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Applied Microbiology and Biotechnology (v.68, #1)

Eco-Efficiency Analysis of biotechnological processes by Peter Saling (pp. 1-8).

Eco-Efficiency has been variously defined and analytically implemented by several workers. In most cases, Eco-Efficiency is taken to mean the ecological optimization of overall systems while not disregarding economic factors. Eco-Efficiency should increase the positive ecological performance of a commercial company in relation to economic value creation—or to reduce negative effects. Several companies use Eco-Efficiency Analysis for decision-making processes; and industrial examples of best practices in developing and implementing Eco-Efficiency have been reviewed. They clearly demonstrate the environmental and business benefits of Eco-Efficiency. An instrument for the early recognition and systematic detection of economic and environmental opportunities and risks for production processes in the chemical industry began use in 1997, since when different new features have been developed, leading to many examples. This powerful Eco-Efficiency Analysis allows a feasibility evaluation of existing and future business activities and is applied by BASF. In many cases, decision-makers are able to choose among alternative processes for making a product.

Biotechnological production and applications of coenzyme Q₁₀ by Jin-Ho Choi; Yeon-Woo Ryu; Jin-Ho Seo (pp. 9-15).

Coenzyme Q₁₀ is widely used as an essential component of ATP generation in the oxidative phosphorylation process and as an antioxidant preventing lipid peroxidation and scavenging superoxide. It is also recommended as a supplement to 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors. Research efforts on the production of coenzyme Q₁₀ by microorganisms focus on the development of potent strains by conventional mutagenesis and metabolic engineering, analysis and modification of the key metabolic pathways and optimization of fermentation strategies. Especially, random mutants with drugs resistance show a high coenzyme Q₁₀ concentration. Metabolic engineering techniques have been applied to improve coenzyme Q₁₀ production. The key enzymes involved in the coenzyme Q₁₀ biosynthesis pathway have been cloned and expressed in Escherichia coli. The rational design of metabolic pathways in combination with engineering optimization of fermentation processes could facilitate the development of viable bioconversion processes.

Biofiltration of volatile organic compounds by Luc Malhautier; Nadia Khammar; Sandrine Bayle; Jean-Louis Fanlo (pp. 16-22).

The removal of volatile organic compounds (VOCs) from contaminated airstreams has become a major air pollution concern. Improvement of the biofiltration process commonly used for the removal of odorous compounds has led to a better control of key parameters, enabling the application of biofiltration to be extended also to the removal of VOCs. Moreover, biofiltration, which is based on the ability of micro-organisms to degrade a large variety of compounds, proves to be economical and environmentally viable. In a biofilter, the waste gas is forced to rise through a layer of packed porous material. Thus, pollutants contained in the gaseous effluent are oxidised or converted into biomass by the action of microorganisms previously fixed on the packing material. The biofiltration process is then based on two principal phenomena: (1) transfer of contaminants from the air to the water phase or support medium, (2) bioconversion of pollutants to biomass, metabolic end-products, or carbon dioxide and water. The diversity of biofiltration mechanisms and their interaction with the microflora mean that the biofilter is defined as a complex and structured ecosystem. As a result, in addition to operating conditions, research into the microbial ecology of biofilters is required in order better to optimise the management of such biological treatment systems.

Evaluation of procedures to acclimate a microbial fuel cell for electricity production by Jung Rae Kim; Booki Min; Bruce E. Logan (pp. 23-30).

A microbial fuel cell (MFC) is a relatively new type of fixed film bioreactor for wastewater treatment, and the most effective methods for inoculation are not well understood. Various techniques to enrich electrochemically active bacteria on an electrode were therefore studied using anaerobic sewage sludge in a two-chambered MFC. With a porous carbon paper anode electrode, 8 mW/m² of power was generated within 50 h with a Coulombic efficiency (CE) of 40%. When an iron oxide-coated electrode was used, the power and the CE reached 30 mW/m² and 80%, respectively. A methanogen inhibitor (2-bromoethanesulfonate) increased the CE to 70%. Bacteria in sludge were enriched by serial transfer using a ferric iron medium, but when this enrichment was used in a MFC the power was lower (2 mW/m²) than that obtained with the original inoculum. By applying biofilm scraped from the anode of a working MFC to a new anode electrode, the maximum power was increased to 40 mW/m². When a second anode was introduced into an operating MFC the acclimation time was not reduced and the total power did not increase. These results suggest that these active inoculating techniques could increase the effectiveness of enrichment, and that start up is most successful when the biofilm is harvested from the anode of an existing MFC and applied to the new anode.

Haematococcus pluvialis cultivation in split-cylinder internal-loop airlift photobioreactor under aeration conditions avoiding cell damage by J. Vega-Estrada; M. C. Montes-Horcasitas; A. R. Domínguez-Bocanegra; R. O. Cañizares-Villanueva (pp. 31-35).

The effects of superficial gas velocity in the riser (U_Gr) and gas entrance velocity (v) on the growth of Haematococcus pluvialis cultivated in a split-cylinder internal-loop airlift photobioreactor were investigated. Cell growth decreased when U_Gr and v were increased above 12 mm s⁻¹ and 22.8 m s⁻¹, respectively. The maximum cell density of H. pluvialis was 110×10⁴ vegetative cells ml⁻¹ and the chlorophyll-a titer was 7 mg l⁻¹. The cell damage in the photobioreactor was greater when v was increased by an increase in U_Gr rather than by a decrease in sparger internal diameter. The overall volumetric mass transfer coefficient (k_La) of the photobioreactor was measured at the same U_Gr (6–24 mm s⁻¹) and v (12–80 m s⁻¹). The k_La values reached in the airlift photobioreactor were between 10 h⁻¹ and 32 h⁻¹.

Lactobacillus reuteri ATCC 53608 mdh gene cloning and recombinant mannitol dehydrogenase characterization by Yoshiharu Sasaki; Maris Laivenieks; J. Gregory Zeikus (pp. 36-41).

A gene encoding mannitol-2-dehydrogenase (E.C. 1.1.1.138) (MDH) was cloned from Lactobacillus reuteri and expressed in Escherichia coli. The 1,008-bp gene encodes a protein consisting of 336 amino acids, with a predicted molecular mass of 35,920 Da. The deduced amino acid sequence of L. reuteri MDH (LRMDH) is 77% and 76% similar to the MDHs from Leuconostoc mesenteroides and Leuconostoc pseudomesenteroides, respectively. The purified recombinant enzyme appears as a single band of 40 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but gel filtration indicates that the native enzyme is a dimer. The optimum temperature for the recombinant enzyme is 37°C, the pH optima for D-fructose reduction and D-mannitol oxidation are 5.4 and 6.2, respectively. The K_m values for NAD (9 mM) and NADH (0.24 mM) are significantly higher than those for NADP (0.35 mM) and NADPH (0.04 mM). The K_m values of LRMDH for D-fructose and D-mannitol are 34 mM and 54 mM, respectively. Contrary to what the enzyme sequence suggests, recombinant LRMDH contains a single catalytic zinc per subunit.

Xylitol production by a Pichia stipitis D-xylulokinase mutant by Yong-Su Jin; Jose Cruz; Thomas W. Jeffries (pp. 42-45).

Xylitol production by Pichia stipitis FPL-YS30, a xyl3-Δ1 mutant that metabolizes xylose using an alternative metabolic pathway, was investigated under aerobic and oxygen-limited culture conditions. Under both culture conditions, FPL-YS30 (xyl3-Δ1) produced a negligible amount of ethanol and converted xylose mainly into xylitol with comparable yields (0.30 and 0.27 g xylitol/g xylose). However, xylose consumption increased five-fold under aerobic compared to oxygen-limited conditions. This suggests that the efficiency of the alternative route of xylose assimilation is affected by respiration. As a result, the FPL-YS30 strain produced 26 g/l of xylitol, and exhibited a higher volumetric productivity (0.22 g xylitol l⁻¹ h⁻¹) under aerobic conditions.

Construction of minimum size cellulase (Cel5Z) from Pectobacterium chrysanthemi PY35 by removal of the C-terminal region by Woo Jin Lim; Su Young Hong; Chang Long An; Kye Man Cho; Byoung Rock Choi; Young Kyun Kim; Jin Mee An; Jung Mi Kang; Sun Mi Lee; Soo Jeong Cho; Hoon Kim; Han Dae Yun (pp. 46-52).

Pectobacterium chrysanthemi PY35 secretes the endoglucanase Cel5Z, an enzyme of the glycoside hydrolase family 5. Cel5Z is a 426 amino acid, signal peptide (SP)-containing protein composed of two domains: a large N-terminal catalytic domain (CD; 291 amino acids) and a small C-terminal cellulose binding domain (CBD; 62 amino acids). These two domains are separated by a 30 amino acid linker region (LR). A truncated cel5Z gene was constructed with the addition of a nonsense mutation that removes the C-terminal region of the protein. A truncated Cel5Z protein, consisting of 280 amino acid residues, functioned as a mature enzyme despite the absence of the SP, 11 amino acid CD, LR, and CBD region. In fact, this truncated Cel5Z protein showed an enzymatic activity 80% higher than that of full-length Cel5Z. However, cellulase activity was undetectable in mature Cel5Z proteins truncated to less than 280 amino acids.

Characterization and potential application of purified aldehyde oxidase from Pseudomonas stutzeri IFO12695 by H. Uchida; T. Fukuda; Y. Satoh; Y. Okamura; A. Toriyama; A. Yamashita; K. Aisaka; T. Sakurai; Y. Nagaosa; T. Uwajima (pp. 53-56).

The molecular weight of purified aldehyde oxidase from Pseudomonas stutzeri IFO12695 was estimated to be 160 kDa by a gel filtration method. SDS-PAGE showed that the enzyme consisted of three non-identical subunits with molecular weights of 18, 38, and 83 kDa. The enzyme exhibited an absorption spectrum with maxima at 277, 325, 365, 415, 450, 480, and 550 nm and possessed molybdenum, CMP, iron, sulfur, and FAD as its cofactors, indicating that it belonged to the xanthine oxidase family. A variety of aliphatic and aromatic aldehydes were oxidized; and among them n-hexylaldehyde gave the most rapidly action. When 10 mM formaldehyde was treated with the aldehyde oxidase in the presence of catalase for 240 min, the formaldehyde concentration was reduced to 0.8 mM, suggesting this enzyme might be effective for the removal of formaldehyde contained in wastewater.

Molecular characterization of a β-1,4-endoglucanase from an endophytic Bacillus pumilus strain by André O. S. Lima; Maria C. Quecine; Maria H. P. Fungaro; Fernando D. Andreote; Walter Maccheroni Jr; Welington L. Araújo; Márcio C. Silva-Filho; Aline A. Pizzirani-Kleiner; João L. Azevedo (pp. 57-65).

Endophytes comprise mainly microorganisms that colonize inner plant tissues, often living with the host in a symbiotic manner. Several ecological roles have been assigned to endophytic fungi and bacteria, such as antibiosis to phytopathogenic agents and plant growth promotion. Nowadays, endophytes are viewed as a new source of genes, proteins and biochemical compounds that may be used to improve industrial processes. In this study, the gene EglA was cloned from a citrus endophytic Bacillus strain. The EglA encodes a β-1,4-endoglucanase capable of hydrolyzing cellulose under in vitro conditions. The predicted protein, EglA, has high homology to other bacterial cellulases and shows a modular structure containing a catalytic domain of the glycosyl hydrolase family 9 (GH9) and a cellulose-binding module type 3 (CBM3). The enzyme was expressed in Escherichia coli, purified to homogeneity, and characterized. EglA has an optimum pH range of 5–8, and remarkable heat stability, retaining more than 85% activity even after a 24-h incubation at pH 6–8.6. This characteristic is an important feature for further applications of this enzyme in biotechnological processes in which temperatures of 50–60°C are required over long incubation periods.

Posttranslational modification of myxobacterial carrier protein domains in Pseudomonas sp. by an intrinsic phosphopantetheinyl transferase by Frank Gross; Daniela Gottschalk; Rolf Müller (pp. 66-74).

We demonstrate the ability of Pseudomonas putida KT2440, Pseudomonas syringae pv. tomato DC3000 and Pseudomonas stutzeri DSM10701 to posttranslationally activate carrier protein (CP) domains of various polyketide synthases, nonribosomal peptide synthetases, and fatty acid synthase by their intrinsic phosphopantetheinyl transferase. The apo-form is modified to the holo-form of the CP by attaching a phosphopantetheine moiety from coenzymeA to a conserved serine residue. The coding regions of the respective domains were cloned in order to generate C-terminal fusions with intein-chitin. The constructs were subcloned into a broad host range vector and transferred into the three pseudomonad hosts. The resulting recombinant pseudomonad strains were cultivated and each fusion protein was purified by affinity chromatography. Each purified CP was analysed using MALDI/TOF for the expected mass increase. Of the seven CPs tested, six could be purified from P. putida, which was chosen as the general host strain. Out of the six domains, five were completely activated, whereas only 5% of the protein of the sixth domain was in holo-form. Four domains were also expressed in the other hosts.

Cell surface display system for Lactococcus lactis: a novel development for oral vaccine by A. R. Raha; N. R. S. Varma; K. Yusoff; E. Ross; H. L. Foo (pp. 75-81).

The food-grade Lactococcus lactis is a potential vector to be used as a live vehicle for the delivery of heterologous proteins for vaccine and pharmaceutical purposes. We constructed a plasmid vector pSVac that harbors a 255-bp single-repeat sequence of the cell wall-binding protein region of the AcmA protein. The recombinant plasmid was transformed into Escherichia coli and expression of the gene fragment was driven by the T7 promoter of the plasmid. SDS-PAGE showed the presence of the putative AcmA′ fragment and this was confirmed by Western blot analysis. The protein was isolated and purified using a His-tag affinity column. When mixed with a culture of L. lactis MG1363, ELISA and immunofluorescence assays showed that the cell wall-binding fragment was anchored onto the outer surface of the bacteria. This indicated that the AcmA′ repeat unit retained the active site for binding onto the cell wall surface of the L. lactis cells. Stability assays showed that the fusion proteins (AcmA/A1, AcmA/A3) were stably docked onto the surface for at least 5 days. The AcmA′ fragment was also shown to be able to strongly bind onto the cell surface of naturally occurring lactococcal strains and Lactobacillus and, with less strength, the cell surface of Bacillus sphericus. The new system designed for cell surface display of recombinant proteins on L. lactis was evaluated for the expression and display of A1 and A3 regions of the VP1 protein of enterovirus 71 (EV71). The A1 and A3 regions of the VP1 protein of EV71 were cloned upstream to the cell wall-binding domains of AcmA protein and successfully expressed as AcmA/A1 and AcmA/A3. Whole-cell ELISA showed the successful display of VP1 protein epitopes of EV71 on the surface of L. lactis. The success of the anchoring system developed in this study for docking the A1 and A3 epitopes of VP1 onto the surface of L. lactis cells opens up the possibilities of peptide and protein display for not only Lactococcus but also for other gram-positive bacteria. This novel way of displaying epitopes on the cell surface of L. lactis and other related organisms should be very useful in the delivery of vaccines and other useful proteins.

Effect of substrate feed rate on recombinant protein secretion, degradation and inclusion body formation in Escherichia coli by Maria Boström; Katrin Markland; Anna Maria Sandén; My Hedhammar; Sophia Hober; Gen Larsson (pp. 82-90).

The effect of changes in substrate feed rate during fedbatch cultivation was investigated with respect to soluble protein formation and transport of product to the periplasm in Escherichia coli. Production was transcribed from the P_malK promoter; and the cytoplasmic part of the production was compared with production from the P_lacUV5 promoter. The fusion protein product, Zb-MalE, was at all times accumulated in the soluble protein fraction except during high-feed-rate production in the cytoplasm. This was due to a substantial degree of proteolysis in all production systems, as shown by the degradation pattern of the product. The product was also further subjected to inclusion body formation. Production in the periplasm resulted in accumulation of the full-length protein; and this production system led to a cellular physiology where the stringent response could be avoided. Furthermore, the secretion could be used to abort the diauxic growth phase resulting from use of the P_malK promoter. At high feed rate, the accumulation of acetic acid, due to overflow metabolism, could furthermore be completely avoided.

Monoxenic production of the entomopathogenic nematode Steinernema carpocapsae using culture media containing agave juice (aguamiel) from Mexican maguey-pulquero (Agave spp). Effects of the contents of nitrogen, carbohydrates and fat on infective juvenile production by Marco-Antonio Islas-López; René Sanjuan-Galindo; Adriana-Inés Rodríguez-Hernández; Norberto Chavarría-Hernández (pp. 91-97).

The production of infective juvenile stages (IJ) of the entomopathogenic nematode Steinernema carpocapsae in the presence of its symbiotic bacterium Xenorhabdus nematophilus was carried out in orbitally agitated bottles. Four complex culture media (M1–M4) were used, containing from 8% to 28% (by vol.) agave juice (aguamiel) from Mexican maguey-pulquero (Agave spp) as the main carbohydrate source. After 20 days of fermentation, a maximum viable IJ concentration of 249,000 IJ/ml and an initial nematode population multiplication factor of ×620 were achieved when medium M4 was used (aguamiel concentration in this medium was 28% by vol.). M4 medium contained (w/v): 0.3% total nitrogen, 3.2% total carbohydrates and 3.0% total fat. According to the results obtained, total carbohydrates concentration appeared to be of great importance in obtaining high IJ concentrations.

A novel synthetic fluoro-containing jasmonate derivative acts as a chemical inducing signal for plant secondary metabolism by Zhi-Gang Qian; Zhen-Jiang Zhao; Yufang Xu; Xuhong Qian; Jian-Jiang Zhong (pp. 98-103).

A novel fluoro-containing jasmonate derivative was chemically synthesized and evaluated as a potential elicitor with respect to the induction of plant defense responses and the biosynthesis of plant secondary metabolites. A bioactive taxuyunnanine C (Tc)-producing cell line of Taxus chinensis was taken as a model plant cell system. The presence of novel synthesized pentafluoropropyl jasmonate (PFPJA) induced two early and important events in plant defense responses, including an oxidative burst and activation of l-phenylalanine ammonia lyase. In addition, PFPJA was found to significantly increase Tc accumulation, without any inhibition of cell growth. Moreover, Tc accumulation was increased more in the presence of PFPJA compared with methyl jasmonate (MJA) and previously reported trifluoroethyl jasmonate (TFEJA). For example, addition of 100 μM PFPJA on day 7 led to a high Tc content (38.2±0.3 mg/g) at day 21, while the Tc content was 29.3±0.3 mg/g and 34.9±0.9 mg/g with the addition of 100 μM MJA and TFEJA, respectively. Quantitative structure–activity analysis of fluoro-containing jasmonates suggests that the increase in the fluoro-groups introduced into the carboxyl side-chain of MJA resulted in a higher stimulatory activity for Tc biosynthesis, which corresponds well with the markedly increased lipophilicity after fluorine introduction. These results indicate that newly synthesized fluoro-containing PFPJA can act as a powerful chemical inducing signal for secondary metabolism in plant cell cultures.

Streptomycete spores entrapped in chitosan beads as a novel biocontrol tool against common scab of potato by Guy Jobin; Geneviève Couture; Claudia Goyer; Ryszard Brzezinski; Carole Beaulieu (pp. 104-110).

Spores of Streptomyces melanosporofaciens EF-76, an actinomycete that inhibits the growth of several plant pathogens, were incorporated in beads of chitosan and polyphosphate using the entrapment technique called complex coacervation. The degradation of spore-loaded beads was monitored by measuring the residual amount of chitosan in soil and by enumerating the S. melanosporofaciens population over time. After the introduction of spore-loaded chitosan beads into soil, the amount of chitosan in sterile soil remained at 1.550 mg/g throughout the first week and diminished to 0.101 mg/g after 7 weeks. Bead degradation proceeded faster in non-sterile soil but a progressive release of both chitosan oligomers and the antagonistic microbial agent was nevertheless observed. Application of these spore-loaded chitosan beads to seed potato tubers protected progeny tubers against common scab.

Isolation and characterization of a novel polychlorinated biphenyl-degrading bacterium, Paenibacillus sp. KBC101 by M. Sakai; S. Ezaki; N. Suzuki; R. Kurane (pp. 111-116).

The biphenyl-utilizing bacterial strain KBC101 has been newly isolated from soil. Biphenyl-grown cells of KBC101 efficiently degraded di- to nonachlorobiphenyls. The isolate was identified as Paenibacillus sp. with respect to its 16S rDNA sequence and fatty acid profiles, as well as various biological and physiological characteristics. In the case of highly chlorinated biphenyl (polychlorinated biphenyl; PCB) congeners, the degradation activities of this strain were superior to those of the previously reported strong PCB degrader, Rhodococcus sp. RHA1. Recalcitrant coplanar PCBs, such as 3,4,3′,4′-CB, were also efficiently degraded by strain KBC101 cells. This is the first report of a representative of the genus Paenibacillus capable of degrading PCBs. In addition to growth on biphenyl, strain KBC101 could grow on dibenzofuran, xanthene, benzophenone, anthrone, phenanthrene, naphthalene, fluorene, fluoranthene, and chrysene as sole sources of carbon and energy. Paenibacillus sp. strain KBC101 presented heterogeneous degradation profiles toward various aromatic compounds.

The mechanism of bacterial indigo reduction by S. K. Nicholson; P. John (pp. 117-123).

The reduction of water-insoluble indigo by the recently isolated moderate thermophile, Clostridium isatidis, has been studied with the aim of developing a sustainable technology for industrial indigo reduction. The ability to reduce indigo was not shared with C. aurantibutyricum, C. celatum and C. papyrosolvens, but C. papyrosolvens could reduce indigo carmine (5,5′-indigosulfonic acid), a soluble indigo derivative. The supernatant from cultures of C. isatidis, but not from cultures of the other bacteria tested, decreased indigo particle size to one-tenth diameter. Addition of madder powder, anthraquinone-2,6-disulfonic acid, and humic acid all stimulated indigo reduction by C. isatidis. Redox potentials of cultures of C. isatidis were about 100 mV more negative than those of C. aurantibutyricum, C. celatum and C. papyrosolvens, and reached −600 mV versus the SCE in the presence of indigo, but potentials were not consistently affected by the addition of the quinone compounds, which probably act by modifying the surface of the bacteria or indigo particles. It is concluded that C. isatidis can reduce indigo because (1) it produces an extracellular factor that decreases indigo particle size, and (2) it generates a sufficiently reducing potential.

Single-stage autotrophic nitrogen-removal process using a composite matrix immobilizing nitrifying and sulfur-denitrifying bacteria by Y. Aoi; Y. Shiramasa; E. Kakimoto; S. Tsuneda; A. Hirata; T. Nagamune (pp. 124-130).

We developed a novel single-stage autotrophic nitrogen-removal process comprised of two composite immobilized biomass layers—one of nitrifying bacteria and one of sulfur-denitrifying bacteria and elemental sulfur—in a Fe-Ni fibrous slag matrix. Nitrification and consumption of dissolved oxygen occurred in the outer part and sulfur denitrification in the anoxic inner part of the composite matrix, thus realizing autotrophic nitrogen removal in a single reactor. The complete conversion of ammonia into N₂ in a single reactor was demonstrated in both batch-mode incubation and continuous-feed operation. The spatial profiles of the ammonia-oxidizing bacteria and denitrifying bacteria were evaluated by real-time PCR, targeting their functional genes, and stratification of these two types was observed in the matrix after several months of incubation. This process does not require any specific reactor type or conditions and thus has the potential to be applied to many different wastewater treatment processes due to its simplicity in both operation and construction.

Bioavailability of phenanthrene in the presence of birnessite-mediated catechol polymers by Fabio Russo; Maria A. Rao; Liliana Gianfreda (pp. 131-139).

Polycyclic aromatic hydrocarbons (PAHs) are widespread environmental contaminants and contribute to the pollution of aquatic and terrestrial environments. In soil, their fate may be affected by interactions with the soil biological community and soil colloids. This study was conducted to investigate the fate of phenanthrene (Phe), selected as a representative PAH, in simplified model systems, which simulate processes naturally occurring in soil. Phe was interacted with catechol (Cat), an orthodiphenol, and common intermediate in the microbial degradation of PAHs, and birnessite (Bir), an abiotic strong oxidative catalyst abundant in soil. Two experimental conditions were investigated: Cat (5 mM)+Bir (1 mg ml⁻¹)+Phe (0.05 mg ml⁻¹) mixed at the same time and incubated for 24 h at 25°C (Cat–Bir−Phe) and Cat+Bir incubated for 24 h at 25°C before Phe addition and then incubated for a further 24 h (Cat–Bir+Phe). After incubation, the systems were analysed for residual Cat and Phe, supplied with a selected Phe-degrading mixed bacterial culture, and then the microbial degradation of Phe and the growth of cells were monitored. Complex phenomena simultaneously occurred. Cat was completely removed after a 24-h incubation with Bir, and no interference by Phe in the Bir-mediated transformation of Cat was observed. Elemental analysis and UV–Vis and Fourier transfer infrared spectra showed that Cat transformation by Bir produced soluble and insoluble polymeric aggregates involving Phe. The hydrocarbon also interacted with the surfaces of Bir either previously coated (Cat–Bir+Phe sample) or not by Cat polymers. When a Phe-degrading bacterial culture was added to the systems after Bir-mediated Cat polymerisation, a different behaviour was observed in terms of Phe consumption and bacterial growth, thus suggesting differentiated availability of Phe to the microbial cells. The hydrocarbon was completely transformed in the presence of Bir and/or Bir covered by Cat polymers. By contrast a reduced degradation was measured when the Phe was involved in the polymerisation of Cat and entrapped in the Cat polymers (Cat–Bir−Phe). Although Cat showed a toxic, lethal effect on the bacterial cells, microbial growth was observed in the presence of Cat and Cat polymers, as the only C source. The mechanism leading to the different availability of Phe in the presence of Cat and Bir is still not clear. Further investigations are requested to provide more insight into such a complex phenomenon.

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Applied Microbiology and Biotechnology (v.68, #1)