Chemotactic Response of Marine Micro-Organisms to Micro-Scale Nutrient Layers

Summary

The fabrication of microfluidic channels and their implementation in experiments for studying the chemotactic foraging behaviour of marine microbes within a patchy nutrient seascape and the swimming behaviour of bacteria within shear flow are described.

Abstract

The degree to which planktonic microbes can exploit microscale resource patches will have considerable implications for oceanic trophodynamics and biogeochemical flux. However, to take advantage of nutrient patches in the ocean, swimming microbes must overcome the influences of physical forces including molecular diffusion and turbulent shear, which will limit the availability of patches and the ability of bacteria to locate them. Until recently, methodological limitations have precluded direct examinations of microbial behaviour within patchy habitats and realistic small-scale flow conditions. Hence, much of our current knowledge regarding microbial behaviour in the ocean has been procured from theoretical predictions. To obtain new information on microbial foraging behaviour in the ocean we have applied soft lithographic fabrication techniques to develop 2 microfluidic devices, which we have used to create (i) microscale nutrient patches with dimensions and diffusive characteristics relevant to oceanic processes and (ii) microscale vortices, with shear rates corresponding to those expected in the ocean. These microfluidic devices have permitted a first direct examination of microbial swimming and chemotactic behaviour within a heterogeneous and dynamic seascape. The combined use of epifluorescence and phase contrast microscopy allow direct examinations of the physical dimensions and diffusive characteristics of nutrient patches, while observing the population-level aggregative response, in addition to the swimming behaviour of individual microbes. These experiments have revealed that some species of phytoplankton, heterotrophic bacteria and phagotrophic protists are adept at locating and exploiting diffusing microscale resource patches within very short time frames. We have also shown that up to moderate shear rates, marine bacteria are able to fight the flow and swim through their environment at their own accord. However, beyond a threshold high shear level, bacteria are aligned in the shear flow and are less capable of swimming without disturbance from the flow. Microfluidics represents a novel and inexpensive approach for studying aquatic microbial ecology, and due to its suitability for accurately creating realistic flow fields and substrate gradients at the microscale, is ideally applicable to examinations of microbial behaviour at the smallest scales of interaction. We therefore suggest that microfluidics represents a valuable tool for obtaining a better understanding of the ecology of microorganisms in the ocean.

Protocol

Preparation 1. Create a Mask  Using a CAD software, design the channel for high-resolution printing on a transparency. This will be the “mask”. In the clean room: 2. Clean and bake the wafer First, squirt the wafer with Acetone, then quickly with Methanol, then with Isopropanol. Finally, dry the wafer using Nitrogen. Bake the wafer in…

Discussion

An understanding of how marine microbes interact with their local chemical and physical environment is imperative for a more complete and precise perception of the role of planktonic microorganisms in the oceans nutrient and carbon cycles (Azam and Malfatti 2007). However, due to the small scales (< mm) over which many important microbial interactions take place, technical limitations have prevented detailed examinations of microbial behaviour within the heterogeneous bio-physico-chemical landscape predicted to be ex…

Materials

Material Name	Type	Company	Catalogue Number	Comment
PDMS, Sylgard 184	Silicone Elastomer Kit	Dow Corning, Midland, MI, USA		http://www.ellsworth.com/sylgard.html
SU8-2100	Photoresist	MicroChem, Newton, MA, USA		www.microchem.com
Nikon Eclipse TE2000-E inverted microscope	Microscope	Nikon, Japan
PEEK tubing (0.762 mm ID, 1.59 mm OD)	Tool	Upchurch Scientific, Oak Harbor, WA, USA		www.upchurch.com
Syringes (Luer-Lok Tip)	Tool	BD, Franklin Lakes, NJ, USA
Fitting Part P-704-01	Tool	Upchurch Scientific, Oak Harbor, WA, USA		To connect tubing to Luer-Lok Tip Syringes
Syringe Pump (PHD 2000 Programmable)	Equipment	Harvard Apparatus, Holliston, MA, USA
CCD Camera (PCO 1600)	Equipment	Cooke, Romulus, MI, USA