RESEARCH ARTICLE Reproducibility ofa microbial river water community to self-organize upon perturbation with the natural chemical enantiomers, R - and S -carvone Katja Lehmann, Andrew Crombie & Andrew C. Singer Centre for Ecology and Hydrology, Oxford, UK Downloaded from https://academic.oup.com/femsec/article/66/2/208/570676 by guest on 30 September 2021 Correspondence: Andrew C. Singer, Centre Abstract for Ecology and Hydrology, Oxford, Mansfield Road, Oxford, OX1 3SR, UK. Tel.: 144 0 1865 A river water microbial community was studied in response to perturbation with 281630; fax: 144 0 1865 281696; e-mail: the monoterpene enantiomers R- and S-carvone. The microbial community [email protected] structure and function was also evaluated after enantiomers exposure was switched. Microbial communities were evaluated by length heterogeneity PCR. Received 16 December 2007; revised 26 April The addition of R- and S-carvone enriched for a range of functionally different 2008; accepted 4 June 2008. communities: enantiomer-selective, racemic and ones that contain both. After 5 First published online 1 August 2008. days incubation, the R- and S-carvone treatments developed a range of dominant microbial communities, which were increasingly dissimilar from the ones in which DOI:10.1111/j.1574-6941.2008.00554.x no carvone degradation had taken place (R-values: R-carvone 0.743, S-carvone 0.5007). There was an increase in the evenness of the microbial community Editor: Riks Laanbroek structure upon carvone depletion. After the cross-over, the rate of carvone Keywords utilization was significantly faster than after the first carvone addition (P=0.008) river water; self organization; microbiology; as demonstrated by concomitant carvone and oxygen depletion. The main R- microbial ecology; length heterogeneity PCR; degrading community (450–456 bp) appeared enantioselective and largely unable carvone. to degrade S-carvone, whereas the S-carvone-degrading community (502–508 bp) appeared to have racemic catabolic capacity. In conclusion, chemical perturba- tions, such as enantiomers, might generate a significant shift in the river microbial ecology that can have implications for the function of a river in both a spatial and temporal context. compounded when microbial ecology is interrogated, as Introduction each member of the community is uniquely responding to The scientific method relies upon the premise that a its individual physical–chemical–biological condition. response from a system can be replicated upon recreation Therefore, complex microbial systems are, arguably, impos- of the precise physical–chemical–biological conditions un- sible to replicate (Huisman & Weissing, 2001). Yet, despite der which the response was generated. In the physical their inherent complexity, the ‘system’ can function with a sciences, this can be easily demonstrated – as a projectile considerable degree of reliability – this is evident in as will precisely and accurately hit its target when all variables disparate operations as sewage treatment works and wine- are reproduced. However, proving such a premise when it ries. However, how natural systems self-organize to perform entails biological systems can be logistically impossible. a function and why on occasion they do not, is largely Curiously, our inability to precisely reproduce physical– unknown. chemical–biological conditions in the natural sciences has The current study aims to investigate the reproducibility not precluded genuine insights into the mechanics of of a river water system to self-organize upon perturbation natural systems. For example, the premise has enabled a with the natural chemical carvone. Carvone is one of over deep understanding of the physiology of microorganisms 400 different naturally occurring monoterpenes constituting despite the lack of control over many of its component parts, the largest class of secondary metabolites in plants (van der e.g. gene instability, mutations, and gene regulation. The Werf et al., 1999a, b). S-(1)-Carvone is the principal con- problems posed by microbial systems are considerably stituent of the oils from caraway (50–70%) and dill c 2008 Federation of European Microbiological Societies FEMS Microbiol Ecol 66 (2008) 208–220 Published by Blackwell Publishing Ltd. All rights reserved Functional diversity of a river water bacterial community 209 (40–60%), while R-( À )-carvone is the principal constituent distinguish enantiomers plays an important role in fields of the oil from spearmint (51%) (de Carvalho & da Fonseca, such as drug effectiveness, insect chemical communication, 2006). Monoterpenes play an important role in chemical taste perception, and bioremediation (Laska et al., 1999; ecology, where they act as attractants, repellents, sex pher- Singer et al., 2002). The varied physical activities and omones, alerting pheromones, and defence secretions, as toxicological risks of enantiomers should be expected given well as antifungals, and biofilm and enzyme inhibitors that the biological receptors (e.g. proteins) are chiral and (Hartmans et al., 1995; Amaral & Knowles, 1998; Amaral thus potentially enantioselective (Laska et al., 1999). et al., 1998; van der Werf & Boot, 2000; Niu & Gilbert, 2004). This study repeatedly monitored residual carvone, Emissions of monoterpene from trees are estimated at dissolved oxygen, protists, and community structure over a 127 Â 1014 g carbon yearÀ1 (Guenther et al., 1995) and are 22-day period to qualify the variability in the resulting com- deemed central to a terpene-based food chain in conifer- munity structure and functional response of a river water Downloaded from https://academic.oup.com/femsec/article/66/2/208/570676 by guest on 30 September 2021 dominated locations (Button, 1984). Few studies have bacterial community after spiking with different carvone investigated the biotransformation of terpenoids. Aerobic isomers. We hypothesized that despite every effort having biodegradation of limonene, the most widespread mono- been made to replicate the physical–chemical–biological terpene in the world (van der Werf et al., 1999a, b), has been conditions within and between treatments, the resulting among the best characterized (Trudgill, 1990; van der Werf community structure and function would be highly variable. et al., 1998; Duetz et al., 2001, 2003). Cumate (Eaton, 1996), We predicted one of three outcomes: (1) enrichment of pinene (Trudgill, 1990; Singh et al., 2006; Smolander et al., enantiomer-selective degraders, (2) enrichment of racemic 2006), cymene (DeFrank & Ribbons, 1977a, b; Eaton, 1997; degraders, or (3) enrichment of both enantiomer-selective Harms et al., 1999; Nishio et al., 2001), and carveol (van der and racemic degraders. The study used a cross-over design, Werf et al., 1999a, b; van der Werf & Boot, 2000) are among where communities exposed to one isomer of carvone were the few additional monoterpenes where microbial biodegra- subsequently exposed to the other isomer, raising the dation studies have been conducted. A number of studies question: how rapidly can the function and structure of a have investigated the role of carvone and other monoter- microbial community respond to fluctuating optical iso- penes in the induction of xenobiotic degradation (Gilbert & mers, and how variable is the response? Crowley, 1997; Hernandez et al., 1997; Singer et al., 2000; Dercova et al., 2003; Rhodes et al., 2007), implicating a Materials and methods fortuitous, and potentially important mechanism for the natural attenuation of contaminated aquatic and terrestrial Experimental design systems (Singer et al., 2003; Singer, 2006). The structural similarity of R- and S-carvone to many of Two experimental treatments, one spiked with (R)-(1)- the other 4001 naturally occurring monoterpenes per- carvone and the other with (S)-( À )-carvone and one mitted its use as a model natural chemical for perturbing control treatment were monitored over 12 days to investi- the river water ecosystem. Using optical isomers as the gate the degradation process of the different carvone iso- perturbation agent, the treatment effect can be isolated to mers. On day 13 the microbial communities from each the interaction between the chiral biomolecules within the treatment were retrieved from their environment and re- river water microorganisms and the chiral nature of carvone suspended in water spiked with the alternative carvone – as all physical properties of enantiomers are identical (e.g. isomer (see Table 1). This cross-over was used to assess the water solubility, log P, Henry’s constant, etc.). The ability to functional diversity of the communities. After the cross-over Table 1. Overview over the sampling regime for both chemical and community analysis Day 0 Addition of Carvone enantiomers and division of water into replicates (12) Day 1 Chemical and community structure sampling Days 2/3 Chemical sampling Day 4 Chemical and community structure sampling Day 5 Chemical and community structure sampling Day 6 Chemical sampling Day 7 Chemical and community structure sampling Day 8/9/10 Chemical sampling Day 11 Chemical and community structure sampling Day 13 Extraction and resuspension of cells in sterile river water containing the carvone isomer that had not been degraded before. Controls were resuspended in sterile river water only. Day 16, 17, 19 Chemical sampling Day 22 Chemical and community structure sampling FEMS Microbiol Ecol 66 (2008) 208–220 c 2008 Federation of European Microbiological Societies Published by Blackwell Publishing