64
5.2 Cultivation
Early studies of marine microorganisms, including N 2 -fixers, relied on isolation,
purification and cultivation on solid or liquid media (Guerinot and Colwell 1985;
Werner et al. 1974) (Table 5.1). About this time there was also growing recognition
that microbial cultures were generally not representative of natural species and
strains (Giovannoni and Rappe 2000). This was ultimately shown to be due to sensitivity of marine strains to contaminating metals, or excessively high concentrations of nutrients in typical microbiological media. Selecting for N 2 -fixing
microorganisms presented an even more difficult challenge, since it is difficult to
eliminate all contaminating N from all reagents or the seawater used for media.
Beginning in the 1960s with the emerging recognition of roles of the
nonheterocyst- forming Trichodesmium and heterocyst-forming diatom symbionts
in the oligotrophic open ocean (see below), studies necessarily relied more on
experiments and measurements at sea (Carpenter 1983; Mague et al. 1977; Saino
and Hattori 1978). Cultures of Trichodesmium and diatom symbionts were eventually obtained (Foster et al. 2011; Ohki and Fujita 1982; Prufert-Bebout et al. 1993;
Villareal 1990). Crocosphaera was isolated from Atlantic Ocean waters (Sargent
et al. 2016; Waterbury and Rippka 1989; Webb et al. 2009) years before its general
distribution and importance were recognized (Zehr et al. 2001). Isolates from estuarine or more eutrophic waters have not been so problematic, and there are cultures
of estuarine cyanobacteria, and marine heterotrophic bacteria (Bentzon-Tilia et al.
2015a, b; Bostrom et al. 2007; Chen et al. 1998, 1999).
Cultures (Fig. 5.1) are important for evaluating physiological capabilities and
nutrient requirements. Many heterotrophic marine diazotrophs have been isolated
over the years (Capone 1988; Guerinot and Colwell 1985 and references therein),
although given the isolation techniques at the time, they may not be representative
of in situ populations. For Trichodesmium several isolates are available (Fu and Bell
2003; Ohki et al. 1986; Prufert-Bebout et al. 1993). Numerous studies have
addressed physiological characteristics such as photophysiology (Chen et al. 1999;
Ohki et al. 1992), the circadian rhythm (Chen et al. 1998), nitrogenase nutrient
regulation (Ohki et al. 1991) as well as its capacity for adaptation and evolution
(Hutchins et al. 2015). Several isolates of the UCYN B organism, Crocosphaera,
are also available (Hutchins et al. 2013; Webb et al. 2001) and have been used extensively in experimental studies as well (Fu et al. 2008; Garcia et al. 2011, 2013). New
isolates for deep sea diazotrophic Archaea have also become available (Mehta and
Baross 2006). Although cultures are available for some common diazotrophs, there
is still question as to how representative they are of uncultivated strains in the environment, and if they change under long-term cultivation.
5 Measurements of Organism Abundances and Activities
5.2 Cultivation
Early studies of marine microorganisms, including N 2 -fixers, relied on isolation,
purification and cultivation on solid or liquid media (Guerinot and Colwell 1985;
Werner et al. 1974) (Table 5.1). About this time there was also growing recognition
that microbial cultures were generally not representative of natural species and
strains (Giovannoni and Rappe 2000). This was ultimately shown to be due to sensitivity of marine strains to contaminating metals, or excessively high concentrations of nutrients in typical microbiological media. Selecting for N 2 -fixing
microorganisms presented an even more difficult challenge, since it is difficult to
eliminate all contaminating N from all reagents or the seawater used for media.
Beginning in the 1960s with the emerging recognition of roles of the
nonheterocyst- forming Trichodesmium and heterocyst-forming diatom symbionts
in the oligotrophic open ocean (see below), studies necessarily relied more on
experiments and measurements at sea (Carpenter 1983; Mague et al. 1977; Saino
and Hattori 1978). Cultures of Trichodesmium and diatom symbionts were eventually obtained (Foster et al. 2011; Ohki and Fujita 1982; Prufert-Bebout et al. 1993;
Villareal 1990). Crocosphaera was isolated from Atlantic Ocean waters (Sargent
et al. 2016; Waterbury and Rippka 1989; Webb et al. 2009) years before its general
distribution and importance were recognized (Zehr et al. 2001). Isolates from estuarine or more eutrophic waters have not been so problematic, and there are cultures
of estuarine cyanobacteria, and marine heterotrophic bacteria (Bentzon-Tilia et al.
2015a, b; Bostrom et al. 2007; Chen et al. 1998, 1999).
Cultures (Fig. 5.1) are important for evaluating physiological capabilities and
nutrient requirements. Many heterotrophic marine diazotrophs have been isolated
over the years (Capone 1988; Guerinot and Colwell 1985 and references therein),
although given the isolation techniques at the time, they may not be representative
of in situ populations. For Trichodesmium several isolates are available (Fu and Bell
2003; Ohki et al. 1986; Prufert-Bebout et al. 1993). Numerous studies have
addressed physiological characteristics such as photophysiology (Chen et al. 1999;
Ohki et al. 1992), the circadian rhythm (Chen et al. 1998), nitrogenase nutrient
regulation (Ohki et al. 1991) as well as its capacity for adaptation and evolution
(Hutchins et al. 2015). Several isolates of the UCYN B organism, Crocosphaera,
are also available (Hutchins et al. 2013; Webb et al. 2001) and have been used extensively in experimental studies as well (Fu et al. 2008; Garcia et al. 2011, 2013). New
isolates for deep sea diazotrophic Archaea have also become available (Mehta and
Baross 2006). Although cultures are available for some common diazotrophs, there
is still question as to how representative they are of uncultivated strains in the environment, and if they change under long-term cultivation.
5 Measurements of Organism Abundances and Activities