MICROBIAL STRUCTURE AND FUNCTIONS OF BIOFILM DURING WASTEWATER TREATMENT IN THE DAIRY INDUSTRY

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1 Article A&EB MICROBIAL STRUCTURE AND FUNCTIONS OF BIOFILM DURING WASTEWATER TREATMENT IN THE DAIRY INDUSTRY Irina Schneider and Yana Topalova Sofia University St. Kliment Ohridski, Faculty of Biology, Sofia, Bulgaria Correspondence to: Irina Schneider Abstract The microbial structure and activity of biofilm are two important parameters for the successful operation and control of fixed film processes in wastewater treatment. However, the constant interaction between fixed and suspended biomass requires the parallel investigation of these two biological components. The aim of the present article is to study the key microbiological and enzymological parameters of biofilm and suspended biomass from an anaerobic sequencing batch biofilm reactor for dairy wastewater treatment. The effectiveness of organic matter (measured as COD) removal and nitrates removal was determined. The microbial structure of biofilm and suspended biomass was investigated by the quantity of anaerobic heterotrophs, anaerobic spore-forming microorganisms and denitrifying bacteria. The total dehydrogenase activity and the nitrate reductase activity were used as indicators for biomass activity. The obtained data showed that the enzyme activity of the fixed biomass was higher in comparison with that of the suspended biomass. This result is related to the more heterogenic media of the inert carrier and more dynamic conditions in the biofilm inner layers. The total dehydrogenase activity of the fixed biomass was four times higher and the nitrate reductase activity of the fixed biomass was two times higher than that of the suspended biomass during stable biofilm functioning. The investigated biological system mineralized the organic matter with high effectiveness (to 90 %) and removed 67 % of the nitrates from dairy wastewater as a result of the controlled water treatment process. Biotechnol. & Biotechnol. Eq. 2013, 27(3), Keywords: dehydrogenase activity, nitrate reductase activity, dairy wastewater, heterotrophic biofilm Introduction Wastewater treatment processes with fixed biomass are widely applicable in the practice for wastewater treatment: with high organic matter concentration (7, 17, 20); with variable organic load (5); with content of toxic substances (18, 23), etc. This is related to: the easier start of the treatment process due to the quicker formation of biofilms in comparison to activated sludge (3); the higher stability of biofilms towards toxic pollutants and sharp variations in the environmental conditions because of the presence of a polysaccharide matrix (12, 15); the quicker recovery of biofilms under stress conditions due to the presence of spore-forming organisms (2); the possibility to keep microorganisms with lower specific rate of growth such as methanogens in the biofilters in comparison to the biobasins (13, 14, 22); the smaller volume of biomass for treatment and, respectively, the lower economic value of the wastewater treatment process (2, 5). All this determines the more detailed study of the biological and functional diversity of the biofilm and, more specifically, of its microbial segment a key factor for the realization of the biotransformation processes. The microbial structure of the biofilm and its activity are two key parameters for the effective functioning of the biofilters and for the control of the wastewater treatment processes. Widely used methods for characterization of the biofilms are thickness, dry weight and the total number of 3782 microbial cells (12). These parameters, however, are not sufficient to provide a precise evaluation of the biofilm activity. This is why there are methods developed for studying and regulating the enzymological profile of the biofilms. These methods include determining total enzymological indicators such as dehydrogenases (8, 12). The total dehydrogenase is an indicator, which determines the total activity of the different dehydrogenases showing activity most often with substrate glucose and having different redox potentials (21). Different compounds are used as indicators: triphenyl tetrazolium chloride (TTC), 2-(р-iodophenyl)-3-(р-nitrophenyl)-5-phenyl tetrazolium chloride (INT) or 5-cyano-2,3-diotil tetrazolium chloride (CTC) (6, 12). All these compounds are colorless and, after reduction by means of the dehydrogenases, are transformed to monophormazans, which are characterized by a stable red color, which is easily determined quantitatively by means of spectrophotometric methods. A high degree of correlation is found between TTC-dehydrogenase and the quantity of ATP, another widely used biochemical indicator for the activity of biofilms (8). Complex approaches are applied as a whole in the characterization of biofilms, including microscopic techniques, microbiological and enzymological profile, as well as molecular genetic methods. Their aim is obtaining more precise and more complete information about the structure and functioning of the immobilized communities. Biofilms have to be studied together with the space around them, including the liquid phase, since the formation of the biofilm and its functioning happen in parallel with the processes which take place in the liquid phase and with the

2 development of the suspended cells in it (16). This is related to the constant exchange of microorganisms, since part of the cells in the water phase attach themselves to the inert carrier and participate in the biofilm formation, while under stress conditions or upon aging of the biofilm, cells or whole aggregates of cells are separated in the water phase, which are included in the content of the suspended biomass. The aim of the present study was to follow the dynamics of key microbiological and enzymological indicators of the biofilm and the suspended biomass in an anaerobic sequencing batch biofilm reactor for dairy wastewater treatment. Materials and Methods Experimental design An anaerobic treatment process was simulated with model wastewater from the dairy processing industry for the period of 282 days. Three control points (CP) were studied in the course of the process: early phase of the process, which is related to the inoculation of the biofilter and the initial biofilm formation (CP1); intermediate phase of the process, related to the transition from batch to semi-continuous mode (CP2) and later phase of the process, which corresponds to the development of a stable functioning biofilm (CP3). Microbial indicator groups and enzyme activities were studied quantitatively in the three control points of the process and were related to: the transformation of carbon anaerobic heterotrophs; anaerobic spore-forming bacteria; total dehydrogenase activity; the transformation of nitrogen and, more specifically, removal of nitrates from the model system denitrifying microorganisms; nitrate reductase activity; the ecological adaptations of the communities to the changes in the surrounding environment fast-growing (r-strategists) and slow-growing microorganisms (K-strategists). The dynamics of the transformation processes were assessed by the indicators effectiveness of COD decreasing and effectiveness of nitrate removal. The process was simulated in glass bioreactors (biofilter type) with a working volume of 0.5 L. The carrier in the anaerobic sequencing batch biofilm reactor was gravel, with particle size of 8 mm to 16 mm, and was presterilized at 160 С. The reactors were placed in a thermostat in the dark at a temperature of 28 C ± 2 С. The model wastewater contained mineral media, which has a buffering role and whey as the only source of carbon. The last was chosen as a target pollutant, since in some dairy processing plants it is not utilized and gets into the wastewater. The content of the model wastewater included: 0.57 g L -1 NH 4 Cl, 0.43 g L -1 KH 2 PO 4, 1.09 g L -1 K 2 HPO 4, 1.33 g L -1 Na 2 HPO 4, g L -1 MgSO 4.7H 2 O, g L -1 CaCl 2, g L -1 FeCl 3.6H 2 O, and 3.65 g L -1 whey. The whey concentration was chosen on the basis of reaching the value of the parameter COD around mgо 2 L -1, which is characteristic of the wastewater from the dairy processing industry (5). The ph of the model wastewater upon starting the process was 7.2. The biofilter was inoculated with a preliminarliy processed and adapted activated sludge (AS), taken from the Sofia Municipal Wastewater Treatment Plant Kubratovo. It was treated with an ultrasonic disintegrator UD-20 automatic (4 10 seconds at a frequency of 22 khz and vibration amplitude of 8 µm) in order to obtain a thick microbial suspension with a high degree of biodiversity. Microscopic control was done simultaneously with the disintegration to avoid breaking the integrity of the separate cells. The treated AS was acclimated to the model wastewater for a period of 135 hours. The concentration of AS at the start of the process was 10 g L -1, measured as dry weight, with 3 g L -1 of the Laktazym preparation added to it. Laktazym contains different species of spore-forming bacteria and is used for treatment of wastewater from the food industry. The structure of the used lyophilized preparation is shown in Fig. 1. Fig. 1. Structure of the microbiological preparation Laktazym observed by scanning electron microscope at magnification Х (a) and X (b). Particles from the preparation and the spores attached to them are seen in the scanning electron microscope images. The preliminary analysis showed that the content of anaerobic heterotrophs and anaerobic spore-forming bacteria in the lyophilized preparation Laktazym was CFU g -1, and that of the denitrifying microorganisms was CFU g -1. Analytical methods The chemical oxygen demand (COD) and concentration of nitrates were measured according to APHA (1). The microbial diversity of the suspended biomass and of the biofilm was determined by plate count techniques (10). The preliminary processing of the sample included ultrasonic disintegration with a UD-20 automatic (3 5 s with a frequency of 22 khz and vibration amplitude of 8 μm) for making a homogenous suspension. The anaerobic heterotrophs (AnH) and the anaerobic spore-forming bacteria (AnS) were cultivated on nutrient agar (Scharlau, Brit. Pharm.). The samples for determining the anaerobic spore-forming bacteria were treated at 80 С for 10 min. The denitrifying microorganisms (DN) were cultivated on solid nutrient media of Giltay (10). All microorganisms were incubated for 7 days at 28 C ± 2 C in anaerobic jars (Merck &Co., Inc.) with Anaerocult A (Merck &Co., Inc.) for creation of anaerobic conditions. The structure characterization of the microbial communities was based on the r/k distribution of microorganisms depending on the growth rate. The cultivation method described by De 3783

3 Leij et al. (4) was used. The fast growing bacteria (r-strategists) were defined as bacteria that form visible colonies on nutrient agar at 28 C to 30 C within 48 hours. In contrast with the r-strategists, the K-strategists are slow-growing bacteria and yield visible colonies at 28 C to 30 C after the 48 th hour of cultivation. The bacterial colonies that appeared on agar plates were quantified daily for a period of seven days. The number of anaerobic colonies at each day was expressed as a proportion (%) of the total count. The Petri dishes in which the number of colonies was between 30 and 300 were used. The total dehydrogenase activity (DHA) was investigated spectrophotometrically according to Gabbita and Huang (6). The nitrate reductase activity (NRA) was measured by a decrease of nitrate concentration per unit of time under anoxic conditions, with an ion selective electrode of ionmeter Jenway (21). The data for both enzymes were presented for unit protein. The protein content was measured by the micro-biuret method (9). The biodegradation effectiveness (Eff) was calculated for the total organic content (measured as COD) and nitrates. The following formula was used:, % where Ct 1 is the concentration of pollutant at moment t 1 ; Ct 2 is the concentration of pollutant at moment t 2. Moment t 1 is just after a feeding and moment t 2 is just before the next feeding. The microbiological parameters were measured in six repetitions for calculation of the standard deviations. The data were analyzed using Sigma Plot. Results and Discussion In the water phase during the three studied CPs, according to the concept of r- and K-strategists, the structure of the community was preserved unchanged. It was found that the slow-growing species from the groups of the anaerobic heterotrophs and the denitrifying bacteria dominated, while the anaerobic spore-forming ones were characterized with quick growth (Fig. 2а,b,c). The obtained results show that the acclimated activated sludge and the microbiological preparation Laktazym added to it, used for the initial inoculation of the biofilter, were a well-structured biological system with mutually complementing metabolic pathways. The heterotrophic microorganisms in the water phase were characterized by high competitiveness in this well populated environment because of the partial colonization of the inert carrier in the early phases. The presence of fast growing anaerobic spore-forming microorganisms was an indicator that the bacteria from this group were characterized with smaller species diversity and lower competitiveness (19). Most probably this is related to the fact that the added preparation Laktazym was the basic source of spore-forming microorganisms in the used inoculum. The low competitiveness of these allochthonous microorganisms in the community suspended in the water phase was a result of the more difficult overcoming of the ecological barrier of the new environment and more specifically their inclusion in the structure of the autochthonic community, which was characterized by high population stability. Fig. 2. Dynamics of the functional diversity of microbial populations in the water phase (a, b, c) and in the biofilm (d, e, f) in: CP1 (a, d), CP2 (b, e), CP3 (c, f); ± SD; n = 6. The only changes in the structure of the microbial community in the water phase were related to the type of distribution of the colonies in time. Uneven distribution was found during CP1 and CP2, whereas during CP3 the colonies were evenly distributed during the whole period of cultivation, which showed stabilization of the environmental conditions. Dynamic restructuring was found in the immobilized community during the three phases of the process (Fig. 2d,e,f) which showed higher dynamics in the environmental conditions. That was related to the colonization of the inert carrier and the formation of diverse microhabitats. The fast-growing anaerobic heterotrophs in the biofilm dominated during CP1 and CP2 (Fig. 2d,e), which is characteristic of the biofilms at an early phase of formation (19). This confirms that the biofilm is still forming and structuring. The presence of fast growing anaerobic spore-forming microorganisms during CP1 was also related to the presence of more space for colonization and the presence of easily degradable substrates for utilization. The slow growing spore-forming bacteria dominated during CP2, which was most probably related to the inclusion of part of the allochthonous microorganisms added with the preparation, in the biofilm structure. The slow growing anaerobic heterotrophs dominated during CP3, which is characteristic of the mature biofilms (19). This showed the biofilm stabilization. When there is sufficient time for increasing the number of the heterotrophic populations and utilization of the available substrates, microorganisms with a K-strategy of growth but with active biodegradation potential are selected. The active cell metabolism of the K-strategists allows them to compete for less quantity of organic matter and nutrients and to utilize substrates that are more difficult to degrade. The dynamic restructuring of the biofilm, because of the more diverse conditions of the environment, ensures a fuller 3784

4 realization of the metabolic potential of the immobilized community in comparison to that in the suspended biomass. This was confirmed by the observation that, during the late phase of the process (CP3), the total metabolic activity of the biofilm was four times higher in comparison to the one of the suspended biomass, and the nitrate reductase activity was two times higher (Fig. 3, Fig. 4). The higher enzyme activity of the biofilm was most probably related to the higher local concentration of substrates and enzymes because of the presence of an inert carrier and of a polysaccharide matrix. Fig. 3. Dynamics of the total dehydrogenase activity (DHA), quantity of anaerobic heterotrophs (AnH) and anaerobic spore-forming bacteria (AnS) in the water phase (a) and the biofilm (b). Fig. 4. Dynamics of nitrate reductase activity (NRA) and of the quantity of denitrifying microorganisms (DN) in the water phase (a) and the biofilm (b). The alteration in the quantity of the anaerobic heterotrophs and the denitrifying bacteria followed one and the same tendency in the water phase and in the biofilm. A peak of the microbial populations formed at the intermediate phase of the process (CP2), followed by a decrease in their quantity during the late phase (Fig. 3, Fig. 4). At the same time, a peak of the total dehydrogenase activity was found in CP3. This apparent paradox between activity and quantity of the microorganisms in the late phase of the process (CP3) was due to the high activity of the thin biofilms (11). The hypothesis for explaining this phenomenon, reported by Nikolov et al. (15), presupposes the presence of highly active cells on the surface of the biofilm under more favorable conditions for mass exchange, respectively, with a better supply of substrates and washing of the intermediate metabolites. Another possible reason could be that in the stabilized mature biofilms there was a high degree of synergetic interrelations and a highly effective polysubstrate cooperation among the microorganisms forming the biofilm (21). The obtained data about the structure and the activity of the biological system confirm that in some cases the enzymological indicators were a much quicker and more reliable indicator for the functional changes in the microbial communities in comparison to the used quantities of the key microbial groups (21). The alteration in the quantity of the anaerobic spore-forming bacteria followed a different tendency: in the water phase their quantity decreased from CFU cm -3 to CFU cm -3 during the process, whereas in the biofilm it increased from CFU cm -2 to CFU cm -2 (Fig. 3). The commercial preparation Laktazym contains spore-forming bacteria, which presupposes that the decrease of the anaerobic spore-forming microorganisms in the water phase was a result of their staying on the inert carrier. The dehydrogenase activity for the two studied microbial communities increased in the course of the process (Fig. 3). This tendency was confirmed indirectly also by the dynamics of the chemical and kinetic indicators (Table 1). It was found that, as the process progresses, the effectiveness of organic matter (measured as COD) biodegradation increases. The effectiveness reached up to 26 % during the early phase (CP1) and up to 90 % during the late phase (CP3). The dehydrogenase activity was lowest during CP1 and CP2, although the highest quantity of anaerobes was registered then. Their increased quantity was a compensatory mechanism of the decreased metabolic activity. The alteration of the nitrate reductase activity followed that of the dehydrogenase one. A peak in the enzyme activity was observed again at the end of the process, despite the low quantities of the denitrifying bacteria (Fig. 4). The effectiveness of nitrate removal was highest at the end of the process (CP3), when it reached up to 67 % (Table 1). The similar tendencies in the alteration of the anaerobic heterotrophs and the denitrifying microorganisms, as well as of the two enzymological indicators, clearly illustrate the relation between the transformation of Concentrations and effectiveness of biotransformation for COD and nitrates TABLE 1 CP Parameter Influent concentration, mg L -1 Effluent concentration, mg L -1 Effectiveness, % CP1 COD Nitrates CP2 COD Nitrates CP3 COD Nitrates

5 the carbon and the nitrogen in the model system, since, for the removal of nitrates, the denitrifying microorganisms need readily biodegradable organic matter. Conclusions The increasing application of biofilm processes in wastewater treatment requires the development of a new analytical approach for fixed biomass characterization. This article presents data on parameters for analysis of the microbiological structure and functions of fixed biomass and their possible application as biological indicators in dairy wastewater treatment. The biomass activity in terms of substrate removal ability is a key parameter in wastewater treatment technologies. However, the obtained data showed that this parameter was not proportional to the quantity of active immobilized cells. Therefore, new indicators for biofilm activity and for better control of the fixed film process effectiveness should be used. The enzymological parameters (total dehydrogenase activity and nitrate reductase activity) were more accurate indicators for biofilm activity in comparison to the investigated physiological groups of microorganisms. Our results are connected with two general directions of environmental biotechnology. First, they contribute to quick and adequate indication of the rate and scale of the bioprocesses that are critical for the effective management of the technologies. Second, these indicators have a potential to monitor events which are important for environmental management, in complex biological systems (biofilm, activated sludge, etc.) including different types of synergy, syntrophy, metabolic cooperation, microbial and enzyme successions. The high level of synergism and the created effective metabolic cooperation and succession in biofilm determined its higher metabolic activity in comparison with the activity of the suspended biomass. This determined the high effectiveness of the investigated biological system at the late phase of the process for mineralized organic pollutants (to 90 %) and for nitrate removal (to 67 %) from dairy wastewater. Acknowledgеments This work was financially supported by the National Scientific Fund of the Bulgarian Ministry of Education, Youth and Science Project DMU03-56: Innovative ecological approaches for dairy wastewater treatment. REFERENCES 1. APHA, AWWA, WEF (1989) Standard Methods for the Examination of Water and Wastewater, American Public Health Association, Washington DC. 2. Bitton G. (2005) Wastewater Microbiology, 3 rd Ed., John Wiley & Sons, Inc, Hoboken, New Jersey. 3. Cresson R., Carrere H., Delgenes J.P., Bernet N. (2006) Biochem. Eng. J., 30, De Leij F.A.A.M., Whipps J.M., Lynch J.M. (1993) Microbial Ecol., 27, Demirel B., Yenigun O., Onay T. (2005) Process Biochem., 40, Gabbita K., Huang J. (1984) Toxicol Env Chem, 8, Haridas A., Suresh S., Chitra K.R., Manilal V.B. (2005) Water Res., 39, Klapwijk A., Drent J., Steenvoorden J. H. A. M. (1974) Water Res., 8, Kochetov A. (1980) Practical Guidance of Enzymology, Science, Moscow. 10. Kuznetzov S. I., Dubinina G. A. (1989) Methods of Investigation of Aqueous Microorganisms, Science, Moscow. 11. Lazarova V.Z., Capdevill B., Nikolov L. (1992) Water Sci. Technol., 6, Lazarova V.Z., Manem J. (1995) Water Res., 29, Liu W.T., Chan O.C., Fang H.H.P. (2002) Water Res., 36, Meier- Schneiders M., Busch C., Diekert G. (1993) Appl. Microbiol. Biotech., 38, Nikolov L., Mamatarkova V., Petrova E., Stoyanov S. (2005) Ecological Engineering and Environment Protection, 2, Nikolov L., Mamatarkova V., Slavchev S., Stoychev S. (2008) Ecological Engineering and Environment Protection, 1, Omil F., Garrido J., Arrojo B., Mendez R. (2003) Water Res., 37, Sen S., Demirer G.N. (2003) Water Res., 37, Sigee D.C. (2005) Freshwater Microbiology, Whiley Press, Manchester. 20. Sirianuntapiboon S., Teeyachok N., Larplai R. (2005) J. Environ. Manage., 76, Topalova Y. (2009) Biological Control and Management of Wastewater Treatment, PublishScieSet-Eco, Sofia. 22. Wijffels R.H., Tramper J. (1995) Enzyme and Microbial Technol., 17, Yun M., Yeon K., Park J., Lee C., et al. (2006) Water Res., 40,