Rapid isolation and identification of fungi from bottled water production system

Analyses of fungi by conventional methods are time consuming. At least two weeks are required for growth and identification of even the most rapidly growing fungi. Where initial contamination levels may be very low as in bottled water, it is difficult to obtain representative samples using traditional sampling and isolation techniques. The major problem report by bottlers is the failure to detect contaminations during routine quality control analysis. The fungi manifests in the bottles when the product has reached the retailer, exactly the point of maximum exposure to the consumer. One way to avoid these problems is to decrease the time of fungal incubation using modified media. In order to adapt the existing mycological media, Penicillium brevicompactum was used to spike bottled water. The recovery of viable fungal was determined. Modifications in strength of Neopeptone Glucose Rose Bengal Aureomycin Agar were assessed. The fluorescent dyes (e.g. calcofluor white) and other substrates for specific enzymatic activities were also used as potentially diagnostic supplements.EU project QLK1-CT-2002-7084

Fungi from a bottled water production system

Fungi may cause off tastes and odours in bottled water and there are potential health problems from mycotoxins. Additionally, fungal spoilage is manifested as visible clumps of growth in the bottles which is unacceptable to consumers. Direct and indirect financial losses for bottlers arise; the former due to product recall and down-time caused by the need for special sanitation, and the latter is the result of bad publicity created by such incidents. An EU project is ongoing in Micoteca da Universidade do Minho in order to control the mycological contamination of bottled water. Only limited data exists on which species may be present, and which can be considered as particularly problematic despite the problems mentioned above. Fungi were isolated and identified. From samples collected in a bottled water plant from seven water sample points. Four were water sources, one was the storage-tank outlet, another the filter outlet and, the finally the end product. Rinse samples of: (a) glass bottles at the washer inlet and outlet, (b) of caps were also analysed. The methods to concentrate fungal biomass in the samples were filtering, baiting and swabbing. Detection of fungal growth was based on plating on the selective media NGRBA, CMA ½ strength, YMG and DRBC. All colonies formed were counted. The fungal ones were transferred and isolated on MEA. Fungal counts from the water source often reached more than 20/L fungal colonies. However, the number of fungi decreases throughout the bottled water production system. These results demonstrate of that filtration offered an effective barrier to prevent the presence of fungi in bottled water. Moreover, it was of observed that glass bottles and caps are an external source of fungal contamination

Uso de técnicas de detecção rápida de fungos filamentosos na água

Dissertação Mestrado em Tecnologia do AmbienteNa Carta Europeia da Água do Conselho da Europa, proclamada em Estrasburgo em 6 de Maio de 1968, encontra-se o seguinte item: “A investigação sobre a água, e especialmente sobre a água já utilizada, deve ser encorajada ao máximo. Os meios de informação devem ser ampliados e as trocas internacionais facilitadas, ao mesmo tempo que se impõe uma formação técnica e biológica de pessoal qualificado para as diferentes disciplinas que interessam.” Passados quase 40 anos e com o presente trabalho procurou-se acrescentar algo à investigação sobre a água, a qual foi o principal objecto desta dissertação. As águas para consumo humano estão sujeitas a um apertado controlo de qualidade microbiológico. Esse controlo focaliza-se essencialmente nas análises bacteriológicas deixando a descoberto o controlo micológico. Os fungos sendo ubíquos, estão também presentes nestes sistemas ecológicos. Contudo, o conhecimento do seu papel ecológico bem como os métodos para a sua detecção, enumeração e isolamento não estão implementados, nem tão pouco regulamentados. No presente trabalho o principal objectivo foi adaptar ou mesmo implementar novos métodos para a detecção, enumeração e isolamento de fungos filamentosos presentes na água. Para alcançar os objectivos inicialmente propostos partiu-se da análise de amostras de água engarrafada. A preocupação de tornar as práticas micológicas simples e acessíveis esteve presente durante o decorrer de todo o trabalho. Neste contexto adaptou-se um meio de cultura (NGRBA – Neopeptona Glucose Rosa de Bengala Agar) alterando-o quer na composição da fonte de carbono e de azoto, quer adicionando sais de tetrazólio (MTT, TTC e CTC). O uso de fluorocromos foi outra técnica utilizada para a detecção da presença de fungos filamentosos na água. Os fungos filamentosos presentes na água em maior número pertencem aos géneros Penicillium (58%), Cladosporium (27%) e Trichoderma (6%). Por sua vez dentro do género Penicillium as três espécies mais representativas são: P. brevicompactum (33%), P. glabrum (23%) e P. chrysogenum (12%). O meio NGRBA é um meio apropriado para enumerar e isolar fungos filamentosos. A sua composição pode ser alterada nas fontes de carbono e azoto tornando-o economicamente mais atractivo sem alterar as suas propriedades. Pode-se ainda encurtar o tempo de detecção / enumeração de fungos filamentosos pela adição do sal de tetrazólio MTT. O uso de técnicas fluorescentes revelou-se uma ferramenta rápida e eficaz para a detecção de fungos em amostras de água. No entanto, a sua implementação requer um investimento avultado e formação técnica específica por parte do operador.The Water European Charter, approved in Strasbourg in 6th of May 1968, the following item is proclaimed: "the research about water, and in particular wastewater, must be encouraged. The information must be extended and the international exchanges facilitated, at the same time must be imposed a biological technique training of qualified staff." Passed almost 40 years, this item remain actual and the main aim of this work is to contribute for water research. The water for human consumption is required to pass throughout a high microbiological quality control. This control is focused essentially in the bacteriological analyses leaving mycological control out. The fungi are present all around places, so they are also present in water ecological systems. However, the knowledge of the ecological role of fungi as well as their methods for its detection, enumeration and isolation are not yet implemented, nor regulated. In the present work the main aim was adapt or even implement new methods for detection, enumeration and isolation of filamentous fungi present in the water. Initially only the analyses of bottled water samples were considered. To achieve our aim the simplicity of mycological procedures established was our main concern. Taking this into account the medium NGRBA (Neopeptone Glucose Rose Bengal Agar) was adapted and modified in its composition: the carbon and nitrogen sources were changed and it was also added tetrazolium salts like MTT, TTC and CTC. The use of fluorocroms it was used as another technique to detect the presence of filamentous fungi in the water. The filamentous fungi were presented in water in high number which belongs to the genera Penicillium (58%), Cladosporium (27%) and Trichoderma (6%). In the Penicillium taxon the three more representative species were: P. brevicompactum (33%), P. glabrum (23%) and P. chrysogenum (12%). The NGRBA medium was the most appropriate one for enumeration and isolation filamentous fungi. Its composition could be modified in order to make it more economic. The time consumption for detection and enumeration could be shortened with the addition of tetrazolium salt MTT to the NGRBA medium. The use of fluorescent techniques have shown be a fast and efficient tool for the detection of fungi in water samples. Notwithstanding, its implementation requires a high capital investment and a very well trained staff

Studies of decolorization of azo dyes by ascomycete yeasts

Poster apresentado no Micro'2001, Póvoa de Varzim, 2001.Azo dyes are the most widely used colored materials in textile industries and its biodegradability is, therefore, an important issue in the biological treatment of dye-containing wastewater. However, these treatments are not totally effective in removing color of textile dye wastewater since dyes are typically resistant to oxidative degradation. Most biodegradation studies on azo dyes involve bacterial species, and anaerobic or microaerophylic conditions are usually refered to as being favorable to a reduction step producing colorless amines. Our group has succeeded in isolating a number of yeast species, which revealed an interesting potential in the color removal of azo dyes in aerated culture media. Three of these species (UM2, UM41 and UM45), where used in the present work. The tested dyes included several sulphonic and carboxylic monoazo dyes, and several reactive textile dyes. The results which will be described refer to (i) the conditions and time-course of the decolorization process, (ii) the optimization of the decolorization medium, aiming at the reduction in yeast extract concentration, (iii) the identification of dye reduction products formed in the course of the decolorization process and (iv) the effect of those reduction products on yeast specific growth rates.BIOEFTEX Project

Patulin penicillia from a bottled water factory

Background The objective of this work is to assess the significance of penicillia isolated from a bottled water factory particularly in relation to mycotoxin production. The annual revenue from sales of bottled water is very large: bottled water has an image of being healthy. There has been an increase in reports of fungal contamination of bottled water and one of mycotoxin production. Patulin is known to be produced by Penicillium expansum and has been reported from P. brevicompactum. The isoepoxydon dehydrogenase (IDH) gene of the patulin metabolic pathway has been used to determine the potential for patulin production in fungi. Methods Fungi were obtained in pure culture from throughout a bottled water factory including from the bottled water and obtained in pure culture to assess where contamination may occur. P. expansum and P. brevicompactum strains were analysed for patulin by TLC and the IDH gene by the PCR. Other mycotoxins were also analysed. Results Penicillia were isolated from sites within the factory including P. expansum. In addition, P. brevicompactum was frequent. Patulin was detected from P. expansum and ambiguously from P. brevicompactum. However, the IDH gene was detected from both in some cases. Other important mycotoxins were detected from the strains. Secondary metabolites in medium used to produce biomass for DNA analysis were observed. Conclusions Fungi are capable of being present in bottled water from the manufacturing process. Important mycotoxin producers are present including patulin ones. The effect of mycotoxins on DNA analysis requires consideration. Further work is needed to determine whether the mycotoxins can be present in bottled water

Paper-Based Biosensors for Analysis of Water

The presence of contaminants in water generates a great concern worldwide. As contaminants, we can refer different classes of chemicals, such as pharmaceuticals, personal care products, heavy metals, and also microorganisms, such as waterborne pathogens. Some of the chemical compounds have the potential to bioaccumulate in the aquatic biota. Hence, the development of simple and portable methods for the detection of contaminants in the aquatic environment can improve their monitoring and, consequently, the study of their environmental impact. In this context, the development of paper-based analytical tools and also of biosensor devices has been exploited for quantitative and semiquantitative analysis of several contaminants in different water matrices. The association of these two analytical strategies can provide the implementation of low-cost, portable, and easily handled methods for detecting chemical and biological contaminations in water. In this chapter, we provide a review of the developed paper-based analytical biosensors, highlighting the features of the paper-based (paper substrate and fabrication procedures) and biosensor devices (transducers and biorecognition elements). Moreover, the application of the referred paper-based biosensors for the detection of different water contaminants (pathogens, pharmaceuticals, and heavy metals) in environmental and wastewater samples is discussed

Economic and environmental assessment of small and decentralized wastewater treatment systems

The aim of the present work was the assessment of economic and environmental aspects of decentralised energy-saving wastewater treatment systems. The formulated investment and operation cost functions were adjusted by a power law function. The different wastewater systems serving population settlements between 50 p.e. and 250 p.e., presented associated investment costs varying from 400 €/p.e. to 200 €/p.e. and annual operation costs in the range of 70 €/p.e. to 20 €/p.e., respectively. A Life Cycle Analysis approach was used to compare the environmental impact alternative wastewater systems. The assessment was focused on two energy-saving systems (constructed wetland and slow rate infiltration) and a conventional one (activated sludge process). The low environmental impact of the energy-saving wastewater treatment systems was demonstrated, being the most relevant the global warming indicator. Options for reduction of life cycle impacts were assessed including materials used in construction and operation lifetime of the systems. A 10 % extension of operation lifetime of constructed wetland and slow rate infiltration systems lead to a 5 % and 7 % decrease in the abiotic depletion indicator, respectively, and to a 1 % decrease in CO2 emissions in both systems. Replacing steel with HDPE in the activated sludge tank resulted in a 1 % reduction in CO2 emission and 1 % in the abiotic depletion indicator. In the case of the Imhoff tank a 1 % reduction in CO2 emissions and 5 % in abiotic depletion indicator were observed when concrete was replaced by HDPE. Therefore, considering the huge potential of energy saving wastewater treatment systems, the overall environmental impact of such design alternatives should not be discarded.EU Program INTERREG III-B Atlantic Arc, Depuranat project (n.º 54)

Life cycle assessment of wastewater treatment options for small and decentralized communities

Sustainability has strong implications on the practice of engineering. Life cycle assessment (LCA) is an appropriate methodology for assessing the sustainability of a wastewater treatment plant design. The present study used a LCA approach for comparing alternative wastewater treatment processes for small and decentralised rural communities. The assessment was focused on two energy-saving systems (constructed wetland and slow rate infiltration) and a conventional one (activated sludge process). The low environmental impact of the energy-saving wastewater treatment plants was demonstrated, the most relevant being the global warming indicator. Options for reduction of life cycle impacts were assessed including materials used in construction and operational lifetime of the systems. A 10% extension of operation lifetime of constructed wetland and slow rate infiltration systems led to a 1% decrease in CO2 emissions, in both systems. The decrease in the abiotic depletion was 5 and 7%, respectively. Also, replacing steel with HDPE in the activated sludge tank resulted in a 1% reduction in CO2 emission and 1% in the abiotic depletion indicator. In the case of the Imhoff tank a 1% reduction in CO2 emissions and 5% in the abiotic depletion indicator were observed when concrete was replaced by HDPE.(undefined

Economic and environmental assessment of small and decentralized wastewater treatment systems

The aim of the present work was the assessment of economic and environmental aspects of decentralized energy-saving wastewater treatment systems. The formulated investment and operation cost functions were adjusted by a power law function. The different wastewater systems serving population settlements between 50 p.e. and 250 p.e., presented associated investment costs varying from €400/p.e. to €200/p.e. and annual operation costs in the range of €70/p.e.– €20/ p.e., respectively. A life cycle analysis approach was used to compare the environmental impact of alternative wastewater treatment systems. The assessment was focused on two energy-saving systems (constructed wetland and slow rate infiltration) and a conventional one (activated sludge process). Low environmental impact of energy-saving wastewater treatment systems was demonstrated, being the most relevant the global warming indicator. Options for reduction of life cycle impacts were assessed including materials used in construction and operation lifetime of the systems. A 10% extension of operation lifetime of constructed wetland and slow rate infiltration systems lead to a 5% and 7% decrease in the abiotic depletion indicator, respectively, and to a 1% decrease in CO2 emissions in both systems. Replacing steel with HDPE in the activated sludge tank resulted in a 1% reduction in CO2 emission and 1% in the abiotic depletion indicator. In the case of the Imhoff tank a 1% reduction in CO2 emissions and 5% in abiotic depletion indicator were observed when concrete was replaced by HDPE. Therefore, considering the huge potential of energy saving wastewater treatment systems, the overall environmental impact of such design alternatives should not be discarded.EU Program INTERREG III-B Atlantic Arc, Depuranat project (No. 54