. As a consequence, almost all of the enzymes leading to solvent formation are known, their genes have been sequenced (in fact, Clostridium acetobutylicum has been recently included in the microbial genome sequencing project), the regulatory mechanisms controlling solventogenesis have begun to emerge and recombinant DNA techniques have been developed for these clostridia to construct specific production strains. In parallel, cheap agricultural-waste-based feedstocks have been exploited for their potential as novel substrates, continuous culture methods have been successfully established and new on-line product recovery technologies are now available, such as gas stripping, liquid/liquid extraction, and membrane-based methods. In combination with these achievements, a reintroduction of acetone/butanol fermentation on an industrial scale seems to be economically feasible, a view that is supported by a new pilot plant in Austria recently coming into operation. Received: 18 December 1997 / Received revision: 27 January 1998 / Accepted: 27 January 1998     

High concentrations of fatty acids affect the lipase treatment of softwood thermomechanical pulps

Triglycerides, a major class of wood extractives, contribute to the colloidal pitch that initiates pitch deposits. Because industrial or pilot-scale treatments with lipolytic enzymes to reduce triglyceride concentrations in pulp have not been successful in North America, we investigated such treatments at a laboratory scale. Different batches of industrial softwood chemithermomechanical pulps (CTMP) were treated with a range of concentrations of two commercial lipases: Resinase A 2X (Novo Nordisk AG) and Lipidase 10 000 (American Laboratories Inc.). A pilot-scale thermomechanical pulp (TMP) made from the same wood as the CTMP, but without the sodium hydrosulfite used in the CTMP, was also treated with the lipases. While triglycerides decreased in all the pulp treatments, the extent of their hydrolysis varied according to the ratio of triglyceride to the fatty/resin acid fraction. As this ratio can vary significantly in softwood TMP and CTMP, the success of industrial treatments of softwood mechanical pulps by commercial lipases may be related to variations in this ratio. Supporting this, adding linoleic acid to an extractives-free pulp that was spiked with olive oil reduced lipase activity by up to 55%. Received: 7 August 1997 / Received last revision: 8 December 1997 / Accepted: 14 December 1997     

Possible Involvement of Cysteine and Histidine Residues in the (NH4 + + Na+)-Activated ATPase of an Anaerobic Alkaliphile, Amphibacillus xylanus

Effect of various inhibitors on the (NH₄ ⁺ + Na⁺)-activated ATPase of an anaerobic alkaliphile, Ep01(a strain of Amphibacillus xylanus), was examined. Among the chemicals tested, the enzyme was drastically inactivated by p-chloromercuribenzoic acid and diethyl pyrocarbonate. The ATPase activity of the enzyme, which was inactivated by p-chloromercuribenzoic acid and diethyl pyrocarbonate, was remarkably restored by β-mercaptoethanol and hydroxylamine, respectively, suggesting the involvement of cysteine and histidine residues in the enzyme activity. Analysis of the inhibition kinetics by diethyl pyrocarbonate indicated that modification of a single histidine residue per ATPase molecule was sufficient to inactivate the enzyme. Received: 2 June 1997 / Accepted: 7 July 1997     

Metal ion accumulation by immobilised cells of Brevibacterium sp

This paper explores the use of an experimental system based on polyacrylamide-entrapped cells of Brevibacterium sp strain PBZ for the removal of metal ions from solutions. Experiments were performed in columns filled with the immobilised cells and challenged with influents containing 20 mg L⁻¹ of lead and 10 mg L⁻¹ of cadmium. The cells were able to accumulate lead (about 40 mg g⁻¹ dry biomass) and, to a lesser extent, cadmium (about 13 mg g⁻¹ dry biomass) from solutions. In the presence of 0.4 g L⁻¹ of glucose, the cells removed up to 53% of lead. Lead competed with cadmium for attachment to the binding sites when a solution containing both the metals was applied. Lead removal occurred by a combination of fast physico-chemical adsorption and prolonged low rate accumulation mediated by cell metabolism. The biosorptive capacity of the cells was sensitive to pH. Desorption of the metal with EDTA restored the binding capability of the cells. Received 07 July 1997/ Accepted in revised form 26 November 1997     

Cloning, sequencing and overexpression of a Rhodothermus marinus gene encoding a thermostable cellulase of glycosyl hydrolase family 12

A gene library from the thermophilic eubacterium Rhodothermus marinus, strain ITI 378, was constructed in pUC18 and transformed into Escherichia coli. Of 5400 transformants, 3 were active on carboxymethylcellulose. Three plasmids conferring cellulase activity were purified and were all found to contain the same cellulase gene, celA. The open reading frame for the celA gene is 780 base pairs and encodes a protein of 260 amino acids with a calculated molecular mass of 28.8 kDa. The amino acid sequence shows homology with cellulases in glycosyl hydrolase family 12. The celA gene was overexpressed in E. coli when the pET23, T7 phage RNA polymerase system was used. The enzyme showed activity on carboxymethylcellulose and lichenan, but not on birch xylan or laminarin. The expressed enzyme had six terminal histidine residues and was purified by using a nickel nitrilotriacetate column. The enzyme had a pH optimum of 6–7 and its highest measured initial activity at 100 °C. The heat stability of the enzyme was increased by removal of the histidine residues. It then retained 75% of its activity after 8 h at 90 °C. Received: 5 August 1997 / Received revision: 6 November 1997 / Accepted: 7 November 1997     

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     

Substrate inhibition under stationary growth conditions – nutristat experiments with Ralstonia eutropha JMP 134 during growth on phenol and 2,4-dichlorophenoxyacetate

Ralstonia eutropha (formerly Alcaligenes eutrophus) JMP 134 was continuously grown on phenol and 2,4-dichlorophenoxyacetate at elevated levels of stationary substrate concentration by using the nutristat principle in order to study the physiological impact exerted by these toxic substrates. Growth at stationary concentrations of both the substrates resulted in the reduction of growth efficiency and growth rate. The growth yield data revealed a pronounced dependence on the substrate concentration, and the growth yield increasingly diminished with rising substrate concentration. Inhibition was more pronounced with 2,4-dichlorophenoxyacetate, which reduced the growth yield coefficient by 50% at a substrate concentration of 0.1–0.25 mM. The same effect was obtained with phenol at about 5 mM. The growth rate profile had two distinct phases: after an initially strong reduction, the rate levelled-off at higher substrate concentrations. Standardizing the inhibition profiles, by taking into account the maximum effect after extrapolating the data to zero growth yield, revealed an almost identical pattern with both substrates, indicating some common mechanism. The growth yield data show that an increased amount of energy is required for both growth and maintenance. Homeostatic work was increased by a factor of 8 at 75% inhibition; growth collapsed once this amount of energy was no longer available. The effects are discussed with respect to the properties of these substrates functioning as potential uncouplers of energy conservation. Received: 5 June 1997 / Received revision: 7 July 1997 / Accepted: 12 July 1997     

Continuous vapor-phase trichloroethylene biofiltration using hydrocarbon-enriched compost as filtration matrix

Two sources of finished compost material were examined for the capacity to support trichloroethylene(TCE)-degrading microbial populations in a gas-phase bioreactor. Gaseous hydrocarbon was passed through the bioreactor to stimulate cometabolic oxidation of TCE. Significant differences in TCE removal efficiencies were observed between the two compost types, and between hydrocarbon-stimulated and non-stimulated compost. At an average column retention time of 5.6 min, deciduous leaf debris compost removed more than 95% of a 5–50 ppm (by vol.) TCE gas stream, whereas less than 15% removal was observed under similar conditions with a woodchip and bark compost. Trichloroethylene removal efficiency varied with the hydrocarbon-stimulation regime employed, although propane and methane stimulated TCE degradation equally well. Amendment of compost with granular activated carbon substantially increased biological TCE removal. Differences in TCE removal efficiencies observed between the two compost types and between hydrocarbon-stimulated and non-stimulated composts were investigated in terms of changes in the overall heterotrophic microbial populations by using community-level physiological profile analysis. Received: 14 April 1997 / Received revision: 21 July 1997 / Accepted: 25 August 1997