Ammonia oxidizing archaea enrichments Abi & Angela Hopkins Microbiology Course nd July 2010

Proposed AOA respiratory pathway. Walker C B et al. PNAS 2010;107: Fig. 1. Proposed AOA respiratory pathway. Text indicates the described possible hydroxylamine (blue text and arrows) and nitroxyl (green) pathways. Red arrows indicate electron flow not involved in ammonia oxidation. Blue shading denotes blue copper-containing proteins. Pink box indicates possible alternative respiratory electron sink. Hexagons containing Q and QH2 represent the oxidized and reduced quinone pools, respectively. NH 4 + NH 2 O NO 2 - NO 3

Sampling sites Elkhorn Slough water (Kirby Park) Aquarium sand filter material Aquarium water Hopkins sediment filter

2 enrichments per row Slury material & DI H 2 O Static bottle, aerobic, dark Modified Synthetic Crenarchaeota Media Growth for 2 weeks 3x 1 ml samples taken every other day Storage at -20°C Enrichment

Selection for AOA Filter 1: No extra organic matter in the culture ( prevent carbon source for other bacteria/archaea) Filter 2: Screening for AOA (< 0.2 µM) NH 4 + and CO 2 (NaHCO 3 -buffer): only energy and carbon source Dark: Crenarchaeota are light sensitive

Chemical analysis of NH 4 + & NO 2 - For both: – Standard curve linearity µM – RT, dark Ammonia (NH 4 + ): mix sample with reagents, incubation for 2 h, extinction at  = 630 nm Nitrite (NO 2 - ): mix with Nitrite color reagent, incubation for 5 min, extinction at  = 540 nm

Elkhorn Slough - Kirby Park (Row 1) Both enrichments grew (NO2- ‘quick test’) but one set of measurements failed

MB Aquarium outflow (Row 2) Both AOA enrichments were the same

MA Aquarium water (Row 3)

Hopkins sand filter material (Row 4) (Row 4)

Final time points (Rows 1-4)

Outlook PCR screening for AOA & AOB with amoA gene FISH Isolation of organism of interest