Indian Journal of Engineering & Material s Sciences Vol. 10. December 2003, pp. 486-49 I

Biofilm formation on structural materials in deep sea environments

R Venkatesan", E S Dwarakadasab & M Ravindran"

"National Institute of Ocean Techno logy, Ve lac hery-Tambaram Road, Pa llikaranai, Chennai 601 302, India "Department of Metallurgy. Indian Institute of Science, Bangalore 560 0 I 2, India

Received 9 April 2003; accepted 16 October 2003

Biofilm formation on st ructural materials in deep-sea environment after long duration of exposure is repmted here for the first time. Some structural materia l specimens such as steel, steel with zinc and aluminum anodes and carbon fibre­reinforced composite were exposed at depths of 500, 1200. 3500 and 5 100 m fo r 174 days. The bacteria l colony formation on these specimens was studi ed after their retrieval from the ocean . Macrofouling was not found in any o f these material s. Carbon fibre-reinforced composite did not support bacterial co lony formation at all the tested depths. Steel supported bacterial colon ies at all depths. Aluminium and zinc anodes did not support bacteri al colonies at 3500 and 5 100 m depths. Thi s could be related to lower temperatures prevailing at these depths. Also, all the identified bacterial colonies were aerobic as disso lved oxygen was present even at 5100 m depth. Hence. anaerobic conditions did not exist during exposure under these structural cond itions. Disso lved oxygen data at the experimental site is also presented and discussed.

Biological organisms are present in virtually all-natural aq ueous environments. In seawater, the tendency for organisms is to attach and grow on all structural materials, resulting in the formation of biological film or biofilm l

, immobilized at a substratum and frequently embedded in an organic polymer matrix of microbial origin2

. The mature biofilm is structurally heterogeneous with large cell clusters and voids. Adjacent to the large cell clusters can be the areas where the biofilm is very thin and nonexistent. The general tendency for microorganisms is to adhere firmly and randomly to any wet surface. They grow, reproduce and produce extra cellular polymeric substances (EPS) or slimes that extend from the cell leading to a dense fibrous network. This network structure is refelTed to as biofilm. The biofilm can range from a microbiological slime film a few micrometers thick to a heavy encrustation of hard­shelled fouling organisms several centimeters thick.

The microbial film deve lopment takes place in stages . The first step is a chemical conditioning of the metal surface by the deposition of a thin layer of small and large organic molecules . This is quickly followed by the colonization of the surface by pioneer bacteria, most of which are mobile gram-negative rods, which become established in a matter of hours. Soon enough other bacteria, stalked ones, budding types, microalgae and protozoa may develop extensively in a matter of days. In the next stage, the biofilm th ickens, accumulating other particles, dead cells and detritus3

.

The biofilm itself can range from a microbiological slime film on fresh water heat transfer surfaces to a heavy encrustation of hard-shell fouling organisms on structures in coastal seawater. The biofilm that forms on the surface of virtually all structural metals and alloys immersed in aqueous environments influences corrosion of metals and alloys4.5 .

At sha llow depths , the oxygen supply is normally at or close to saturation . Biological activity likewise is at a maximum at these shallow depths and includes both plant and animal life. Any biofouling beyond depths of 20 to 35 m is largely animal, since plants cannot exist in the absence of sun light ). Usually there is less fouling as one proceeds away from shore, since major source of animal embryos is from breeding activities near the shore. Fouling is also known to decrease at any given distance from the shore.

As more and more effort is devoted to offshore drilling, ocean mining, etc ., the best poss ible materials must be developed for use in new technologies. Fundamental to the development of such materials is a requirement of the knowledge of how they will behave in deep-sea environment 3. Hence in designing deep water systems, the performance of the materials at such an environment is required. Such in situ test data are scarce and are not generally avai lable for the public domain. Such work involves very expensive deep-sea mooring system and ship-time for long-term observations. Hence there is little or no information available on the behaviour of these materials in deep sea. The on ly such study conducted by the US Naval

VENKATESAN el af.:BIOFILM FORMATION ON STRUCTURAL MATERIALS IN DEEP SEA ENVIRONMENTS 487

Civil Engineering Laboratory and the US Naval Research Laboratory is limited up to 2060 m3

.

Cathodic protection (CP) uses sacrificial anodes and the principles of galvanic coupling to protect structures from corrosion. Galvanic corrosion becomes a complex process, which support the growth of microscopic organisms in biofilm 5.

In the present study, biofilm formation and taxonomical studies on materials have been studied in Deep Ocean up to a depth of S100 m. The specimens of mild steel , mild steel coupled with zinc and aluminium anodes and carbon fibre reinforced composite were exposed at depths of SOO, 1200, 3S00 and SIOO m in the Indian Ocean . The exposed coupons were studied for the presence of biofilm as the Colony Forming Units were enumerated. Taxonomical studies were also conducted. Though lesser fouling is expected in deeper ocean, a biofilm can create conditions at the interphase accelerating corrosion parameters. Hence this study is undertaken to under their effect on structural materials

Experimental Procedure Materials used for this study were selected based

on their applicability for structural applications. The chemical composition of the materials was: Mild steel AISI 1020 (sheet) [Fe 99.S; C 0.034; Si 0.006], Zinc anode (bar) [Zn 99.62; Mg 0.0014; Mn 0.0018; Fe 0.28], Aluminium anode (bar) [AI 96.82; Zn 2.68; Fe 0.31; Si 0.089; Mn 0.003] (wt%).

Carbon fibre reinforced composite coupons of lSxlS0x2 mm3 were cut from laminate prepared from Fibredux -914C-803-40% (bi-directional Carbon­Epoxy pre-preg) supplied by Hexcel Composites Ltd., U.K, which is an aerospace certified material.

Steel coupons of SOxSOx2 mm3 were cut from sheets and circular anode coupons 2S mm dia x S mm thick were machined from rods. The steel and anode coupons were attached together by plastic fasteners with a cathode to anode area ratio of 1 :25 . Metallic specimens were initially polished with 600-grade emery, degreased with methanol, cleaned with distilled water and dried before exposure to the corrosive environment. The composite specimens were not polished. All specimens were attached to a deep sea mooring as shown in Fig. 1 and moored at depths 500, 1200, 3500 and 5100 m for 174 days. The location of the experimental site was at latitude 14° 58.3' S and longitude 76° 58.8' E in the Indian Ocean. During these exposures, care was taken to ensure that

the specimens did not come in contact with any other metal and were attached only with nylon rope and plastic fasteners. The sub-surface deep sea mooring system with coupons was recovered from the sea after the exposure by releasing the mooring system anchored on to the seabed. After careful visual observations, the specimens were removed from the mooring. Data on seawater current speed and direction were collected by current meters attached to this deep sea mooring. Ship borne oceanographic instruments were used to collect data on physical and chemical oceanographic parameters. The research vessel A.A Sidorenko was used for this experiment. The experimental procedure on details of exposure in deep ocean is explained elsewhere6

.7

.

Scrapings from suspended plates obtained using sterile scalpel were streaked on nutrient agar plates. Carbon steel coupons were scraped with a sterile nylon brush to remove the biofilm from the surface of the coupons. Total viable counts, heterotrophic plate count in the biofilm on carbon steel coupons were

Subsurface Floats

Floats

Current Meter

Current Meter

Current Meier

Floats

Current Meter

Acoustic Release

Material Specimens 500m

Material Specimens I 200m

Material Specimens 3500m

Material Specimens 5100m

5

Fig. 1-Typical diagram of a mooring system deployed in the Indian Ocean to test coupons in three phases for duration of 174 days

488 INDI AN J. ENG. MATER. SCI.. DECEMBER 2003

enumerated. Total viable bacterial counts were enumerated by pour plate method using 20 1.1.1 of sample and transferring it into a sterile petriplates using micropipettes. The plates were incubated at room temperature 27±1 °C for 48 h. The number of visible colonies was cou nted at the end of the incubation period usi ng Quebec colony counter and the counts were expressed as colony forming units per mL (cfu/mL) . Zobel marine agar medium was used Heterotrophic bacteria were isolated by pour plate method. In this, 20llL of sample was taken in a sterile petripl ates using micropipette followed by heterotrophic agar medium base. The sample was

allowed to mix thoroughly with the medium by swirling the petriplates. Then, the petri plates were incubated at room temperature for 48 h. The number of visible colonies were counted after 48 h using Quebec colony counter. Colony Forming Units (CFU) of culturabk bacteria were isolated from steel, cathodically protected steel plates with zinc and aluminium anodes and carbon fi bre reinforced composite specimens from depths studied here.

Results and l[)iscussion Results of qualitative microbiological work done

on plates suspended in oceanic water at depths of 500,

Table 1- Micro-biological studi es on the surface of the exposed Carbon fibre reinforced composite specimens

Depth (m) Material Growth in replicates Colony morphology

500 CFRC Absent Absent Nil 1200 CFRC Absent Absent Nil 3500 CFRC Absent Absent Nil 5100 CFRC Absent Absent Nil 500 Mild Steel Present Present Two types of colonies yellow pigmented and

colourless 1200 Mild Steel Present Present Yellow pigmented colonies. 3500 Mild Steel Present Absent Yellow pigmented colonies 5100 Mild Steel Present Absent Yellow pigmented colonies 500 MS +AI anode Present Present Co lourless colon ies

Sample from MS 1200 MS +AI anode Present Present Co lourless colonies.

Sample from MS 3500 MS +AI anode Present Present Colourless colonies.

Sample from MS 5100 MS +AI anode Absent Absent Nil

Sample from MS 500 MS +AI anode Present Present Heavy growth of non-pigmented bacteria.

Sample from AI 1200 MS +AI anode Absent Absent Nil

Sample from AI 3500 MS +AI anode Absent Absent Nil

Sample from AI 5100 MS +AI anode Absent Absent Nil

Sample from AI 500 MS+Zn anode Present Present Two types o f colonies yellow pigmented and

Sample from MS colourless. 1200 MS+Zn anode Present Present Ye llow pi gmented colon ies.

Sample from MS 3500 MS+Zn anode Absent Absent Nil

Sample from MS 5100 MS+Zn anode Absent Absent Nil

Sample from MS 500 MS+Zn anode Present Present Two types of colonies yellow pigmented and

Sample fro m Zn colourless. 1200 MS+Zn anode Present Present Ye llow pigmented colonies.

Sample from Zn 3500 MS+Zn anode Absent Absent Nil

Sample from Zn 5100 MS+Zn anode Absent Absent Nil

Sample from Zn

VENKATESAN et ai.:BIOFILM FORMATION ON STRUCTURAL MATERIALS IN DEEP SEA ENVIRONMENTS 489

Table 2 - Bacterial isolates from various depths on metal surfaces are given along with substrate on which they formed

500m

Mild steel - Flavobacterium ; Micrococcus; Enterobacter

Styphylococcus

Mi ld steel and zi nc couple - Vibrio

Mild steel and aluminium Couple - Pseudomonas

Enterobacter; Pseudomonas

1200 m

Mild steel- Pseudomonas; Acinetobacter

1200, 3500 and 5 100 m in the Indian Ocean are discussed here. The Colony Forming Units (CFU) of culturable bacteria were isolated from mild steel plates, cathodically protected mild steel plates with zinc and aluminium anodes and carbon fibre reinforced composite (CFRC) specimens. Data are presented for two replicates in Tables 1 and 2.

Carbon fibre reinforced composite The results show that carbon fibre reinforced

composite did not support formation of biofilm. It was noticed accidentally that the bottom of synthetic water-proof tape (make:3M) used for attaching coupons to nylon twine horboured bacterial colonies. The surface characteristics are usually related with the formation of first layers of biofouling. In fact, the formation of the organic conditioning film that triggers the adhesion of the first layer depends naturally on the surface characteristics, namely on its critical surface tension . Generally, for surface tension in the range of 20-30 dyne/cm, the adhesion of microorgani sms is less favourable8

. For these conditions, it was proved by Mayer et al.9 that the adsorbed biofouling retracts and rolls back from the surface, indicating low adhesion capacity. However, most engineering materials and coatings show high capacity for absorbing the first layers of the biofilm, presenting values of critical surface tension above the mentioned interval. As regards the roughness of the deposition surface, higher surface roughness favours the anchorage of microorganisms due to the increase in the contact areas. However, this effect is limited to the first layer of deposit. But, the final thickness of the deposit is not affected by these surface characteristics. As such, for bacterial colonies to form on any surface a substrate is sufficient. Carbon fibre­reinforced composite being an inert material should have supported these colonies. This is rather inexplicable and calls for more studies.

3500m

Mild steel and zi nc couple - Pseudomonas

Mild steel and aluminium couple - Ellterobacter

5100m

Mild steel and aluminium couple - Enterobacter

Mild steel with zinc and aluminium anodes Mild steel with aluminium anodes horboured

bacterial colonies at 500, 1200 and 3500 m depths and not at 5100 m depth as shown in Table 1. Zinc supported bacterial colonies only at 500 and 1200 m and not at other depths viz. 3500 and 5100 m. Aluminium supported bacterial colonies only at 500 m and not at other three depths viz 1200, 3500 and 5100 m. This could be due to the fact that zinc and aluminium anodes would have faced uniform corrosion leading to continuous dissolution of metals into seawater and hence could not have supported the bacterial colonies.

Effect of toxicity of aluminium and zinc - Toxicity of some metallic ions is related to the formation of biological deposit. However, the results published in the interactive are not conclusive, since they appear to depend on the dominant microbial species and on the environmental condition. Aluminium surfaces are sometimes referred as the less fou led substratum, due to the toxic effest of Al 3+ ion acting on the polysaccharide adhesive structure. Pinheiro et at. 8

showed that when measuring thickness and mass of deposits formed after 48 h exposure using copper, aluminium and brass surfaces, toxic effect of the metallic ions ceases after several layers of biofouling are formed . But, they may be responsible for retarding the overall process without eliminating completely. Hence, it is evident that although toxicity of aluminium and zinc ions may affect the polyssacride layer formation, their role is not clearly known . Also aluminium and zinc supported bacterial colonies at 500 and 1200 m are not supported at 3500 and 5100 m depths. Hence, toxicity cannot be considered. Dexter4 reported the effect of natural biofilms on galvanic corrosion of steel with zinc anodes and showed that biofilms did not increase consumption of zinc anodes . The presence of a biofilm on a metal surface can create chemical conditions vastly different

490 INDI AN J. ENG . MATER. SCI. , DECEMBER 2003

from those of the ambient environment. A microbial fil m can act as a diffusion barrier as well as a source or sink for chemical species that are important to corrosion processes. Factors that vary little in the open ocean (such as pH, di ssolved oxygen, peroxide, and the heavy metals manganese and iron) can vary dramatically at the metal-biofilm barrier.

Effect of pH - Also influence of pH could be di scussed here. The influence of fl uid pH is related, not on ly with the growth rate of the microorganis ms, but also with the adhesion forces at the surfaces . As to the growth rate, the majority of mi croorgan isms in contami nated waters has a preference fo r growth at a neutral pH. In deep waters, pH of seawater remains close to neutral and the variation is fro m 7.8 to 7.2 only from surface to 5100 m depth in Indi an Ocean?

Effect surface potential - Fletcher and Loeb 10

fo und a higher number of adherent bacteria Pseudomonad Marin.a in pl atinum than in glass or mica surfaces. In these, it is found that less fouled surfaces have negative zeta potenti als, while other res ted surfaces have positi ve zeta potenti als" . It can be said that the coating of solid surfaces with materi als that increase the electronegativity would reduce the possibilities of adhesion of the greater majority of bacteria. The adhesion of macromolecules and microbial cells is also dependent on the pH. Since, it can affect the distribution of electrical surface charges of materials, if present, and thus their zeta potentials. The adhesion between these materials is increased when the corresponding zeta potentials have opposite si gns. Majority of marine bacteria have negati ve electrical surface charges, thus presenting higher facility for the adhesion to solid surfaces with positi ve zeta potentials l2

.

Effect of seawater temperature - Other reason for the absence of biofi lm at 3500 and 5100 m depths

o .. -r------=::=.o---, j I

1000 . ... rJ) i ~ 1 Q) 2000 . . ~ E ~ £ 3000 •. j a. I Q) ..

o i 4000 · ·1

~ I

5000 . . 1j-· --,----,---r----; I o 20 40 Seawater temperature deg C

Fig. 2 - Seawater temperature profile with respect to depth in Indian Ocean at the experimenta l site

could be very low temperature prevai ling in deeper waters. Temperature constitutes a critical parameter in the development of biofouling . The maj ority of microbial species present has its optimu m temperature range 20-40°C. Low temperature leads to drastic reduction in formation and growth of bi ofilm. Below 1200 m depth , temperature drops down drastically as shown in Fig. 2. Jeffrey and Melchers 14 reported that bacteria do not appear to be signi ficantly involved for immersion corrosion at lower water temperatures . For example, for specimens exposed to an average water temperature of about 4°C, it took some 5 years before corrosion was no longer clearly controlled by oxygen di ffu sion as evidenced from the corrosion time studies l3

. Hence, at low temperature, dissolu tion of anodes due to electrochemical reactions would have resulted in absence of bacterial coloni es on zinc and aluminium anodes.

Mild steel Biofilms were present on mild steel specimens at

all depths. Attachment of mild stee l at all depths appears to point to the fact that the special requirement of iron ions by these bacterial species in these extreme environment. In deep waters, aerobic conditions do not prevail from the graph shown in Fig. 3 as dissolved oxygen is present is seawater even at 5000 m depth.

Anaerobic condition in deeper waters The role of anaerobic bacteria in the marine

corrosion of steels appears to be particularly important for higher water temperatures and longer durations as than anaerobic conditions appear to govern, estimating service life of steel in seawater l4

.

Current velocity and seawater temperature decreases as the water depth increases in the ocean as shown in the Table 3. This data is recorded from respective

0

1000 rJ) (J)

-E5 2000 E li 3000 Q)

0 4000

" 5000

o 200 400 Dissolved Oxygen urn/kg

Fig. 3 - Dissolved oxygen profile with respect to depth in Indian Oeean at the experimental site

VENKATESAN et al.:BIOFTLM FORMATION ON STRUCTURAL MATERIALS IN DEEP SEA ENVIRONMENTS 491

Table 3 - Maxi mum current ve locity of seawater collected at the experimenta l site

Depth Maximum velocity Maximum Temperature (m) (mJs) (0C)

500 0.49 8.99 1200 0.20 5.02

3500 0.15 1.50

5100 0.03 1.00

depth measured using current meters. At 5100 m depth, close to sea bed (5180 m), still some movement of water is recorded which cou ld be due to cold Antarctic water flowing in that region as reported by many workers's. These data further ascertain that anaerobic condition could not have not have prevailed on the surface of coupons under normal exposure conditions.

Temperature, pH, salinity, nutrients , microbial population and nature of the solid surface influence this build-up of slime. Any surface exposed to natural fresh or saline waters provide an opportunity for the settlement and subsequent growth of organisms, as a very great di versity of aquatic species. It may be noted that the data presented here are evidently not the result of a planned experiment. For this reason, there are a number of aspects, which could have been investigated In more detail or with different techniques .

Conclusions It was observed in the present investigations that

carbon fibre reinforced composite did not support bacterial colonies at all depths. Also, slime formation was observed on the metallic specimens. Aerobic bacteria were observed on material coupons at the four tested depths, as anaerobic condition did not prevail in deeper water on coupons under the present exposure conditions. Absence of bacterial colonies on aluminium and zinc anodes at 3500 and 5100 m depths could be due to lower temperatures and electrochemical dissolution of these anodes would have resulted in the absence of bacterial colonies on zinc and aluminium anodes. Macro fouling was absent on steel coupled with zinc, aluminium and

magnesium anodes and carbon fibre reinforced composite in deeper waters as such larvae are absent in deeper waters far away from shore. Microbiologically induced corrosion on these materials was also not observed in deep-sea water column.

Acknowledgements Our sincere thanks are due to the Department of

Ocean Development, and the Council of Scientific & Industrial Research, for providing financial support. Thanks are due to Dr. Ehlrich Desa (Director NIO) and Dr Lata Raghukumar (NIO) for their help during the work. Our sincere thanks are also due to the Master and crew of research ship M .V.A.A. Sidorenko.

References I Biojilllls, edited by W G Charackli s & K C Marshall (John

Wiley and Sons Inc, New York), 1990 pp. 22-45. 2 Lewandowski Z, Stoodley S D & Roe F, NACE, 2 (1995) 22-

26. 3 Schumacher M, Seawater corrosion handbook (Noyes Data

Corporation, New Jersey, USA), 1979, pp. 107-964. 4 Dexter S C, Biological effects - Specific industries and

environments (Corrosion-ASM Metal s handbook) , 1988, p. . 13, pp. 41-43.

5 John Morgan J H, Cathodic protection (Second edn), (Leonard Hill , Texas, USA), 1959, pp. 35-55.

6 Venkatesan R, Dwarakadasa E S & Ravindran M, Offshore Technol Con/(OTC 14325), (2002) 1-5.

7 Venkatesan R, Venkatasamy M A, Bhaskaran T A, Dwarakadasa E S & Ravindran M, British Corros J, 4(2002) 257-266.

8 Pinheiro M M V P S, Melo L F, Bott T R & Pinheiro J D, Fouling Sci Techno:, (1988) 223-232.

9 Mayer A E, Baier R E & King R W, Fouling of non-tox ic coatings in fresh brackjsh and seawater, Thirty sixth Can Chem Eng Con/, Sarmia, Canada, (1986) 23.

10 Fletcher.M & Loeb G I, Appl Environ Microbiol , 37 (I) ( 1979) 67.

II Neihof R.A. , Loeb G.I, Lim/lology Oceanography, 17(7 ) ( 1972) 198.

12 Harden V P & Harris J 0 , J Bacteriol, ( 1953) 65 . 13 Southweel C R, Bultman J D & Hummer C W, Estimating

service life of steel in sea water, M Schumacher (Ed) , (Seawater Corrosion Handbook, Noyes Data Corporation, New Jersey), 1979, pp. 374-387.

14 Jeffrey R & Melchers R E, Corros Sci, 45(2003) 693-714. J 5 Warren A B, Deep Sea Res, 28 (A8) (J 981), 759-788.