Planta

, Volume 219, Issue 4, pp 561–578

Exploitation of marine algae: biogenic compounds for potential antifouling applications

Authors

  • Punyasloke Bhadury
    • Centre for Marine Biodiversity and Biotechnology, School of Life SciencesHeriot Watt University
    • Plymouth Marine Laboratory
    • Biological and Environmental Systems Group, Department of Chemical and Process EngineeringUniversity of Sheffield
Review

DOI: 10.1007/s00425-004-1307-5

Cite this article as:
Bhadury, P. & Wright, P.C. Planta (2004) 219: 561. doi:10.1007/s00425-004-1307-5

Abstract

Marine algae are one of the largest producers of biomass in the marine environment. They produce a wide variety of chemically active metabolites in their surroundings, potentially as an aid to protect themselves against other settling organisms. These active metabolites, also known as biogenic compounds, produced by several species of marine macro- and micro-algae, have antibacterial, antialgal, antimacrofouling and antifungal properties, which are effective in the prevention of biofouling, and have other likely uses, e.g. in therapeutics. The isolated substances with potent antifouling activity belong to groups of fatty acids, lipopeptides, amides, alkaloids, terpenoids, lactones, pyrroles and steroids. These biogenic compounds have the potential to be produced commercially using metabolic engineering techniques. Therefore, isolation of biogenic compounds and determination of their structure could provide leads for future development of, for example, environmentally friendly antifouling paints. This paper mainly discusses the successes of such research, and the future applications in the context of understanding the systems biology of micro-algae and cyanobacteria.

Keywords

AntifoulingBiofoulingBiogenic compoundsMarine algaeMetabolic engineering

Abbreviations

AHL

Acylated homoserine lactone

TBT

Tributyl tin

Introduction

An immense number of organisms in the marine environment, including the marine algae, produce a variety of chemical deterrents for defence purposes, which have served as the basis for ecological studies (Paul and Puglisi 2004; Kubanek et al. 2003; Steinberg and de Nys 2002; Engel et al. 2002; Ren et al. 2001; Nagle and Paul 1999; Hay 1996; Hay and Steinberg 1992; Bakus et al. 1986). There have been numerous studies, across the globe, reporting the bioactivities of marine-sourced organisms (e.g. Tziveleka et al. 2003; Peters et al. 2003; Yim et al. 2004; Iken and Baker 2003; Tsoukatou et al. 2002; Bhosale et al. 2002; de Nys and Steinberg 2002; Pereira et al. 2002; Wilsanand et al. 2001; Armstrong et al. 2000; Abarzua et al. 1999; König et al. 1999a; Siddhanta et al. 1997; Badria et al. 1997; Bernan et al. 1997; Carte 1996; Sato 1996; Dhawan et al. 1993; Sridhar and Vidyavathi 1991). Biogenic compounds belonging to several classes of marine micro- and macro-algae have been identified over the last few decades, and their chemical constitution and pharmacological activity have been studied in detail (e.g. Umemura et al. 2003; Takamatsu et al. 2003; Mayer and Gustafson 2003; Blunt et al. 2003; de Nys and Steinberg 2002; Hellio et al. 2002; Harada et al. 2002; Burja et al. 2001; Ren et al. 2001; Vairappan et al. 2001; Hellio et al. 2001; Borchardt et al. 2001; Etahiri et al. 2001; König et al. 1999b; de Nys et al. 1998; Kleinkauf and von Döhren 1997; Carte 1996; Falch 1996; Sato 1996; Borowitzka 1995; Abarzua and Jakubowski 1995; Teuscher et al. 1992). These biogenic compounds have proven their potential in several fields, particularly as new therapeutic agents for a variety of diseases (Umemura et al. 2003; Takamatsu et al. 2003; Mayer and Gustafson 2003; Harada et al. 2002; Romanos et al. 2002; Burja et al. 2001; Burgess et al. 1999; Bernan et al. 1997; Riguera 1997; Moore 1996; Gerwick et al. 1994; Pietra 1990; Gerwick 1990; Carmichael et al. 1990; Moore et al. 1988). An examination of the MarinLit database reveals that ca. 3,600 compounds have been identified from marine macro- and micro-algae (including cyanobacteria), from a marine natural products pool of 15,000 natural products, thus representing ca. 24%. Extension into non-therapeutic areas is also possible, e.g. due to their antibacterial, antimacrofouling, antifungal and antiprotozoan properties these biogenic compounds could also be used effectively for future development of antifouling paints (Fusetani 2004; Kubanek et al. 2003; Greer et al. 2003; Da Gama et al. 2002; de Nys and Steinberg 2002; Hellio et al. 2000b, 2002; Steinberg and de Nys 2002; Ali et al. 2002; Dobretsov and Qian 2002; Hellio et al. 2001; Dworjanyn 2001; Rittschof 2001; Kjelleberg and Steinberg 2001b; Jaki et al. 2000; Abarzua et al. 1999; Manefield et al. 1999; Dworjanyn et al. 1999; Maximilien et al. 1998; Clare 1996; Abarzua and Jakubowski 1995).

The scope of this review is to report on the present status of biogenic compounds isolated so far from marine micro- and macro-algae with antialgal, antibacterial, antiprotozoan and antimacrofouling properties. Antifouling coatings are presented as a case study. We also discuss the future scope and prospects of commercial biotechnological production of these biogenic and related compounds using metabolic engineering/systems biology approaches.

Biofouling

Biofouling is one of the more serious problems currently facing worldwide maritime domains (Abarzua et al. 1999; Clare 1996). Solid surfaces, when exposed to sea water, undergo a series of changes leading to the generation of a complex layer formed as a result of adhesion by marine organisms, mainly consisting of microbial slimes, diatoms, barnacles, tunicates, bryozoans and spores of marine algae (Rasmussen and Østgaard 2003; Bhosale et al. 2002; Elvers and Lappin-Scott 2000; John et al. 1999; Abarzua et al. 1999; Abarzua and Jakubowski 1995; Mary et al. 1993; Szewzyk et al. 1991; Henschell and Cook 1990; Characklis 1981). This phenomenon is known as biofouling. These fouling organisms can cause extensive damage to commercially important marine structures (Bhosale et al. 2002; Shin and Smith 2001; Dexter 1996). Being a prolific source of bioactive compounds, prokaryotic and eukaryotic marine algae (Kubanek et al. 2003; Burgess et al. 2003; Da Gama et al. 2002; de Nys and Steinberg 2002; Bhosale et al. 2002; Armstrong et al. 2001; Burja et al. 2001; Abarzua et al. 1999; de Nys et al. 1995; Abarzua and Jakubowski 1995) show potential for solving this issue by inhibiting the process of fouling (Hellio et al. 2002).

Causes of biofouling

Biofouling is considered to have four distinct stages. The fouling process starts from the moment a man-made object is immersed in water. The surfaces of these objects quickly accumulate dissolved organic matter, and molecules such as polysaccharides and protein fragments. This phase is sometimes known as the accumulation or conditioning phase (Abarzua et al. 1999; Abarzua and Jakubowksi 1995). It is regarded as the first stage of fouling, and it sets the scene for later events. Gradually, bacteria (usually species of Bacillus and Pseudomonas) and single-cell diatoms then sense the surface and start settling on it, forming a microbial film (Costerton et al. 1995). The ability of a bacterial cell to perform this initial attachment is controlled by both environmental and genetic factors, such as nutrient levels, temperature, pH, the presence of genes encoding motility functions, environmental sensors, adhesions (O’Toole et al. 2000; Costerton 1995). Klausen et al. (2003) reported bacterial migration in the development of complex multicellular structures in Pseudomonas aeruginosa biofilms. The first description of specific genes that regulate bacterial biofilm formation was made using transcriptional lacZ reporter-gene fusions, and it was believed that bacterial attachment results in gene expression (Sauer 2003; Costerton et al. 1995; Davies et al. 1993; Dagostino et al. 1991). Staphylococcus epidermis, a bacterium, has provided vital information regarding the formation of biofilms (Dalton and March 1998). It has been shown that a clone of the unmutated operon, which is involved in the synthesis of a polysaccharide known to be essential for biofilm formation, from S. epidermis (Mack et al. 1996; Heilmann et al. 1996) when transformed into a non-biofilm-forming species, like S. carnosus, converts it to a biofilm-forming species (Dalton and March 1998). It has been also speculated that an autolysin (Heilmann et al. 1997) may be important to a bacterium for attachment to a surface, since autolysins play an important role in cell division and cell wall turnover (Dalton and March 1998). Rohde et al. (2001) have found that IcaADBC-encoded proteins help in the synthesis of the intercellular polysaccharide adhesin, which is involved in Staphylococcus epidermis biofilm formation. Brõzel et al. (1995) also reported changes in global gene expression in Pseudomonas aeruginosa and alteration of 11 types of protein during different stages of its attachment. Genes like slp and ompC have been found to be associated with the initial steps of Escherichia coli biofilm formation on abiotic surfaces (Otto et al. 2001; Prigent-Combaret et al. 1999). It has been shown that quorum sensing (Purevdorj and Stoodley 2003; Fuqua et al. 1997), based on cell-to-cell communication, is important for the development of swarming colonies, which dominate biofilms (Ren et al. 2001; Davies et al. 1998; Eberl et al. 1996; Lawrence et al. 1991). Quorum sensing is dependent on small extracellular molecules called autoinducers; in Gram-negative bacteria they are acylated homoserine lactone (AHL) molecules (Ren et al. 2001; Eberl 1999), while in Gram-positive bacteria they are peptide signalling molecules (Bassler 1999; Kleerebezem et al. 1997). In Vibrio cholerae, multiple quorum-sensing circuits work in parallel to control biofilm formation and virulence (Hammer and Bassler 2003). Hammer and Bassler (2003) have identified the quorum-sensing circuits that control the transcription of genes engaged in exopolysaccharide (EPS) production in V. cholerae during biofilm formation. Quorum sensing has been shown to be vital in biofilm formation, e.g. in Pseudomonas aeruginosa (Davies et al. 1998); however, Schembri et al. (2003) reported no regulation of genes in response to quorum sensing in E. coli biofilm formation. Sauer et al. (2002), using two-dimensional gel electrophoresis and reporter-gene analysis, have shown that P. aeruginosa displays five distinct physiologies during biofilm development. Similarly, Oosthuizen et al. (2002) revealed, by proteomic analysis, differential protein expression in Bacillus cereus during biofilm development.

After initial attachment, the cells begin to grow and start forming colonies. During this period, major changes are noticed, e.g. the formation of the exopolysaccharide (EPS) layer, one of the distinct features of a developed biofilm (Costerton 1995; Danese et al. 2000). It is believed that the extracellular polymeric substances (EPS) play an important role in the adhesion of bacteria to the surfaces (Rasmussen and Østgaard 2003; Danese et al. 2000; Costerton 1995). The presence of adhesive substances and rough irregular microbial colonies gradually starts trapping more particles and organisms. Spores of algae, e.g. species of Enteromorpha intestinalis, Ulothrix zonata, marine fungi and ciliate protozoa soon appear on the film (Abarzua and Jakubowski 1995; Zahuranec 1991; Cuba and Blake 1983; Caron and Sieburth 1981). The transition from a microbial film into a much more complex community is regarded as the third stage of fouling. The final stage involves the settlement of other marine organisms, such as barnacles, tunicates, mussels, bryozoans, polychaetes and tubeworms, together with the growth of algae (Abarzua et al. 1999; Clare 1996; Takasawa et al. 1992; Wahl 1989).

The formation of this complex layer on submerged structures (mainly vessels such as ships and platforms, and other marine infrastructures such as ocean thermal-energy conversion systems, offshore installations) in sea water, has important economic implications. For example, fouling of ships results in increased drag and surface corrosion, a decrease in fuel efficiency, a loss of speed and an increase in pollution (de Nys and Steinberg 1999, 2002; Abarzua et al. 1999; Hall et al. 1999; Hattori and Shizuri 1996; Callow 1996; Miki et al. 1996; Vanelle and Le Gal 1995; Fletcher 1989; Gitlitz 1981). It has been estimated that the growth of marine organisms on submerged surfaces costs the shipping and other marine industries over $6.5 billion per year (Blanke and Yang 2002). According to Callow and Callow (2002), biofouling costs the US Navy an estimated $1 billion per annum.

In due time, the submerged structures become corroded, followed by rusting due to the intense actions of the fouling organisms (Corpe 1970). The fouling of hulls of ships is a real problem, and various technologies have been applied in the past, including the use of antifouling paints like tributyl tin (TBT) and organotins (Evans et al. 2000; Boxall et al. 2000; Hall et al. 1999; Ponasik et al. 1998; Isensee et al. 1994; Willemsen and Ferrari 1993). Recently, increasing concerns about the negative effects of TBT on several marine organisms and the environment have led to a ban on TBT-containing coatings in different countries, including Japan, Canada and those within the European community (Stupak et al. 2003; Burgess et al. 2003; Omae 2003; Hunter and Anderson 2000; Abarzua et al. 1999; Fischer et al. 1999; Hashimoto et al. 1998; Grinwis et al. 1998; Fent 1996; Burridge et al. 1995; Horiguchi et al. 1995; Ruiz et al. 1995; Suzuki et al. 1992). It has been estimated that antifouling paints when submerged into water release toxic compounds at levels of 4 μg cm−2 of surface per day causing serious environmental effects (Ponasik et al. 1998). The International Maritime Organisation (IMO) and the Marine Environment Protection Committee (MPEC) have recently proposed a ban on use of TBT in antifouling paints (Konstantinou and Albanis 2004).

Work is under way in many academic laboratories and companies worldwide to develop alternative environmentally friendly antifouling compounds. Several groups of workers, including Burgess et al. (2003), Callow and Callow (2002), Youngblood et al. (2003) and Steinberg et al. (see Anisul et al. 2003), are actively involved in developing natural products as replacements for chemicals currently used in antifouling coatings. However, development times and costs may often be prohibitive, e.g. Sea-Nine 211, a biocide developed by Rhom and Haas, took 11 years for EPA registration and the cost was around US $11,000,000 (Rittschof 1999). Currently, the economics associated with registering, licensing and developing compounds like Sea-Nine appear to be very unfavourable.

An alternative to TBT and Sea-Nine is the development of antifouling coatings using biogenic compounds or secondary metabolites that are present in marine organisms and operate as natural anti-settlement agents (Burgess et al. 2003; de Nys and Steinberg 2002; Hellio et al. 2002; Kjelleberg and Steinberg 2001a; Abarzua et al. 1999; Rittschof 2001; Holmström and Kjelleberg 1994; Kjelleberg and Steinberg 1994).

Projects like BRITE/EURAM 3 (environmentally compatible antifouling coatings for the protection of ships, water systems, fish cages and other immersed structures against aquatic growth), also known as ‘Camellia’ within the European Union, have focused on the development of natural products as commercial antifoulants (de Nys and Steinberg 2002) while an EU CRAFT project examined the use of enzymes as antifouling agents in marine coatings for ships and other marine installations (see http://www.biolocus.com/public). In Japan, several antifouling compounds have been already discovered from the marine environment under the ERATO antifouling program (Fusetani et al. 1996).

Marine algae, as well as other benthic organisms, are relatively free from settlement by fouling organisms (Kubanek et al. 2003; Hellio et al. 2001, 2002; Steinberg and de Nys 2002; Paul 1992) due to the production of biogenic compounds that possess antibacterial, antialgal, antifungal, antiprotozoan and antimacrofouling properties. These biogenic agents are generally produced as secondary metabolites by marine algal species (Da Gama et al. 2002; de Nys and Steinberg 2002; Hellio et al. 2001, 2002; Bhosale et al. 2002; Etahiri et al. 2001; Abarzua et al. 1999; Abarzua and Jakubowski 1995). Therefore, the isolation and production of these natural products from marine algae could be used effectively for the prevention of biofouling.

Teuscher and Lindequist (1994) defined biogenic compounds as chemical compounds which are synthesised by living organisms and which, if they exceed certain concentrations, cause temporary or permanent damage or even death of other organisms by chemical or physicochemical effects.

A huge number of compounds with antiviral, antibacterial, antialgal and antifungal properties have been reported from marine algae (Kubanek et al. 2003; de Nys and Steinberg 2002; Bhosale et al. 2002; Dobretsov and Qian 2002; Steinberg and de Nys 2002; Hellio et al. 2001; Burja et al. 2001; Steinberg et al. 2001; Vairappan et al. 2001; Abarzua et al. 1999; Banker and Carmeli 1998; Clare 1996; Padmakumar et al. 1993; Mizukoshi et al. 1992; Bernard and Pesando 1989; Wahidulia et al. 1987). The principal groups that have been shown to possess the biogenic compounds are members of the prokaryotic Cyanobacteria, eukaryotic Chlorophyceae, Rhodophyceae and Phaeophyceae, besides Dinophyceae and Chrysophyceae. Most of these compounds inhibit the growth of bacteria, protozoa, algae and other macrofouling organisms (Smyrniotopoulos et al. 2003; de Nys and Steinberg 2002; Steinberg and de Nys 2002; Bhosale et al. 2002; Steinberg et al. 2001; Abarzua et al. 1999; König and Wright 1997; Clare 1996; Abarzua and Jakubowski 1995). The following sections detail the types of compound encountered, and their biological source.

Biogenic compounds from marine Cyanophyceae, a group of marine microalgae

Marine cyanobacteria (Cyanophyceae), a group of prokaryotic marine (blue-green) algae, have been identified as a new and rich source of bioactive compounds (Shimizu 2003; Burja et al. 2001; Abarzua et al. 1999; Banker and Carmeli 1998; Namikoshi and Rinehart 1996; Abarzua and Jakubowski 1995; Teuscher et al. 1992). Several biogenic compounds, such as bromophenols, malyngolides, aponin, cyanobacterin, hapalindoles, fischerellin and scytophycins have been isolated from a range of species of cyanobacteria containing antibacterial, antialgal, antifungal or antiprotozoan properties (Burja et al. 2001; Jaki et al. 2000; Abarzua et al. 1999; Abarzua and Jakubowski 1995, 1996) (Fig. 1). Most of the isolated substances belong to groups of polyketides, amides, alkaloids, fatty acids, indoles and lipopeptides (Burja et al. 2001; Kleinkauf and von Döhren 1997; Falch 1996; Borowitzka 1995). A list of some of the biogenic compounds isolated from cyanobacterial members has been compiled (Table 1).
Fig. 1a,b

Chemical structures of calothrixin-A (a) and hapalindole (b) isolated from cyanobacterial strains

Table 1

Biogenic compounds isolated from Cyanobacteria

Source

Biogenic compound

Activity

References

Lyngbya majuscula

Malyngolide

Antibacterial

Burja et al. (2001)

Hapalosiphon fontinalis

Hapalindole

Antifungal

Burja et al. (2001)

Scytonema hofmanni

Cyanobacterin

Antifouling (Nitzschia pusilla)

Abarzua et al. (1999)

Calothrix brevissima

Crude extract

Antifouling (Nitzschia pusilla)

Abarzua et al. (1999)

Scytonema ocellatum

Tolytoxin (Scytophycin)

Antifungal

Patterson and Carmeli (1992)

Scytonema pseudohofmanni

Scytophycins

Antifungal

Burja et al. (2001), Ishibashi et al. (1986)

Tolypothrix tenuis

Toyocamycin

Antifungal

Burja et al. (2001), Moore (1982)

Nostoc spongiaeforme var. tenue

Methanolic extract

Antibacterial

Banker and Carmeli (1998)

Nostoc spongiaeforme

Nostocine A

Antialgal

Hirata et al. (1996)

Nostoc commune

Nostodione

Antifungal

Böhm et al. (1995)

Nostoc muscorum

Aqueous extracts

Antibacterial

Bloor and England (1991)

Nostoc muscorum

Methanolic extracts

Antifungal

De Mule et al. (1991)

Nostoc sp.

Nostocyclamide

Antifungal

Moore et al. (1988), Yang et al. (1993)

Nostoc linckia

Cyanobacterin LU-1

Antialgal

Gromov et al. (1991)

Hyella caespitose

Carazostatin

Antifungal

Burja et al. (2001)

Tolypothrix tjipanasensis

Tjipanazoles

Antifungal

Bonjouklian et al. (1991)

Fischerella muscicola

Fischerellin

Antialgal

Gross et al. (1991)

Phormidium tenue

Galactosyldiacylglycerols

Antialgal

Murakami et al. (1991)

Oscillatoria sp.

Ether extracts

Antialgal

Baghi et al. (1990)

Gomphosphaeria aponina

Aponin

Antialgal

Eng-Wilmot et al. (1979)

Synechocystis leopoliensis

Methanolic extracts

Antibacterial

Cannell et al. (1988)

Lyngbya majuscula, a marine cyanobacterium from the family Oscillatoriaceae, for example, has been shown to be a diverse source of bioactive compounds, some of which possess antifungal and antimicrobial properties (Burja et al. 2001). According to Burja et al. (2001) more than 75% of the 113 biogenic compounds isolated from Lyngbya majuscula possess some sort of biological activity, out of which more than 10% have been reported to have antifungal and antimicrobial properties (Nagle et al. 1996). Compounds like majusculamide A-D (see Fig. 4), malyngolide and laxaphycin A-B isolated from L. majuscula have shown promising results as antifungal and antibacterial agents (Burja et al. 2001).

As an example from other organisms, cyanobacterin, a biogenic compound isolated from Scytonema hofmanni, has been found to deter populations of the fouling benthic diatom Nitzschia pusilla (Abarzua et al. 1999; Abarzua and Jakubowski 1996). Comnostins (see Fig. 4), a class of novel extracellular diterpenoids isolated from the cyanobacterium Nostoc commune have shown promising antibacterial activity against Staphylococcus epidermis, a biofilm-forming species (Jaki et al. 2000).

Most of the biogenic compounds isolated so far from marine cyanobacteria tend to be lipopeptic in nature, i.e. they consist of an amino acid fragment linked to a fatty acid portion. Lipopeptides are an interesting class of compounds, 85% of which are bioactive; of these, around 4% show antifungal properties, 12% show antibiotic properties and a few are antialgal, in addition to having other pharmacological properties (Burja et al. 2001).

Compounds such as linolenic acid, isolated from the marine cyanobacterium Synechococcus sp. and bastadin from Anabaena basta have also shown encouraging antibacterial traits (Franklin et al. 1996; Pettit et al. 1995; Ohta et al. 1994; Dexter et al. 1993; Miao et al. 1990).

Biogenic compounds from the Dinophyceae and Chlorophyceae (microalgae)

Biogenic compounds that also possess interesting antibacterial, antialgal and antifouling properties have been isolated from members of the marine micro-algae belonging to the Dinophyceae and Chlorophyceae (Table 2).
Table 2

Biogenic compounds isolated from Dinophyceae and Chlorophyceae

Source

Biogenic compound

Activity

References

Dinophyceae

  Peridinium bipes

Water-soluble extract

Antialgal

Wu et al. (1998)

  Goniodoma pseudogoniaulax

Goniodomin A

Antifungal

Abe et al. (2002)

  Gambierdiscus toxicus

Polyether compounds (gambieric acid A and B)

Antifungal

Nagai et al. (1993)

  Prorocentrum lima

Polyether compounds

Antifungal

Nagai et al. (1990)

  Dinophysis fortii

Polyether compounds

Antifungal

Nagai et al. (1990)

Chlorophyceae

  Staurastrum gracile

Methanol extracts

Antibacterial

Cannell et al. (1988)

  Pleurastrum terrestre

Methanol extracts

Antibacterial

Cannell et al. (1988)

  Dictyosphaerium pulchellum

Methanol extracts

Antibacterial

Cannell et al. (1988)

  Klebsormidium crenulatum

Methanol extracts

Antibacterial

Cannell et al. (1988)

  Chlorococcum sp.

Aqueous extract

Antibacterial

Ohta et al. (1993)

  Chlorococcum HS-101

α-Linolenic acid

Antibacterial

Ohta et al. (1993)

  Chlorokybus atmophyticus

Acetone extracts

Antibacterial

Cannell et al. (1988)

Biogenic compounds from the Rhodophyceae, Phaeophyceae and Chlorophyceae (macro-algae)

The marine macro-algal species are dominated mainly by members of the Rhodophyceae, Phaeophyceae and some of the Chlorophyceae. Compounds exhibiting antifouling, antialgal, antibacterial and antifungal properties have been isolated from these algae (Kubanek et al. 2003; Smyrniotopoulos et al. 2003; de Nys and Steinberg 2002; Steinberg and de Nys 2002; Vairappan et al. 2001; Borchardt et al. 2001; Kjelleberg and Steinberg 2001a; Steinberg et al. 1998; Abarzua and Jakubowski 19951996; Walters et al. 1996; de Nys et al. 1995; Schmitt et al. 1995; Clayton and King 1990; Ragan and Glombitza 1986; McLachlan and Craige 1964; Langlois 1975). Most of the compounds belong to the polar and non-polar classes of secondary metabolites (Kjelleberg and Steinberg 2001a; Steinberg et al. 1998; de Nys et al. 1995). Table 3 provides an overview of the biogenic compounds isolated so far from different members of marine macro-algae.
Table 3

Biogenic compounds isolated from the Chlorophyceae, Rhodophyceae and Phaeophyceae

Source

Biogenic compounds

Activity

References

Rhodophyceae

  Laurencia majuscula

Elatol, iso-obtusol

Antibacterial

Vairappan (2003)

  Laurencia nidifica

Halogenated metabolites

Antibacterial

Vairappan et al. (2001)

  Laurencia yonaguniensis

Brominated diterpene

Antibacterial

Takahashi et al. (2002)

  Laurencia mariannensis

Halogenated metabolites

Antibacterial

Vairappan et al. (2001)

  Laurencia pinnatifida

Dichloromethane fraction

Antibacterial

Hellio et al. (2001)

  Laurencia obtusa

Laurencienyne

Antibacterial

Caccamese et al. (1981)

5β-Hydroxyaplysistatin

Antifouling (Ulva sp., Balanus amphitrite

Steinberg et al. (1998)

Palisol

Balanus neretina)

Steinberg et al. (1998)

Palisadin A

Antifouling (Bugula neretina, Balanus amphitrite, Ulva sp.)

Steinberg et al. (1998)

Antifouling

Da Gama et al. (2002)

  Laurencia rigida

Elatol, deschloroelatol

Antibacterial, antialgal, anti-macrofouling (Bugula amphitrite, Balanus neretina)

de Nys et al. (1996a)

  Rhodomela confervoides

Bromophenols

Antibacterial

Xu et al. (2003)

  Ceramium rubrum

Methylene chloride extract

Antialgal

Hellio et al. (2002)

  Polysiphonia lanosa

Dichloromethane fraction

Antibacterial

Hellio et al. (2001)

  Chondrus crispus

Ethanolic fraction

Antibacterial

Hellio et al. (2001)

  Plocamium costatum

Halogenated monoterpenes

Antifouling (B. amphitrite)

König et al. (1999a)

  Plocamium hamatum

Polyhalogenated monoterpenes

Antialgal

König et al. (1999b)

  Gracilaria corticata

Chloroform extract

Antibacterial

Sastry and Rao (1994)

  Sphaerococcus coronopifolius

Lipid extract

Antibacterial, Antifungal

Caccamese et al. (1981)

Phaeophyceae

  Lobophora variegata

Lobophorolide

Antifungal

Kubanek et al. (2003)

  Ectocarpus siliculosus

Dichloromethane fraction

Antibacterial

Hellio et al. (2001)

  Laminaria pinnatifida

Ethanolic extract

Antialgal

Hellio et al. (2002)

  Laminaria ochroleuca

Methylene chloride extract

Antialgal

Hellio et al. (2002)

  Laminaria agardhii

Methanolic and Chloroform extract

Antibacterial

Lustigmann et al. (1992)

  Laminaria saccharina

Unsaturated fatty acids

Antibacterial

Rosell and Srivastava (1987)

  Sargassum muticum

Ethanolic extract

Antialgal

Hellio et al. (2002)

  Sargassum vestitum

Phlorotannins

Antifouling (Ulva lactuca)

Jennings and Steinberg (1997)

  Sargassum wightii

Chloroform extract

Antibacterial

Sastry and Rao (1994)

  Sargassum hornei

Mucilage extract

Antialgal

Tanaka and Asakawa (1988)

  Sargassum natans

Phlorotannins

Antifouling (bacteria, marine worms, copepods)

Sieburth and Conover (1965)

  Ecklonia radiata

Phlorotannins

Antifouling (Ulva lactuca)

Jennings and Steinberg (1997)

  Padina tetrastromatica

Chloroform extract

Antibacterial

Sastry and Rao (1994)

  Ascophyllum nodosum

Phlorotannins

Antifouling (Laminaria cloustoni, Vorticella marina)

Langlois (1975), Hellio et al. (2001)

Ethanolic fraction

Antibacterial

  Dictyota menstrualis

Diterpene alcohols e.g. dictyol E, pachydictyol A

Antimacrofouling (Bugula neretina)

Schmitt et al. (1995)

  Costaria costata

Galactosyl and sulfo-quinovosyl-diacylglycerols

Antimacrofouling (Mytilus edulis)

Katsuoka et al. (1990)

  Undaria pinnatifida

Galactosyl and sulfo-quinovosyldiacylglycerols

Antimacrofouling (Mytilus edulis)

Katsuoka et al. (1990)

  Fucus vesiculosus

Methanolic extract

Antibacterial

Lustigmann and Brown (1991)

Phlorotannins

Antialgal

McLachlan and Craigie (1964)

  Fucus endentatus

Methanolic extract

Antibacterial

Lustigmann and Brown (1991)

  Fucus spiralis

Phlorotannins

Antifouling (against Vorticella marina)

Fletcher, (1975), Langlois (1975)

  Ralfsia spongiocarpa

Phlorotannins

Antifouling (red algae)

Fletcher (1975)

  Pelvetia canaliculata

Phlorotannins

Antibacterial

Glombitza and Klapperich (1985)

  Scytosiphon lomentaria

Phlorotannins

Antifouling (Vorticella marina)

Langlois (1975)

  Desmarestia ligulata

Unsaturated fatty acids

Antibacterial

Rosell and Srivastava (1987)

  Cytoseira balearica

Lipid extract

Antibacterial, antifungal

Caccamese et al. (1981)

  Zanardinia prototypus

Lipid extract

Antibacterial, antifungal

Caccamese et al. (1981)

Chlorophyceae

  Caulerpa prolifera

Acetylene sesquiterpenoid esters

Antibacterial, antialgal

Smyrniotopoulos et al. (2003)

  Codium iyengarii

Steroidal glycosides

Antibacterial

Ali et al. (2002)

  Ulva reticulata

Benzene, diethylether and petroleum ether extracts

Antibacterial

Charles and Sivalingam (1994)

  Enteromorpha flexulosa

Sulphono-glycolipid

Antibacterial

Siddhanta et al. (1991)

  Enteromorpha linza

Methanol extract

Antibacterial

Lustigmann and Brown (1991)

  Dilophus okamurai

Diterpene

Antimacrofouling

Kurata et al. (1990)

  Codium coralloides

Lipid extract

Antibacterial, antifungal

Caccamese et al. (1981)

  Caulerpa ashmeadii

Terpenoids

Antibacterial

Paul et al. (1987)

  Halimeda sp.

Diterpenoid trialdehyde

Antibacterial, antifungal

Paul and Van Alstyne (1988)

Fascinating discoveries of biogenic compounds with antifouling potential from marine macro-algae

From the benthic marine macro-alga Delisea pulchra (Class Rhodophyceae), an unusual class of secondary metabolite, halogenated furanones (see Fig. 4) or fimbrolides, has been identified. This class of compound acts as a specific antagonist of the acylated homoserine lactone (AHL) regulatory system (quorum sensing) present in bacteria, thereby inhibiting bacterial colonization through a non-toxic and non-growth mechanism (de Nys and Steinberg 2002; Manefield et al. 2002; Hentzer et al. 2002; Steinberg et al. 2001; Kjelleberg and Steinberg 2001b; Kjelleberg et al. 1997). Furanones inhibit AHL-regulated phenotypes in a wide range of Gram-negative bacteria by shutting down the biofilm development process (Manefield et al. 1999, 2002; Givskov et al. 1996). Halogenated furanones have been tested against fouling species like the barnacle Balanus amphitrite (Steinberg et al. 1998), the macroalga Ulva lactuca (Maximilien et al. 1998) and a marine bacterium (strain SW 8; De Nys et al. 1995). Ren et al. (2002) have shown that furanone can inhibit the growth of Bacillus subtilis biofilm formation and its swarming motility in a concentration-dependent way. At a concentration of 40 μg ml−1, furanone decreased the thickness of biofilm by 25%, indicating that it can control the multicellular behaviour of Gram-positive bacteria (Ren et al. 2002).

Two antifouling compounds have been successfully identified from D. pulchra. These are 4-bromo-3-butyl-5-(dibromoethylene)-2(5H)-furanone and 4-bromo-5-(bromomethylene)-3-butyl-2(5H)-furanone (Rasmussen et al. 2000; see Fig. 2). As an example of their application, the quorum-sensing disruptor, (5Z)-4-bromo-5-(bromomethylene)-3-butyl-2(5H)-furanone inhibited the swarming motility of E. coli completely at 13 μg cm−2 (also at 20 μg ml−1; Ren et al. 2001). Using confocal scanning-laser microscopy it was shown that furanone inhibits biofilm formation of E. coli at a concentration of 60 μg ml−1 (Ren et al. 2001). Furanone was also found to inhibit by 3,300-fold the quorum sensing of Vibrio harveyi via autoinducer 1 (AI-1), by 5,500-fold that of V. harveyi via autoinducer 2 (AI-2) and by 26,600-fold in E. coli via AL-2 at a concentration of 10 μg ml−1 (Ren et al. 2001). Manefield and his co-workers (2002) demonstrated that halogenated furanones in D. pulchra can also regulate the LuxR protein responsible for the reception and response to AHLs in Vibrio fischeri. Furanone, being a non-specific intercellular signal antagonist, could be used for controlling bacterial biofilm formation without any level of toxicity (Ren et al. 2001).
Fig. 2a,b

Chemical structures of 4-bromo-3-butyl-5-(dibromoethylene)-2(5H) furanone (a) and 4-bromo-5-(bromomethylene)-3-butyl-2(5H)-furanone (b) isolated from the red alga Delisea pulchra and possessing antifouling properties (Rasmussen et al. 2000)

Work is underway using the Delisea model system to investigate inhibition of biofilms on artificial substrates, and the possibility of using it for the development of environmentally friendly antifouling compounds (de Nys and Steinberg 2002; Kjelleberg and Steinberg 2001b). Furanones have shown inhibitory effects on fouling organisms, in both field and laboratory experiments, when they have been incorporated into polymers (de Nys and Steinberg 2002).

Phlorotannins (see Fig. 4), polymers of phenolic phloroglucinol (1,3,5-trihydroxy-benzene) isolated from the Australian brown algae Ecklonia radiata and S. vestitum, have been shown to inhibit the settlement and growth of propagules of the fouling green alga Ulva sp. at concentrations of 100 mg l−1 (Jennings and Steinberg 1997).

Polar metabolites such as 5-bromo-3,4-dihydroxy-benzaldehyde, 2,3-dibromo-4,5-dihydroxyphenyl-ethylamine and 3,4-dihydroxyphenyl-ethylamine isolated from the red algae Polysiphonia morrowii, P. lanosa and Monostroma fuscum have been found to be toxic to a variety of fouling unicellular algae (McLachlan and Craigie 1996).

Many studies have been undertaken on the activities of crude non-polar extracts from marine algae against bacteria (Devi et al. 1997; Caccamese et al. 1985), invertebrates and algal spores (Steinberg et al. 1998; Martin and Uriz 1993). Two non-polar secondary metabolites, dictyol E and pachydictyol A (see Fig. 4), isolated from the brown alga Dictyota menstrualis, were found to inhibit the settlement of fouling barnacle larva, B. neretina (Schmitt et al. 1995).

Extracts from the marine algae Sargassum muticum (Class Phaeophyceae) and Polysiphonia lanosa (Class Rhodophyceae) were found to inhibit the development of fouling organisms, including bacteria, fungi and mussels, without any level of toxicity (Hellio et al. 2000a, 2000b). According to de Nys et al. (1995), broad-spectrum activities of compounds against fouling organisms, as mentioned above, are very important for future development of antifouling compounds.

Furthermore, extracts of Chondrus crispus (Class Rhodophyceae) and Laminaria pinnatifida (Class Rhodophyceae) have shown promising inhibitory activities on the germinating spores of fouling macroalgae (Hellio et al. 2002). These extracts have also been found to inhibit diatoms, especially from the genus Amphora (Jackson 1991).

Elatol and deschlorelatol (see Fig. 4), two secondary metabolites isolated from the marine red alga Laurencia rigida, were found to exhibit strong deterrence against fouling invertebrate larvae derived from Bugula neritina and Balanus amphitrite (inhibitory concentration of 100 ng cm−2 against B. amphitrite; König and Wright 1997; de Nys et al. 1996). Both these metabolites belong to the sesquiterpene alcohol group. In addition, antifungal and antialgal activities have been reported from the extracts of Laurencia rigida (König and Wright 1997).

Plocamium costatum, a red alga from the Tasmanian region of Australia, has a deterrent effect against the macrofouling barnacle, B. amphitrite at concentrations varying between 10 and 100 μg cm−2 (König et al. 1999a). Two biogenic compounds belonging to the halogenated monoterpene (Fig. 3), have been isolated and purified from this alga, both of which deter fouling organisms at concentrations of 10 and 1 μg cm−2, suggesting a possible role as natural antifoulants (König et al. 1999b). However, further investigation of these compounds and their activity against ecologically relevant fouling organisms is necessary before ascribing such a role (de Nys et al. 1998; Dworjanyn et al. 1999).
Fig. 3

Chemical structure of one of the metabolites with antifouling properties isolated from the alga Plocamium costatum (König et al. 1999a)

Elatol and iso-obtusol, two secondary halogenated metabolites isolated from the Malaysian red alga Laurencia majuscula, have been shown to inhibit six species of bacteria, including the biofilm-forming species, Staphylococcus epidermis (Vairappan 2003).

Compounds belonging to the acetylene sesquiterpenoid esters [1,2-dihydro-(2a-2i) or 1,2,3,3′-tetrahydro-2,3-didehydro(3a-3f) caulerpenyne carbon backbone, Fig. 4] have been successfully isolated from the green alga Caulerpa prolifera (Smyrniotopoulos et al. 2003). These compounds show moderate to substantial inhibitory effects against a wide range of marine bacteria, and also against the marine microalga Phaeodactylum tricornatum, indicating its role as a biocide candidate (Smyrniotopoulos et al. 2003). The inhibition zone around the disks on these bacterial strains ranged between 17 and 83% in comparison to TBT oxide (Smyrniotopoulos et al. 2003).
Fig. 4

Chemical structures of secondary metabolites isolated from marine micro and macro-algae and showing antibacterial, antifungal and antifouling properties

From the brown alga Lobophora variegata, a new compound, lobophorolide (Fig. 4), has been identified. Lobophorolide, a 22-membered cyclic lactone has shown promising antifungal activities against marine fungi (Kubanek et al. 2003). Compounds like lobophorolide can be used in future for developing environmentally friendly antifouling compounds.

It has been also shown that the haloperoxidase systems present in the seaweed Laminaria digitata are capable of mediating the deactivation of acylated homoserine lactones in Chromobacterium violaceum, thus suggesting that haloperoxidase systems could potentially be used to control fouling (Borchardt et al. 2001).

Methanolic extracts from Padina tetrastromatica (Phaeophyceae), a brown alga from the Indian coast, have been found to inhibit several strains of bacteria, including Pseudomonas vesicularis and Bacillus pumilus, indicating its potential use as a future antifoulant (9–11 mm inhibition at 500 μg/disk; Bhosale et al. 2002). Similarly, macroalgae such as Cryptopleura ramosa (Hellio et al. 2000b), Laminaria ochroleuca, Ascophyllum nodosum, Chondrus crispus and Zanardinia prototypes are well documented as inhibitors of marine bacterial strains.

New avenues of research

There is no doubt that marine algae are a rich source of biogenic compounds with antifouling potential. However, research and isolation of many of the biogenic compounds are still in the early stages. Also, isolation of these potentially important biogenic compounds from marine algae is currently very expensive. Many researchers believe that combinatorial genetic or metabolic engineering (see Bailey 1991), or hybrids (Tietze et al. 2003), might be possible remedies for this problem. In addition to offering a secure supply of naturally occurring metabolites, such technologies could be used to produce more-diverse chemicals (Burja et al. 2001). Although research is relatively new within this area, with only a few studies published to date, it seems that soon it will be possible to transfer the genes responsible for the production of these active secondary metabolites from one organism to another in order to develop more-productive organisms. Once such technology is put into force, sustainable production of these compounds will become cheaper. Some steps towards this goal for antifouling compounds are presented below. Of course, such studies do need to be benchmarked carefully against the potential alternatives such as sustainable aquaculture or culturing (e.g. Fowler et al. 1990; Marwick et al. 1999; Mendola 2003), or a facile and efficient/cheap chemical synthesis (e.g. Reid et al. 2003).

Currently, within the cyanobacteria, such levels of post-genomic work are primarily restricted to particular species of Anabaena, Synechococcus PCC7002 and Synechocystis sp. PCC6803 (from which cyanobacterial products have been produced within recombinant E. coli, see, for example, Frey et al. 2002). Importantly, we are now beginning to be at a stage of progress with cyanobacteria such that systems biology approaches to optimise product formation are becoming possible (Burja et al. 2003). Importantly, metabolic pathway constructions for the model cyanobacterium Synechocystis sp. PCC6803 are underway [see, for example: Integrated Genomics’ Bioinformatics Platform, freely available using ERGO Light (http://www.ergo-light.com/ERGO/) and The Microbial Cell Project (http://lsweb.la.asu.edu/Synechocystis/index.htm)]. Although Synechocystis sp. PCC6803 may not be an extremely interesting compound generator in its own right, there are genetic tools available that may facilitate its use as a microalgal “cell factory”. To aid in this, it was shown recently that there are 17 cyanobacterial genomes available at various stages of completion/annotation (Burja et al. 2003).

As a lead-on to this kind of production, Tillett and co-workers (2000) have shown, via gene disruption and knockout mutant analysis, that a cluster of ten bidirectional transcribed open reading frames (ORFs) are involved in microcystin production within the cyanobacterium Microcystis aeruginosa PCC7806. Gerwick et al. (2000) have targeted the lipoxygenes responsible for the production of curacins 73–76 in Lyngbya majuscula. Moffitt and Neilan (2001) reported putative peptide synthetase and polyketide synthetase genes in the cyanobacterial species Nodularia spumigena that are possibly responsible for the biosynthesis of nodularin. Kaebernick et al. (2002) reported, possibly for the first time, that multiple alternate transcripts direct the synthesis of microcystin in the mcyABCDEFGHIJ gene cluster of Microcystis aeruginosa. Recently Hoffmann et al. (2003) have identified the biosynthetic gene clusters in Nostoc sp. GSV224 that are involved in the production of nostopeptolides. Therefore, important work on the genetic machinery likely required for generation of natural antifouling products from cyanobacteria is now appearing.

As an example with regard to plant/macroalgal post-genomic exploitation, sesquiterpenes and related compounds are internationally the focus of many metabolic engineering programmes, for example, the groups of Jay Keasling at the University of California, Berkeley (Martin et al. 2003), and Greg Rorrer at Oregon State University (Polzin et al. 2003). With regards to products generation in algae, a main focus has been on production of the omega-3 polyunsaturated fatty acid, eicosapentaenoic acid (EPA) or H2, for example. One of the limitations has been the culturing of macroalgae and their manipulation for enhanced products biosynthesis, although strong progress is currently being made in this regard (see, for example, Barahona and Rorrer 2003).

Metabolic engineering has the potential to be used simultaneously with rational biochemical engineering design for large-scale production of biogenic compounds using photobioreactors. There is a need for more effective/efficient photobioreactor design and development, such as enclosed outdoor photobioreactors, which can be used for large-scale production of phototropic microorganisms, e.g. microalgae and cyanobacteria (Janssen et al. 2002; Miguel 2000; Pulz and Scheibenbogen 1998). Furthermore, modifications such as the application of optical fibres with lenses or parabolic mirrors can be used for efficiently collecting and guiding solar irradiation into photobioreactors for the production of high-value secondary metabolic compounds (Janssen et al. 2002; Shimizu 2000).

In terms of the exploitation of macro- and micro-algae, Chetsumon et al. (1998) have used seaweed-type photobioreactors constructed from polyurethane for production of a novel cyclic dodecapeptide antibiotic from the filamentous cyanobacterium, Scytonema sp. by immobilization. The antibiotic produced from the reactor was purified and examined with regard to its mode of action, and was found to effectively inhibit Gram-positive bacteria, pathogenic yeasts and filamentous fungi (Chetsumon et al. 1998). Huang and Rorrer (2003) have used microplantlet suspension cultures of the marine red alga Agardhiella subulata within a stirred tank photobioreactor for production of biomass. Barahona and Rorrer (2003) have shown that bioreactor tissue culture can be used successfully for production of secondary metabolites from marine algae within a controlled environment using species of Ochtodes secundiramea and Portieria hornemannii. Muller-Feuga et al. (2003) have implemented swirling flow in a photobioreactor for batch cultures of the red alga Porphyridium cruentum. Since light is one of the principal factors for the growth of photoautotrophic organisms such as marine algae, swirling-flow modifications help in moving biomass into areas with higher photon density more regularly. A balance between mixing and over-shearing of theses cultures needs to be found.

Yim et al. (2003) have used airlift balloon-type photobioreactors for production of sulphated exopolysaccharides from the marine microalga Gyrodinium impundicum. This is important because sulfated exopolysaccharides show strong antiviral activities against the encephalomyocarditis virus (EMCV; Yim et al. 2004). It has been shown, using axenic cultivation, that it is possible to grow phototrophic cyanobacteria and microalgae, such as Synechocystis sp. and Chlorella sp., respectively, in tubular glass photobioreactors (Hai et al. 2000). It is clear that production of secondary metabolites from marine algae is also feasible under a controlled environment (Barahona and Rorrer 2003). Large scales are also possible because Miguel (2000) has used a 25,000-l enclosed and outdoor photobioreactor for the commercial production of astaxanthin from the microalga Haematococcus pluvialis.

In the future, implementation of novel engineering designs such as pneumatically agitated vertical column reactors (Sánchez Mirón et al. 1999), tubular reactors (Yang et al. 2003; Tsygankov et al. 2002; Morita et al. 2001, 2002; Molina et al. 2001) and flat-panel reactors (Degen et al. 2001; Tredici and Zittelli 1998), coupled with metabolic engineering, could dramatically change the production of metabolites from cultured marine organisms (Janssen et al. 2002). As another example, Carlozzi (2003) has used a tubular undulating-row photobioreactor for efficient utilization of solar radiation to produce biomass from the cyanobacterium Arthrospira platensis.

Development of multiple sensors and microprocessor control for real-time monitoring of photobioreactors will also be important for commercial enclosed bioreactors. It is likely that metabolic engineering will be required to improve the efficiency of compound production to levels suitable for industrial exploitation, e.g. metabolic engineering is increasingly being used for sustainable production of hydrogen from phototrophic organisms in photobioreactors (Kosourov et al. 2002; Tsygankov et al. 1999, 2002; Nandi and Sengupta 1998), although commercial production is still way off.

Conclusion

In the future, progress in the field of isolation and production of marine algal bioactive compounds is expected to involve the integration of biochemistry-validated post-genomic methods and techniques, and intelligent bioprocess design.

One of the most important things for developing antifouling compounds from biogenic metabolites is that they must meet the standard of the EC Biocide Directive (98/8/EC) for registered toxins. There is a need to investigate the levels of toxicity and capacities for biological degradation in the aquatic environment of these compounds before they are used in antifouling paints and polymers for the prevention of fouling. Moreover, it is necessary to monitor over a long-term period once the biogenic compounds become incorporated into antifouling paints. This is beginning, as trials have shown that paints into which extracts of Pseudomonas sp. strain NUDMB50-11, Renilla reniformis, etc. have been incorporated are effective in controlling fouling in the marine environment (Burgess et al. 2003; Price et al. 1994; Willemsen and Ferrari 1993). However, the identification of these biogenic compounds with antifouling properties requires a wide range of expertise from the fields of biology as well as chemistry. Due to this, antifouling compounds from marine algae with commercial potential will take time to develop. Metabolic engineering approaches are the possible way forward for future exploitation of secondary metabolites with antifouling properties from marine cyanobacteria (Burja et al. 2003) and algae (Polzin et al. 2003).

Acknowledgements

Punyasloke Bhadury acknowledges the Department for International Development (DFID), Heriot Watt University and the Association of Commonwealth Universities for the provision of a DFID Scholarship. Phillip Wright acknowledges the Engineering and Physical Sciences Research Council for an Advanced Research Fellowship (GR/A11311/01). We thank Professor Thomas K. Wood of The University of Connecticut for helpful advice on furanones.

Copyright information

© Springer-Verlag 2004