IDENTIFICATION OF FILAMENTOUS MICROORGANISMS IN ORGANIC INDUSTRIAL ACTIVATED SLUDGE PLANTS. E. Rodríguez1*, L. Isac 1, N. Fernández1, A. Zornoza1-2 and Mas, M3. 1
Grupo Bioindicación Sevilla (GBS), EDAR La Ranilla. Bda San José de Palmete s/n. Sevilla 41006. Dirección Postal: AP 7279. Sevilla 41080. Teléfono/Fax: 955020847.
[email protected]; www.grupobioindicacionsevilla.com 2 EDAR Quart-Benager (Valencia). Entidad Pública de Saneamiento de la Generalidad Valenciana (EPSAR). E-mail:
[email protected] 3 Hydrolab Microbiologica. c/ Blanco. Barcelona. 3808028.
[email protected]
ABSTRACT The microscopic observation and identification of filamentous bacteria, as well as its distribution and quantification in industrial activated sludge, provide the plant operator with useful information. In fact, most industrial wastewater treatment plants which suffer from Bulking have operational disorders that can severely disrupt liquid-solid separation. Keywords: Filamentous microorganisms; industrial wastewater treatment plant; Bulking sludge; industrial wastewater; microscopic analysis; types of contaminants.
INTRODUCTION In industrial wastewater treatment plants (IWTP), the use of microorganisms (bacteria, protists or metazoans) as indicators of effluent quality is a practical control system that is getting increasingly popular in recent years. If an IWTP performance is good, its effluent can be re-used or released into the natural ecosystems without running environmental risks. The capability of microorganisms population as indicators of effluent quality has often been described in the literature. The different types of industrial wastewaters effluents require different strategies to remove the contaminants. It is known that the characteristics of industrial wastewaters from agriculture or food industry are different from the characteristics of municipal wastewaters. On the ground, industrial wastewaters are biodegradable and nontoxic, but they have higher concentrations of biochemical oxygen demand (BOD) and suspended solids (SS). The results shown are based on a comparative study carried out by GBS in several Spanish IWTPs. It has been established that filamentous bacteria populations in industrial activated sludge differ significantly from those in urban wastewater treatment plants. Out of the twenty-seven samples analysed in this study, the presence of bulking was observed in 17 of them. 17 morphotypes were determined as dominant, according to the classification system proposed by Eikelboom (2006) and Jenkins et al. (2004), based on the classification of the bacteria according to morphotype, amongst which particularly noticeable were, in order of most frequent appearance: Type 021N, Thiotrix sp. and Haliscomenobacter hydrossis. Out of the remainder of the plants analysed, 18% presented problems of deflocculation and 4% presented phenomena of viscosity.
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MATERIAL AND METHODS The populations of filamentous bacteria and the floccular structures in 27 Spanish IWTPs were studied. The samples were taken from different industries, predominantly from the agroalimentary industry, as shown in Table 1. Table 1: No. of IWTPs studied, according to industrial sector. MANUFACTURERS Milk production Terpene manufacturer Paper production Beer production Fat production Metallurgy Wine production Citrus fruit production Snack food production Preserved vegetables Flour production Juice production Tanneries TOTAL
No OF IWTPs STUDIED 5 1 2 3 1 1 4 1 1 1 3 1 3 27
RESULTS AND DISCUSSION Out of the 27 samples analysed, those from the agroalimentary industry predominated (85%). In general, after different pre-treatments, industrial effluents are treated in the same way that urban wastewater. However, the chemical industries, which are represented in this study by tanneries and metallurgical industries (15%), produce toxic elements that make biological degradation difficult. As for the agroalimentary industry, both the typical nutritional deficiency (Agridiotis et al., 2007), in which nitrogen and phosphorous can limit degradation of organic material, and the chemical effluents loaded with toxic agents, produce very poor flocculation; this poor flocculation affects both the respiration and metabolism of the floc-forming bacteria. This process can be seen in the evaluation of the sludge samples shown in figure 1 and in the microscopic evaluation of the sludge described in Table 2.
A
B
C
D
Figure 1: The development of the active industrial sludge samples decanted into test tubes over successive periods of 3 (A), 10 (B), 20 (C) and 30 (D) minutes.
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The results obtained indicate that the totality of the samples analysed, to a greater or lesser extent, presented problems of deflocculation associated with toxins or nutritional deficiencies, which resulted in the phenomena of filamentous bulking (63% of the samples studied), viscous bulking (4% of the samples studied) and pin-point floc (27% of the samples studied).
Table 2: Microscopic evaluation of the structure of the flocs in the samples studied.
INDUSTRY Milk production Terpene manufacture Paper manufacture Beer production Fat production Metallurgy Wine production Citrus fruit production Snack food production Canned vegetables Flour production Tanneries Juice production
FLOCULAR STRUCTURES
No OF SAMPLES OBSERVED
Totally destructuration, pin-point floc, open-structured flocs and large-sized flocs Defloculation and pin-point floc No flocculation and large-sized flocs in a mesh structure Open-structured flocs and large flocs in a mesh structure Large-sized flocs in a mesh structure Pin-point floc and bad performance of the biological process Large or medium-sized flocs, openstructured flocs or dispersed flocs. Viscous bulking. Medium-sized flocs in a mesh structure Pin-point floc Medium-sized flocs and open-structured flocs in a mesh structure Open-structured flocs, dispersed flocs. Predominantly, small-sized flocs. Dispersed flocs or lightly compacted flocs Large-sized flocs in a mesh structure
5 1 2 3 1 1 4 1 1 1 3 3 1
In addition, the high biodegradability of the majority of the influents, practically all the samples (except those from the metallurgical and tannery industry), created a situation of nutritional stress that made possible the growth of filamentous bacteria to such an extent that flocculation was clearly inhibited, as illustrated by the development of a slimy film, typical of T 0041, in conditions of nutritional stress. This situation can be seen in Figure 2. The presence of these formations or slime coatings external to the filaments, were detected in the samples analysed from the beer and citrus fruit producers.
A
B
C
Figure 2: (A) Indian ink test in which the filament type 0041 was observed, associated with nutritional deficiencies (N/P); phase contrast 400x. (B) Abundant bacillary forms dispersed and near the structure of the flocs, retained by abundant extra-cellular material; clear field, 1000x. (C) Appearance of the filament T 0041 (occasionally with a Neisser positive coating) indicative of processes of nutritional deficiency. Citrus samples. Neisser stain. 1000x.
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The protozoan population present in the samples studied, which are affected to a great extent by floccular formation, has been variable. Most of them presented a low diversity of species associated with small-sized and open-structured flocs that only allowed the development of some swimming ciliates and impeded the development of other groups such as crawling or sessile ciliates. The definition of the dominant and secondary filamentous morphotypes has been the key feature in this study. The results obtained are set out in Table 3.
Table 3: Dominant and secondary filament morphotypes observed, as well as the associated category numbers. INDUSTRY Milk production 1 Milk production 2 Milk production 3 Milk production 4 Milk production 5 Terpen manufacture Paper manufacture 1 Paper manufacture 2 Beer production 1 Beer production 2 Beer production 3 Fat production Metallurgy Wine production 1 Wine production 2 Wine production 3 Wine production 4 Citrus fruit production Snack food production Canned vegetables Flour production 1 Flour production 2 Flour production 3 Tannery 1 Tannery 2 Tannery 3 Juice production
DOMINANT FILAMENT
SECONDARY FILAMENT
Type 021N, Type 0675 and Type 1702 Type 0961 Type 0675, Type 021N and H. hydrossis Thiothrix and Type 1863 N. limicola Thiothrix, Type 0803 and Type Type 1863, N. limicola and 0211 Type 021N Microthrix parvicella, Type GALO 1863, Type 021N, N. limicola Fibres/Deflocculation Fibres Type 021N Type 021N and Type 0041 H. hydrossis and Type 0211 Type0211, H. hydrossis and Type 0041 GALO Deflocculation N. limicola and Type 021N and Thiothrix sp. Haslicomenobacter hydrossis Thiothrix sp. and H. Type 021N hydrossis Viscous bulking Thiothrix sp. Type 021N, N. limicola H. hydrossis, Type 0041, Type 021N Type 1701, N. limicola, Thiothrix sp. and GALO Deflocculation N. limicola, Thiothrix sp. and Type 021N GALO H. hydrossis and Type t021N Thiothrix sp. Thiothrix, Type 021N Haliscomenobacter hydrossi and Type 0411 Thiothrix sp. and Type 0041 Type 021N and H. hydrossis Thiothrix sp., Type 021N, GALO and Type 0041 GALO, Thiothrix sp., and N. Type 021N limicola Thiothrix sp., H. hydrossis N. limicola Fungi
CATEGORY NUMBER 5 4 4 5 4 5 3 4 3 4 3 4 4 5 5 5 4 4 3 2 4 5
The morphotype that appeared as dominant on most occasions was the Type 021N, dominating in 31% of the samples as can be seen in Table 5, followed by Thiothrix sp. in 17% of the samples.
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CONCLUSIONS Although the biological treatment of industrial wastewater plays an essential role in minimising the impact on the recipient rivers, it is still an unresolved issue for many industries. We have established that changes in the flocculation process are widespread in the biological treatments of most IWTPs and they have a lot to do with bulking and solids separation problems. So it is our belief that trained professionals in microbiological control techniques (Bio-control) are needed to solve this sort of problems. Treatments such as chlorination, ozonisation, etc. can be useful to promptly control the biological problems associated with these industrial wastewaters, but it is necessary to be aware of the effects of these treatments and the rest of operational factors on the activated sludge, as well as the effects of nutrients and micronutrients limitations. The predominant morphotypes found were Type 021N and Thiotrix sp., as well as Haliscomenobacter hydrossis, GALO and Type 0041, which usually make worse the problems caused by nutritional deficiency and lack of oxygen in these IWTPs. In order to optimise the treatment process of industrial effluents at the lowest cost, it is necessary to improve our knowledge of the biota and the process of floculation in these IWTP. Further research is needed.
ACKNOWLEDGEMENTS We would like to express our gratitude to the companies, organizations and associations to which we belong and, especially, we thank EMASESA for their support.
REFERENCES 1. Agridiotis, V, Forster, C.F. y Cartiell-Marquet, C. (2007). Addition of Al and Fe salts during treatment of paper mill effluents to improve activated sludge settlements characteristics. Bioresource Technology 98, 15, 2926-2934. 2. Eikelboom, D. (2006). Identification and Control of Filamentous Micro-organisms in Industrial Wastewater Treatment Plants. Multi-Media Training CD. IWA Publisingh. ISBN: 1843390965. 3. Jenkins, D., Richard, m. G. y Daigger, G. T. (2004). Manual on the Causes and Control of Activated Sludge Bulking and Foaming. Lewis Publishers (Michigan). 4. Liu, Y. y Tay J-H. (2004). State of the art of biogranulation technology for wastewater treatment. Biotechnology Advances 22, 533-563.
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