33. "Modeling of the simultaneous hydrolysis–ultrafiltration of whey permeate by a thermostable β-galactosidase from Aspergillus niger".
D. G. Hatzinikolaou, E. Katsifas, D. Mamma, A. D. Karagouni, P. Christakopoulos and D. Kekos.
Biochemical Engineering Journal, vol. 24, pages 161-172, (2005).  View at publisher's site  Request copy
Abstract: A wild type strain of Aspergillus niger, denoted as BTL, produced elevated levels of β-galactosidase when grown in a low cost medium that contained wheat bran as the sole carbon and energy source. The enzyme was collected, concentrated and partially purified from the culture supernatant. Its kinetic and stability properties were thoroughly examined towards its potential use for the hydrolysis of acid whey permeate lactose. The β-galactosidase of A. niger BTL showed increased pH and thermal stability, with activation energy for the first order deactivation constant equal to 180 kJ/mol at pH 3.5. Lactose hydrolysis by the enzyme was described by Michaelis–Menten kinetics with competitive inhibition only from galactose. An integrated process, concerning the simultaneous hydrolysis–ultrafiltration of whey lactose that incorporated the specific kinetic properties of the β-galactosidase was developed and modeled. The model proved very successful in predicting the behavior of a continuous laboratory hydrolysis–ultrafiltration set up, specifically designed for that purpose. The validated model was finally used in a number of computer simulations in order to investigate the effect of the various process parameters on the overall system performance.

32. "PCR detection of oxytetracycline resistance genes otr(A) and otr(B) in tetracycline resistant streptomycete isolates from diverse habitats. "
T.L. Nikolakopoulou, S. Egan, L.S. van Overbeek, G. Guillaume, H. Heuer, E.M.H. Wellington, J.D. van Elsas, J.M. Collard, K. Smalla and A.D. Karagouni.
Current Microbiology, vol. 5, pages 211-215, (2004).  View at publisher's site  Request copy
Abstract: A range of European habitats was screened by PCR for detection of the oxytetracycline resistance genes otr(A) and otr(B), found in the oxytetracycline-producing strain Streptomyces rimosus. Primers were developed to detect these otr genes in tetracycline-resistant (Tc(R)) streptomycete isolates from environmental samples. Samples were obtained from bulk and rhizosphere soil, manure, activated sludge and seawater. The majority of Tc(R) streptomycetes originated from bulk and rhizosphere soil. Fewer Tc(R) streptomycetes were isolated from manure and seawater and none from sewage. By PCR, three out of 217 isolates were shown to contain the otr(A) gene and 13 out of 217 the otr(B) gene. Surprisingly, these genes were detected in taxonomic groups not known as tetracycline-producing strains. The majority of the otr gene-carrying strains was assigned to S. exfoliatus or S. rochei and originated from all habitats from which Tc(R) streptomycetes were obtained. Our results indicated that the occurrence of otr(A) and otr(B) genes in natural environments was limited and that otr(B), in comparison to otr(A), seemed to be more common.

31. "Chromium recycling of tannery waste through microbial fermentation. "
A.E. Katsifas, E.P. Gianoutsou, M. Lbraki, M. Barla and A.D. Karagouni.
Journal of Industrial Microbial Biotechnology, vol. 31, pages 57-62, (2004).  View at publisher's site  Request copy
Abstract: An Aspergillus carbonarius isolate, selected from an established microbial culture collection, was used to study the biodegradation of chromium shavings in solid-state fermentation experiments. Approximately 97% liquefaction of the tannery waste was achieved and the liquid obtained from long-term experiments was used to recover chromium. The resulting alkaline chromium sulfate solution was useful in tanning procedures. A proteinaceous liquid was also obtained which has potential applications as a fertilizer or animal feed additive and has several other industrial uses. The A. carbonarius strain proved to be a very useful tool in tannery waste-treatment processes and chromium recovery in the tanning industries. .

30. "The effectiveness of commercial animicrobial compounds, against saccharolytic microorganisms isolated from a beet sugar production line. "
N. Arnanitis, C. Kotzamanidis, N.G. Skaracis and A.D. Karagouni.
World Journal of Microbiology and Biotechnology, vol. 20, pages 291-296, (2004).  View at publisher's site  Request copy
Abstract: The antimicrobial activities of five commercial disinfectants containing quaternary ammonium compoundisopropanol (D1), sodium methyl dithiocarbamate (D2), sodium thiocarbamate (D3), sodium dimethyl dithiocarbamate (D4) and formaldehyde (D5) were studied against three main saccharolytic indigenous isolates (Bacillus cereus, Lactobacillus plantarum and Leuconostoc mesenteroides) from a beet sugar extraction line. Preliminary studies suggested that although all the disinfectants were effective against those isolates, the high economic cost in combination with large amounts of the disinfectants D2, D3 and D4 weaken their possibility for industrial use. Therefore, the minimum inhibitory concentration (MIC) of the other two examined disinfectants D1 and D5 was determined and survivor curves were obtained, for a period of 7 days. Bacterial counts against time (h) suggested that D1 was more effective than D5 against the microbial population. In particular, D1 was bacteriolytic above 7 mg/l for B. cereus and bactericidal above 80 mg/l for Lc. mesenteroides and above 100 mg/l for L. plantarum. The disinfectant D5 was bacteriolytic above 25 mg/l for B. cereus and bactericidal above 500 mg/l for Lc. mesenteroides and L. plantarum. Taking into consideration both features, i.e. high concentration and very low cost, the use of D5 (formaldehyde) appeared more suitable to the concerned beet sugar processor.

29. "Identification of yeast strains isolated from a two-phase decanter system with olive oil waste and investigation of their ability for its fermentation."
E.P. Gianoutsou, C. Meintanis and A.D. Karagouni.
Bioresource Tecnology, vol. 93, pages 301-306, (2004).  View at publisher's site  Request copy
Abstract: Adynamic fed-batch microcosm system is described which permits assessment of the progressive growth of yeasts through olive oil waste. We report on its application to measure the effects of the growth of yeast strains upon the chemical composition of ‘‘alpeorujo’’, the waste of a two-phase decanter system used for the extraction of olive oil. Six phenotypically distinct groups of yeasts were isolated. Three selected isolates were identified as being most closely related to Saccharomyces sp., Candida boidinii and Geotrichum candidum using biochemical tests and partial 18S rDNAgene sequence analysis. This is the first report of yeast growth on ‘‘alpeorujo’’ by the use of a fed-batch microcosm system, resulting in the change of the initial chemical composition of ‘‘alpeorujo’’ and in the decrease of the toxic substances such as phenols.

28. "A novel improved method for Aspergillus nidulans transformation. "
M. Koukaki, E.P. Gianoutsou, A.D. Karagouni and G. Diallinas.
Journal of Microbiological Methods, vol. 55, pages 687–695, (2003).  View at publisher's site  Request copy
Abstract: We systematically investigated the efficiency of Aspergillus nidulans transformation using protoplasts prepared from different stages of conidiospore germination and young mycelium. Using standard integrative plasmids, increased transformation yields were obtained with protoplasts isolated from a specific stage coincident with germ tube emergence. This increase ranged, on the average, from two- to eightfold depending on different plasmids used. Transformation efficiencies with a replicative plasmid were similar to those obtained using previously described methods. Although this observation suggests that elevated transformation efficiencies might be due to increased efficiency of recombination between plasmid and genomic sequences, we cannot exclude other factors associated with the particular developmental stage used. In the course of this study, we also examined the effect of other parameters that might enhance transformation yields. The method described is also significantly easier and faster than other current methods.

27. "Prevalence of streptomycin-resistance genes in bacterial populations in European habitats."
L.S. van Overbeek, E.M.H. Wellington, S. Egan, K. Smalla, H. Heuer, J.M. Collard, G. Guillaume, A.D. Karagouni, T.L. Nikolakopoulou and J.D. van Elsas.
FEMS Microbiology and Ecology, vol. 42, pages 277-288, (2002).  View at publisher's site  Request copy
Abstract: The prevalence of selected streptomycin (Sm)-resistance genes, i.e. aph (3''), aph (6)-1d, aph (6)-1c, ant (3'') and ant (6), was assessed in a range of pristine as well as polluted European habitats. These habitats included bulk and rhizosphere soils, manure from farm animals, activated sludge from wastewater treatment plants and seawater. The methods employed included assessments of the prevalence of the genes in habitat-extracted DNA by PCR, followed by hybridisation with specific probes, Sm-resistant culturable bacteria and exogenous isolation of plasmids carrying Sm-resistance determinants. The direct DNA-based analysis showed that aph (6)-1d genes were most prevalent in the habitats examined. The presence of the other four Sm-modifying genes was demonstrated in 58% of the tested habitats. A small fraction of the bacterial isolates (8%) did not possess any of the selected Sm-modifying genes. These isolates were primarily obtained from activated sludge and manure. The presence of Sm-modifying genes in the isolates often coincided with the presence of IncP plasmids. Exogenous isolation demonstrated the presence of plasmids of 40-200 kb in size harbouring Sm-resistance genes from all the environments tested. Most plasmids were shown to carry the ant (3'') gene, often in combination with other Sm-resistance genes, such as aph (3'') and aph (6)-1d. The most commonly found Sm-modifying gene on mobile genetic elements was ant (3''). Multiple Sm-resistance genes on the same genetic elements appeared to be the rule rather than the exception. It is concluded that Sm-resistance genes are widespread in the environmental habitats studied and often occur on mobile genetic elements and ant (3'') was most often encountered.

26. "Occurrence and reservoirs of antibiotic resistance genes in the environment."
N. Seveno, K. Smalla, J.D. van Elsas, J.M. Collard, G. Guillaume, A.D. Karagoumi, D. Kalifidas and E.M.H. Wellington.
Reviews in Medical Microbiology, vol. 13, pages 1-13, (2002).  View at publisher's site  Request copy
Abstract: Antibiotic resistance genes have become highly mobile since the development of antibiotic chemotherapy. A considerable body of evidence exists proving the link between antibiotic use and the significant increase in drug-resistant human bacterial pathogens. The application of molecular detection and tracking techniques in microbial ecological studies has allowed the reservoirs of antibiotic resistance genes to be investigated. It is clear that the transfer of resistance genes has occurred on a global scale and in all natural environments. The considerable diversity of bacteria and mobile elements in soils has meant that the spread of resistance genes has occurred by all currently known mechanisms for bacterial gene transfer. Trans-kingdom transfers from plants to bacteria may occur in soil. Hot spots for gene transfer in the soil/plant environment have been described and colonized niches such as the rhizosphere and other nutrient-enriched sites, for example manured soil, have been identified as reservoirs of resistance genes. Although exposure and selection for tolerance of antibiotics is considerable in clinical environments there is increasing evidence that selection for resistant phenotypes is occurring in natural environments. Antibiotic-producing bacteria are abundant in soil and there is evidence that they are actively producing antibiotics in nutrient-enriched environments in soil. In addition there is clear evidence that the self-resistance genes found within antibiotic gene clusters of the producers have transferred to other non-producing bacteria. Perhaps most important of all is the use of antibiotics in agriculture as growth promotants and for treatment of disease in intensively reared farm animals. These treatments have resulted in gut commensal and pathogenic bacteria acquiring resistance genes under selection and then, due to the way in which farm slurries are disposed of, the spread of these genes to the soil bacterial community. Integrons with multiple resistance gene cassettes have been selected and disseminated in this way; many of these cassettes carry other genes such as those conferring heavy metal and disinfectant resistance which have been co-selected in bacteria surviving in effluents and contaminated soils, further maintaining and spreading the antibiotic resistance genes.

25. "Gentamicin resistance genes in environmental bacteria: prevalence and transfer."
H. Heuer, E. Krogerrecklenfort, S. Egan, L. van Overbeek, G. Guillaume, T.L. Nikolakopoulou, E.M.H. Wellington, J.D. van Elsas, J.M. Collard, A.D. Karagouni and K. Smalla.
FEMS Microbiology and Ecology, vol. 42, pages 289-302, (2002).  View at publisher's site  Request copy
Abstract: A comprehensive multiphasic survey of the prevalence and transfer of gentamicin resistance (Gm(r)) genes in different non-clinical environments has been performed. We were interested to find out whether Gm(r) genes described from clinical isolates can be detected in different environmental habitats and whether hot spots can be identified. Furthermore, this study aimed to evaluate the impact of selective pressure on the abundance and mobility of resistance genes. The study included samples from soils, rhizospheres, piggery manure, faeces from cattle, laying and broiler chickens, municipal and hospital sewage water, and coastal water. Six clusters of genes coding for Gm-modifying enzymes (aac(3)-I, aac(3)-II/VI, aac(3)-III/IV, aac(6')-II/Ib, ant(2'')-I, aph(2'')-I) were identified based on a database comparison and primer systems for each gene cluster were developed. Gm-resistant bacteria isolated from the different environments had a different taxonomic composition. In only 34 of 207 isolates, mainly originating from sewage, faeces and coastal water polluted with wastewater, were known Gm(r) genes corresponding to five of the six clusters detected. The strains belonged to genera in which the genes had previously been detected (Enterobacteriaceae, Pseudomonas, Acinetobacter) but also to phylogenetically distant bacteria, such as members of the CFB group, alpha- and beta-Proteobacteria. Gm(r) genes located on mobile genetic elements (MGE) could be captured in exogenous isolations into recipients belonging to alpha-, beta- and gamma-Proteobacteria from all environments except for soil. A high proportion of the MGE, conferring Gm resistance isolated from sewage, were identified as IncPbeta plasmids. Molecular detection of Gm(r) genes, and broad host range plasmid-specific sequences (IncP-1, IncN, IncW and IncQ) in environmental DNA indicated a habitat-specific dissemination. A high abundance and diversity of Gm(r) genes could be shown for samples from faeces (broilers, layers, cattle), from sewage, from seawater, collected close to a wastewater outflow, and from piggery manure. In the latter samples all six clusters of Gm(r) genes could be detected. The different kinds of selective pressure studied here seemed to enhance the abundance of MGE, while an effect on Gm(r) genes was not obvious. .

24. "Enzymes of Entner-Doudoroff and pyruvate decarboxylation pathway in Zymomonas mobilis mutant grown in continuous culture. "
A.L. Savvides, K.I. Chalkou, M.A. Typas and A.D. Karagouni.
Antonie van Leeuwenhooek, vol. 80, pages 287-295, (2001).  View at publisher's site  Request copy
Abstract: The osmotolerant Zymomonas mobilis strain suc40, (containing plasmid pDS3154-inaZ), which is capable of producing simultaneously ethanol and ice nuclei protein, was cultivated in a chemically defined complete sucrose medium, as well as in a sugar beet molasses medium in continuous culture. The strain exhibited the normal Monod’s relationship between biomass and dilution rate, and between growth substrate concentration and dilution rate. Specific activities of a number of enzymes that appear to control important steps in the metabolic flux of the Entner–Doudoroff and pyruvate decarboxylation pathways were investigated over a range of growth rates in steady state cultures. With the exception of glucose-6-phosphate dehydrogenase and gluconate kinase, all of the enzymes exhibited a very similar pattern for the wild type Z. mobilis CP4 and for the osmotolerant mutants, independent of the media used; the enzyme patterns remained relatively constant over the studied growth range. The specific activity of glucose-6-phosphate dehydrogenase was increased 2-fold by decreasing dilution rate in sugar beet molasses. The specific activity of gluconate kinase was 2-fold lower at medium growth rates compared with that at either low or high growth rates. Pyruvate kinase, pyruvate decarboxylase and alcohol dehydrogenase activities were significantly higher compared with those of the enzymes governing the early steps of the Entner– Doudoroff pathway. The present study, which was designed to determine the behaviour of important enzymes in sucrosemetabolismof Z. mobilis suc40/pDS3154-inaZ grown in continuous culture showed that the microorganism required regulation of specific enzyme activities at the transcriptional level when sugar beet molasses were used as the growth medium.