World Journal of Microbiology & Biotechnology 12,391-394
Biofilm formation in water cooling systems M.T.S. Lutterbach and F.P. de Fran(;:a* Biofilm formation on stainless steel samples immersed in cooling water has been evaluated by exposing metal samples to cooling seawater for 30 days. Anaerobic bacteria were then at 1.6 × 106/cm z, with sulphate-reducing species predominating• Aerobic bacteria and fungi were 2600 and 140/cm 2, respectively• After 60 days, numbers of aerobic microorganisms remained constant whereas the count of anaerobic microorganisms had increased to 1.8 x 109/cm *. Scanning electron microscopy showed the presence of morphologically different microorganisms in deposits and as a mucilaginous net. No signs of corrosion were detected on the stainless steel surface. Key words: Biofilm, cooling water, microbiologically influenced corrosion, stainless steel.
Biofilms are complex structures made up of microbial cells and of extracellular matter derived from their metabolic activities. Microbial colonization is initiated when organic substances are first transported from the liquid medium to the solid metal surface, thereby leading to local accumulation of nutrients. Bacteria present in the water column come into contact with these rich surfaces, settle on the substratum, and then grow and replicate. The bacteria secrete exopolysaccharides which facilitate attachment of further bacteria and of microorganisms like fungi, microalgae, protozoa, macroalgae and even invertebrates (Characklis & Marshall 1990). Due to the thickness and complex microbial composition of a biofilm, an uneven aeration is established through the various layers of its structure, generating at the base of the biofilm conditions for the growth of anaerobic microorganisms (Stupak et al. 1990) The formation of a biofilm and its properties are influenced by various factors including water quality, type of substratum for colonization, temperature and seasonality. The aqueous environment and the substratum influence the selection of microorganisms that will predominate in the forming biofilm. Within the biofilm structure, that is, within an established microenvironment, changes in the The authors are with the Departamento de Engenharia Bioquirnica Centro de Tecnologia, Bloco E, Universidade Federal do Rio de Janeiro Ilha do Fund&o, 21941-900 Rio de Janeiro, Brazil; fax: (021) 590 4991. • Corresponding author.
substratum depend on microbial metabolism. In some cases of corrosion induced by microbial colonization, the morphology and distribution of deposits are indicative of the type of fouling microorganism (Characklis & Marshall 1990). Cooling systems are of great importance, being used in a vast array of industrial sectors such as the petrochemical and oil branches. Due to their particular location, several Brazilian plants employ an open cooling system with no circulation and water directly drawn from the sea. Sea water analysis reveals fairly high concentrations of chlorides (19 g/l), sulphates (2.7 g/l), bicarbonates (0.4 g/l) and magnesium (1.3 g/l). Due to its content of organic matter, sea water also allows the proliferation of a great variety of microorganisms; in addition, it contains high concentrations of solid matter in suspension which favours the corrosion of metal surfaces (Videla et al. 1992). Metal corrosion by cooling water is mainly caused by the presence of chlorides. However, the role of microorganisms as accelerators of the corrosion process has to be considered. The standard austenitic stainless steel type AISI-304 (I8% Ni; i8% Cr) is susceptible to the action of seawater. Yet it is still used in the cooling equipment of petrochemical plants. Its replacement by special stainless steel is not viable due to the high costs involved. The aim of the present work was to evaluate microfilm formation and its effect on coupons of stainless steel (SS) type AISI-304.
(~) 1996 Rapid Science Publishers Warld ]ournal of Microbiology & Biotechnalogy, Vo112, 1996
391
M. T.S. Lutterbach and F.P. de Franca and the samples were evaluated after 60 days. As a control, steel samples were placed inside flasks with 100 ml of sterile seawater. SYSTEM
COOLING SEA WATER ROM THE SYSTEM
c°?;g2,
WASTE WATER
Figure 1, Diagram of a by-pass duct installed on the industrial cooling system (bar = 50 cm).
Materials and Methods Field (in situ) Testing Field experiments were carried out in a petrochemical plant beside Guanabara Bay, Rio de Janeiro, Brazil. The seawater utilized by the plant as coolant fluid for the heat exchanger system was not pretreated despite it incorporating the waste discharge from neighbouring plants. Seawater analysis revealed the following composition: Mg 2+ 740 rag/l; AP + < 1.0 rag/l; Mn 2+ 0.1 rag/l; K + 253 rag/l; Na + 6.4 g/l and C1- I2.85 g/1. During the experimental period, the water temperature varied from 30°C to 32°C and the pH from 7.7 to 7.9; the mean industrial water flow was 1.9 mUh. Having considered the various kinds of materials employed in the manufacture of industrial equipment, 2.0 x 1.7 cmz pieces of stainless steel type AISI-304 were chosen as metal samples and placed at the coolant water outlet of the heat exchangers (Figure 1). The stainless steel samples were degreased with acetone and kept in a desiccator before being fixed to the ducts. To avoid possible interference with the attachment of microorganisms to the metal surface, samples were not submitted to any abrasive pretreatment (Videla & de Mele 1988; Walsh et al. 1992). Samples were withdrawn at intervals of 30 and 60 days for evaluation of biofilm formation.
Laboratory Testing Steel samples were placed inside flasks (160 ml) containing 100 ml of seawater which was collected at the plant's water intake site. Fresh seawater was added to the flasks at 15 days intervals for a steady supply of nutrients to the microorganisms, with concomitant outflow of old water through a lateral outlet to keep a constant level of water. The flasks were incubated at 30 + 1°C
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World Journal of Microbiology & Biotechnology, Vo112, 1996
Quantitative Determinations Sampling. For determination of aerobic microorganisms, steel samples were aseptically removed from the flasks and flushed with sterile distilled water containing 2% (w/v) NaC1 to remove nonadherent microorganisms. Microorganisms were recovered in 30 ml 2% (w/v) NaCI by scraping one of the surfaces of the metal with a sterile spatula under aseptic conditions, as described by Cook & Gaylarde, I988. The scraping method adopted allowed the detachment of cells and other components responsible for the formation of the biofilm. For determination of anaerobic bacteria, the removal of biofilms was carried out as described, except that a reducing solution (Postgate 1984) previously degassed with N2 was used in place of NaCI, in order to establish conditions appropriate for anaerobiosis. The respective biofllm suspensions were then used to determine the numbers of microorganisms present as well as the total sulphide content.
Quantification of Aerobic Bacteria. Aerobic bacteria were quantified according to the technique of the most probable number (MPN) (Koch 1981). Nutrient broth (Merck) was used as culture medium and tubes were incubated at 32 + I°C for 48 h. Quantification of Anaerobic Bacteria. For quantification of anaerobic bacteria, the MPN technique was also applied using fluid thioglycollate medium (Difco) supplemented with ferric ammonium sulphate and degassed with N2. The assay was carried out inside tubes sealed airtight with a rubber stopper and a metal ring belt, each tube containing 10 ml of medium. After addition of the medium, the flasks were degassed once more with N2 to ensure total removal of O5 prior to use. Incubation was carried out at 32 + 1°C for 28 days. Quantification of Sulphate-Reducing Bacteria. Essentially the same technique described above for quantification of anaerobic bacteria was used to evaluate the number of sulphate-reducing bacteria (SRB) present, using the Postgate B medium (Postgate 1984). Quantification of Fungi. The amount of fungi present was quantified by enumeration of colony-forming units on Petri dishes containing 20 ml of Sabouraud medium (Merck) each. Incubation was carried out at 25 +_ 1°C for 7 days. Quantification of Total Sulphides. Total sulphide content was quantified by a colorimetric method (APHA 1972) after treating the various biofilm suspensions with concentrated HCI. Evaluation by Scanning Electron Microscopy (SEM) SEM analysis was carried out on coupons containing biofilms after the stated exposure times. Specimens were fixed immediately after in situ collection with 2.5% glutaraldehyde solution and 0.1 M cacodylate buffer, 1:1 (v/v) in sea water, for 24 h at 4°C. Further processing of samples for SEM was done according to Coutinho (1991).
Results The biofilms formed on the stainless steel surface during field experiments contained an extremely low number of aerobic microorganisms with respect to the number of
Biofilm formation in water cooling systems Table 1. Number of microorganisms and sulphide concentrations of biofilms formed on the surface of stainless steel samples, Determination
Field tests 30 d -t
Aerobic bacteria (log No/cm 2) Anaerobic bacteria (log No/cm 2) SRB (log No/cm 2) Fungi (log c.f.u./cm 2) Sulphides (mg/cm 2)
3.41" 6.19 4.12 2.13 173.6
_+ 2.4 _+ 5.36 _+ 3,2 + 1.3 _+ 9.4
Laboratory tests 60 d
60 d 3.98 9.26 7.15 2.3 87.2
+ + 4" + +
2.85 8.45 6.15 1.41 5.6
9.15 11.1 11.1 3.89 848.3
+ + + + +
8.23 10.2 10.2 2.6 16.7
Control tests 60 d ND ND ND ND 3.6 _+ 0.2
* Mean value of 4 experiments; ND - not detected. 1 days of exposure to cooling water.
Figure 2. SEM microphotograph showing microfouling of the stainless steel surface after 30 days of in situ exposure to cooling water (bar -- 1 ,um).
cantly higher than on day 30 (Table 1). The elevated levels of sulphides compared to the controls indicate the active participation of SRB. Biofilms obtained under laboratory conditions exhibited greater numbers of microorganisms and consequently a higher total sulphide content (Table 1) compared to in situ conditions. These differences between field derived and laboratory derived data were expected due to the distinct experimental procedures in each case. SEM evaluation of in situ microfouling (field experiments) revealed the presence of several types of microorganism after 30 days of testing, including microalgae of variable morphology and extracellular polymers (Figures 2 and 3). The base of the substratum could still be seen due to the irregular distribution of deposits adhered to the metal surface. On day 60 the number of bacteria had increased and were now linked by a net, possibly of exopolysaccharides (EPS), together with other deposits (Figure 4). SEM analysis of laboratory samples confrmed the attachment of microorganisms to the stainless steel samples.
Discussion
Figure 3. SEM microphotograph showing microfouling of the stainless steel surface with colonization by algae after 30 days of in situ exposure to cooling water (bar = 1/lm).
anaerobic bacteria present (Table 1). No increase in the numbers of aerobic bacteria and fungi was observed between 30 and 60 days exposure to the cooling water. However, on day 60 of testing, the settlement of anaerobic bacteria and sulphate-reducing species (SRB) was signifi-
Significant numbers of anaerobic bacteria were found attached to the stainless steel samples (Table 1), indicating that the conditioning layer provided by the cooling water and the growth of aerobic bacteria allowed the formation of anaerobic sites. The anaerobic microenvironments were generated by the growth of aerobic bacteria and fungi that, besides consuming oxygen, secreted extracellular polymers (Figures 3 and 4) with limited diffusion of oxygen to the base of the biofilm. The low numbers of aerobic microorganisms detected on the surface of steel colonized in situ suggests that the biofilm must have been formed before 30 days of exposure and that there was probably a sloughing effect caused by water turbulence, as these organisms settle preferentially on the outer surface of the biofilm. Several investigators have demonstrated the presence of metabolites, cells and cellular components in the aqueous phase as a consequence
World]oumal of Microbiology& Biotechnology,Vol I2, I996
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M. T.S. Lutterbach and F.P. de Fran¢a
extracellular polymeric substances, possibly synthesized by the Gram-negative bacteria. The surface of the steel from which the biofilms had been removed were subsequently observed through a stereoscopic microscope. Despite the corrosive potential of sea water and the occurrence of microfouling, no signs of corrosion were visible on the surface of the stainless steel. As stainless steel type AISI-304, is an austenitic metal it contains relatively high concentrations of chromium (an inert element) which may have contributed to its resistance to corrosion.
Acknowledgements Figure 4. SEM microphotograph showing microfouling of the stainless steel surface after 60 days of in situ exposure to cooling water (bar = lO/~m).
of biofilm sloughing caused by the water flux of cooling systems (Characklis & Marshall 1990; Marshall & Walker 1990). It should be pointed out that the number of microorganisms transported in the aqueous phase varies depending on temperature fluctuation caused by heat exchange within the cooling towers. Sulphide content was generated essentially by the metabolic activity of SRB that reduce sulphur compounds, thus producing biogenic sulphides. The seawater used contained only 36/zg sulphides/ml and this value remained constant throughout the experimental period, as revealed by the control tests (sterile seawater and sterile steel samples). The greater number of microorganisms detected in the laboratory experiments compared to field experiments can be explained by the absence of a sloughing effect in the former situation, since the aqueous phase remained stationary. Additionally, the constant temperature under laboratory conditions favoured the appearance of a high number of microorganisms due to their initially greater replication rate on the surface of the steel, possibly associated with a decrease in sloughing due to reduced turbulence. The elevated sulphide levels confirm the metabolic activity of SRB, present in high numbers in the biofilms. Among the microalgae shown by SEM, cellular forms belonging to the group of Diatomaceae appeared to be predominant. These algae are characterized by a porous morphology, a feature that probably facilitates and brings about the trapping of other smaller cells, such as bacteria. Furthermore, the microalgae produce organic compounds that can be used as nutrients by the bacteria (Characklis & Marshall 1990), establishing a partnership between the two microorganisms. The rod-shaped cells of different dimensions present in the biofilms are Gram-positive and Gram-negative bacteria, with a predominance of the latter. SEM analysis also revealed the presence of mucilaginous nets composed of
394
World Journal of Microbiology & Biotechnology, Vol 12. 1996
The authors wish to thank the Conselho Nacional de Desenvolvimento Cientlfico e Tecnol6gico/Brazil (CNPq) for financial support; CENPES/Petrobr~s for the use of SEM equipment; Petroflex for the use of the industrial system; CETEC - Vassouras/SENAI for making the samples and holders and Dr Vicente Gentil, Lecturer at the UFRJ, for his valuable suggestions.
References American Public Health Association, APHA. 1972 Tentative me[hod of analysis for hydrogen sulphide content of the atmosphere. In: Methods of air sampling and analyses, pp. 420-432. Washington. Characklis, W.G. & Marshall, K.C. 1990 Biofilms. New York: Wiley-Interscience Publication. Cook, P.E. & Gaylarde, C.C. 1988 Biofilm formation in aqueous metal working fluids. International Biodeterioration 24, 265-270. Coutinho, C.M.L.M. I99I Caracterizac/io ultra estrutural e citoquimica de biofilmes bacterianos, M.Sc. dissertation. Dept. Biologia Celular e Molecular. Fund. Oswaldo Cruz. Rio de Janeiro, Brazil. Koch, A.L. 1981 Growth Measurement. In: Manual of Methods for General Bacteriology, ed. Gerhardt, P., pp. 188-207. Washington: American Society for Microbiology. Marshall, A. & Walker, G.N. 1990 Increased awareness of biological control and its implications. Corrosion/90, paper no. 354, pp. 1-21. Houston: NACE. Postgate, J.R. I984 The sulphate-reducing bacteria. Cambridge: Cambridge University Press. Stupak, M.E., P6rez, M.C. & di Sarli, A.R. 1990 Relaci6n entre la fijaci6n de micro y macrofouling y los procesos de corrosi6n de estructuras met~ilicas.Revista Iberoamericana Corrosion y Protecion 21, 219-225. Videla, H.A. & de Mele, M.F.L. 1988 Assessment of corrosion and microfouling of several metals in polluted seawater. Corrosion Science 44, 423-426. Videla, H.A., G6mez de Saraiva, S.G. & de Mele, M.F.L. 1992 MIC of heat exchanger materials in marine media contaminated with sulphate-reducing bacteria. Corrosion/92, paper no. 189, pp. 1-9. Houston: NACE. Walsh, D., Seagoe, J. & Williams, L. 1992 Microbiologically influenced corrosion of stainless steel weldments, attachment and film evolution. Corrosion/92, paper no. 165, pp. 1-20. Houston: NACE. (Received in revised form 8 February 1996; accepted 10 February 1996)
Biofilm formation in water cooling systems M.T.S. Lutterbach and F.P. de Fran(;:a* Biofilm formation on stainless steel samples immersed in cooling water has been evaluated by exposing metal samples to cooling seawater for 30 days. Anaerobic bacteria were then at 1.6 × 106/cm z, with sulphate-reducing species predominating• Aerobic bacteria and fungi were 2600 and 140/cm 2, respectively• After 60 days, numbers of aerobic microorganisms remained constant whereas the count of anaerobic microorganisms had increased to 1.8 x 109/cm *. Scanning electron microscopy showed the presence of morphologically different microorganisms in deposits and as a mucilaginous net. No signs of corrosion were detected on the stainless steel surface. Key words: Biofilm, cooling water, microbiologically influenced corrosion, stainless steel.
Biofilms are complex structures made up of microbial cells and of extracellular matter derived from their metabolic activities. Microbial colonization is initiated when organic substances are first transported from the liquid medium to the solid metal surface, thereby leading to local accumulation of nutrients. Bacteria present in the water column come into contact with these rich surfaces, settle on the substratum, and then grow and replicate. The bacteria secrete exopolysaccharides which facilitate attachment of further bacteria and of microorganisms like fungi, microalgae, protozoa, macroalgae and even invertebrates (Characklis & Marshall 1990). Due to the thickness and complex microbial composition of a biofilm, an uneven aeration is established through the various layers of its structure, generating at the base of the biofilm conditions for the growth of anaerobic microorganisms (Stupak et al. 1990) The formation of a biofilm and its properties are influenced by various factors including water quality, type of substratum for colonization, temperature and seasonality. The aqueous environment and the substratum influence the selection of microorganisms that will predominate in the forming biofilm. Within the biofilm structure, that is, within an established microenvironment, changes in the The authors are with the Departamento de Engenharia Bioquirnica Centro de Tecnologia, Bloco E, Universidade Federal do Rio de Janeiro Ilha do Fund&o, 21941-900 Rio de Janeiro, Brazil; fax: (021) 590 4991. • Corresponding author.
substratum depend on microbial metabolism. In some cases of corrosion induced by microbial colonization, the morphology and distribution of deposits are indicative of the type of fouling microorganism (Characklis & Marshall 1990). Cooling systems are of great importance, being used in a vast array of industrial sectors such as the petrochemical and oil branches. Due to their particular location, several Brazilian plants employ an open cooling system with no circulation and water directly drawn from the sea. Sea water analysis reveals fairly high concentrations of chlorides (19 g/l), sulphates (2.7 g/l), bicarbonates (0.4 g/l) and magnesium (1.3 g/l). Due to its content of organic matter, sea water also allows the proliferation of a great variety of microorganisms; in addition, it contains high concentrations of solid matter in suspension which favours the corrosion of metal surfaces (Videla et al. 1992). Metal corrosion by cooling water is mainly caused by the presence of chlorides. However, the role of microorganisms as accelerators of the corrosion process has to be considered. The standard austenitic stainless steel type AISI-304 (I8% Ni; i8% Cr) is susceptible to the action of seawater. Yet it is still used in the cooling equipment of petrochemical plants. Its replacement by special stainless steel is not viable due to the high costs involved. The aim of the present work was to evaluate microfilm formation and its effect on coupons of stainless steel (SS) type AISI-304.
(~) 1996 Rapid Science Publishers Warld ]ournal of Microbiology & Biotechnalogy, Vo112, 1996
391
M. T.S. Lutterbach and F.P. de Franca and the samples were evaluated after 60 days. As a control, steel samples were placed inside flasks with 100 ml of sterile seawater. SYSTEM
COOLING SEA WATER ROM THE SYSTEM
c°?;g2,
WASTE WATER
Figure 1, Diagram of a by-pass duct installed on the industrial cooling system (bar = 50 cm).
Materials and Methods Field (in situ) Testing Field experiments were carried out in a petrochemical plant beside Guanabara Bay, Rio de Janeiro, Brazil. The seawater utilized by the plant as coolant fluid for the heat exchanger system was not pretreated despite it incorporating the waste discharge from neighbouring plants. Seawater analysis revealed the following composition: Mg 2+ 740 rag/l; AP + < 1.0 rag/l; Mn 2+ 0.1 rag/l; K + 253 rag/l; Na + 6.4 g/l and C1- I2.85 g/1. During the experimental period, the water temperature varied from 30°C to 32°C and the pH from 7.7 to 7.9; the mean industrial water flow was 1.9 mUh. Having considered the various kinds of materials employed in the manufacture of industrial equipment, 2.0 x 1.7 cmz pieces of stainless steel type AISI-304 were chosen as metal samples and placed at the coolant water outlet of the heat exchangers (Figure 1). The stainless steel samples were degreased with acetone and kept in a desiccator before being fixed to the ducts. To avoid possible interference with the attachment of microorganisms to the metal surface, samples were not submitted to any abrasive pretreatment (Videla & de Mele 1988; Walsh et al. 1992). Samples were withdrawn at intervals of 30 and 60 days for evaluation of biofilm formation.
Laboratory Testing Steel samples were placed inside flasks (160 ml) containing 100 ml of seawater which was collected at the plant's water intake site. Fresh seawater was added to the flasks at 15 days intervals for a steady supply of nutrients to the microorganisms, with concomitant outflow of old water through a lateral outlet to keep a constant level of water. The flasks were incubated at 30 + 1°C
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World Journal of Microbiology & Biotechnology, Vo112, 1996
Quantitative Determinations Sampling. For determination of aerobic microorganisms, steel samples were aseptically removed from the flasks and flushed with sterile distilled water containing 2% (w/v) NaC1 to remove nonadherent microorganisms. Microorganisms were recovered in 30 ml 2% (w/v) NaCI by scraping one of the surfaces of the metal with a sterile spatula under aseptic conditions, as described by Cook & Gaylarde, I988. The scraping method adopted allowed the detachment of cells and other components responsible for the formation of the biofilm. For determination of anaerobic bacteria, the removal of biofilms was carried out as described, except that a reducing solution (Postgate 1984) previously degassed with N2 was used in place of NaCI, in order to establish conditions appropriate for anaerobiosis. The respective biofllm suspensions were then used to determine the numbers of microorganisms present as well as the total sulphide content.
Quantification of Aerobic Bacteria. Aerobic bacteria were quantified according to the technique of the most probable number (MPN) (Koch 1981). Nutrient broth (Merck) was used as culture medium and tubes were incubated at 32 + I°C for 48 h. Quantification of Anaerobic Bacteria. For quantification of anaerobic bacteria, the MPN technique was also applied using fluid thioglycollate medium (Difco) supplemented with ferric ammonium sulphate and degassed with N2. The assay was carried out inside tubes sealed airtight with a rubber stopper and a metal ring belt, each tube containing 10 ml of medium. After addition of the medium, the flasks were degassed once more with N2 to ensure total removal of O5 prior to use. Incubation was carried out at 32 + 1°C for 28 days. Quantification of Sulphate-Reducing Bacteria. Essentially the same technique described above for quantification of anaerobic bacteria was used to evaluate the number of sulphate-reducing bacteria (SRB) present, using the Postgate B medium (Postgate 1984). Quantification of Fungi. The amount of fungi present was quantified by enumeration of colony-forming units on Petri dishes containing 20 ml of Sabouraud medium (Merck) each. Incubation was carried out at 25 +_ 1°C for 7 days. Quantification of Total Sulphides. Total sulphide content was quantified by a colorimetric method (APHA 1972) after treating the various biofilm suspensions with concentrated HCI. Evaluation by Scanning Electron Microscopy (SEM) SEM analysis was carried out on coupons containing biofilms after the stated exposure times. Specimens were fixed immediately after in situ collection with 2.5% glutaraldehyde solution and 0.1 M cacodylate buffer, 1:1 (v/v) in sea water, for 24 h at 4°C. Further processing of samples for SEM was done according to Coutinho (1991).
Results The biofilms formed on the stainless steel surface during field experiments contained an extremely low number of aerobic microorganisms with respect to the number of
Biofilm formation in water cooling systems Table 1. Number of microorganisms and sulphide concentrations of biofilms formed on the surface of stainless steel samples, Determination
Field tests 30 d -t
Aerobic bacteria (log No/cm 2) Anaerobic bacteria (log No/cm 2) SRB (log No/cm 2) Fungi (log c.f.u./cm 2) Sulphides (mg/cm 2)
3.41" 6.19 4.12 2.13 173.6
_+ 2.4 _+ 5.36 _+ 3,2 + 1.3 _+ 9.4
Laboratory tests 60 d
60 d 3.98 9.26 7.15 2.3 87.2
+ + 4" + +
2.85 8.45 6.15 1.41 5.6
9.15 11.1 11.1 3.89 848.3
+ + + + +
8.23 10.2 10.2 2.6 16.7
Control tests 60 d ND ND ND ND 3.6 _+ 0.2
* Mean value of 4 experiments; ND - not detected. 1 days of exposure to cooling water.
Figure 2. SEM microphotograph showing microfouling of the stainless steel surface after 30 days of in situ exposure to cooling water (bar -- 1 ,um).
cantly higher than on day 30 (Table 1). The elevated levels of sulphides compared to the controls indicate the active participation of SRB. Biofilms obtained under laboratory conditions exhibited greater numbers of microorganisms and consequently a higher total sulphide content (Table 1) compared to in situ conditions. These differences between field derived and laboratory derived data were expected due to the distinct experimental procedures in each case. SEM evaluation of in situ microfouling (field experiments) revealed the presence of several types of microorganism after 30 days of testing, including microalgae of variable morphology and extracellular polymers (Figures 2 and 3). The base of the substratum could still be seen due to the irregular distribution of deposits adhered to the metal surface. On day 60 the number of bacteria had increased and were now linked by a net, possibly of exopolysaccharides (EPS), together with other deposits (Figure 4). SEM analysis of laboratory samples confrmed the attachment of microorganisms to the stainless steel samples.
Discussion
Figure 3. SEM microphotograph showing microfouling of the stainless steel surface with colonization by algae after 30 days of in situ exposure to cooling water (bar = 1/lm).
anaerobic bacteria present (Table 1). No increase in the numbers of aerobic bacteria and fungi was observed between 30 and 60 days exposure to the cooling water. However, on day 60 of testing, the settlement of anaerobic bacteria and sulphate-reducing species (SRB) was signifi-
Significant numbers of anaerobic bacteria were found attached to the stainless steel samples (Table 1), indicating that the conditioning layer provided by the cooling water and the growth of aerobic bacteria allowed the formation of anaerobic sites. The anaerobic microenvironments were generated by the growth of aerobic bacteria and fungi that, besides consuming oxygen, secreted extracellular polymers (Figures 3 and 4) with limited diffusion of oxygen to the base of the biofilm. The low numbers of aerobic microorganisms detected on the surface of steel colonized in situ suggests that the biofilm must have been formed before 30 days of exposure and that there was probably a sloughing effect caused by water turbulence, as these organisms settle preferentially on the outer surface of the biofilm. Several investigators have demonstrated the presence of metabolites, cells and cellular components in the aqueous phase as a consequence
World]oumal of Microbiology& Biotechnology,Vol I2, I996
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M. T.S. Lutterbach and F.P. de Fran¢a
extracellular polymeric substances, possibly synthesized by the Gram-negative bacteria. The surface of the steel from which the biofilms had been removed were subsequently observed through a stereoscopic microscope. Despite the corrosive potential of sea water and the occurrence of microfouling, no signs of corrosion were visible on the surface of the stainless steel. As stainless steel type AISI-304, is an austenitic metal it contains relatively high concentrations of chromium (an inert element) which may have contributed to its resistance to corrosion.
Acknowledgements Figure 4. SEM microphotograph showing microfouling of the stainless steel surface after 60 days of in situ exposure to cooling water (bar = lO/~m).
of biofilm sloughing caused by the water flux of cooling systems (Characklis & Marshall 1990; Marshall & Walker 1990). It should be pointed out that the number of microorganisms transported in the aqueous phase varies depending on temperature fluctuation caused by heat exchange within the cooling towers. Sulphide content was generated essentially by the metabolic activity of SRB that reduce sulphur compounds, thus producing biogenic sulphides. The seawater used contained only 36/zg sulphides/ml and this value remained constant throughout the experimental period, as revealed by the control tests (sterile seawater and sterile steel samples). The greater number of microorganisms detected in the laboratory experiments compared to field experiments can be explained by the absence of a sloughing effect in the former situation, since the aqueous phase remained stationary. Additionally, the constant temperature under laboratory conditions favoured the appearance of a high number of microorganisms due to their initially greater replication rate on the surface of the steel, possibly associated with a decrease in sloughing due to reduced turbulence. The elevated sulphide levels confirm the metabolic activity of SRB, present in high numbers in the biofilms. Among the microalgae shown by SEM, cellular forms belonging to the group of Diatomaceae appeared to be predominant. These algae are characterized by a porous morphology, a feature that probably facilitates and brings about the trapping of other smaller cells, such as bacteria. Furthermore, the microalgae produce organic compounds that can be used as nutrients by the bacteria (Characklis & Marshall 1990), establishing a partnership between the two microorganisms. The rod-shaped cells of different dimensions present in the biofilms are Gram-positive and Gram-negative bacteria, with a predominance of the latter. SEM analysis also revealed the presence of mucilaginous nets composed of
394
World Journal of Microbiology & Biotechnology, Vol 12. 1996
The authors wish to thank the Conselho Nacional de Desenvolvimento Cientlfico e Tecnol6gico/Brazil (CNPq) for financial support; CENPES/Petrobr~s for the use of SEM equipment; Petroflex for the use of the industrial system; CETEC - Vassouras/SENAI for making the samples and holders and Dr Vicente Gentil, Lecturer at the UFRJ, for his valuable suggestions.
References American Public Health Association, APHA. 1972 Tentative me[hod of analysis for hydrogen sulphide content of the atmosphere. In: Methods of air sampling and analyses, pp. 420-432. Washington. Characklis, W.G. & Marshall, K.C. 1990 Biofilms. New York: Wiley-Interscience Publication. Cook, P.E. & Gaylarde, C.C. 1988 Biofilm formation in aqueous metal working fluids. International Biodeterioration 24, 265-270. Coutinho, C.M.L.M. I99I Caracterizac/io ultra estrutural e citoquimica de biofilmes bacterianos, M.Sc. dissertation. Dept. Biologia Celular e Molecular. Fund. Oswaldo Cruz. Rio de Janeiro, Brazil. Koch, A.L. 1981 Growth Measurement. In: Manual of Methods for General Bacteriology, ed. Gerhardt, P., pp. 188-207. Washington: American Society for Microbiology. Marshall, A. & Walker, G.N. 1990 Increased awareness of biological control and its implications. Corrosion/90, paper no. 354, pp. 1-21. Houston: NACE. Postgate, J.R. I984 The sulphate-reducing bacteria. Cambridge: Cambridge University Press. Stupak, M.E., P6rez, M.C. & di Sarli, A.R. 1990 Relaci6n entre la fijaci6n de micro y macrofouling y los procesos de corrosi6n de estructuras met~ilicas.Revista Iberoamericana Corrosion y Protecion 21, 219-225. Videla, H.A. & de Mele, M.F.L. 1988 Assessment of corrosion and microfouling of several metals in polluted seawater. Corrosion Science 44, 423-426. Videla, H.A., G6mez de Saraiva, S.G. & de Mele, M.F.L. 1992 MIC of heat exchanger materials in marine media contaminated with sulphate-reducing bacteria. Corrosion/92, paper no. 189, pp. 1-9. Houston: NACE. Walsh, D., Seagoe, J. & Williams, L. 1992 Microbiologically influenced corrosion of stainless steel weldments, attachment and film evolution. Corrosion/92, paper no. 165, pp. 1-20. Houston: NACE. (Received in revised form 8 February 1996; accepted 10 February 1996)
