Sylvan and Jacobson Basalt Enzyme Incubation experiments- FeMO 2

J2-477 Ula Nui Rock Enzyme Assays, 08-09 october 2009

Rocks harvested- R2 and R3- collected by ROV Jason near Ula Nui mats:

Note the beautiful pillow basalts (top left). Rocks were put in Jason’s port biobox to prevent mixing with seawater from other sights and transported to the surface. On the ship, a sterile hammer and chisel was used to harvest rock chips from the two rocks, which were placed in a clean glass jar. The jar was filled with Sigma sterile seawater and put in the refrigerator overnight.

(15) 125 mL polypropylene bottles were filled with approximately 20 cm3 of rock chips. This was enough to completely cover the bottom of the bottle. After filling with rocks, 30 mL of Trisma buffer was added to the bottles, parafilm was used to seal off the top of the bottle, and then they were placed at 4°C until addition of substrate after all bottles were filled. Substrate was added to each bottle as indicated in Table 1. Bottles 16-21 are blanks with 30 mL Trisma base but no rock chips. Figure 1 shows the five MUF-P bottles and two MUF-P blanks.

At time 0, 1 mL was sampled from bottles 16-21 into 2 mL eppendorf tubes and frozen at -80°C. No rocks were sampled. These numbers should serve as initial background fluorescence.

Table 1- Amount of substrate added to each bottle. Bottles 1-15 contained rock chips, 16-21 did not. All bottles contained 30 mL Trisma base.

Bottles from experiment with rocks, buffer and substrate in them. No rocks in negative controls (16 and 17).

At t1 = 88 minutes, 1 ml was collected into 3 eppendorf tubes per bottle (epi tube #s 19-63). Blanks were not collected from t1.

At t2 = 190 minute, 2 ml was collected into single eppendorf tubes for the 15 experimental bottles (epi tube #s 64-78) and 1 ml into 3 eppendorf tubes was collected from the blanks (tubes 79-96).

At t3 = 6 hrs, 10 min, samples were collected exactly as t2­ (epi tube #s 97-129).

At t4 = 12 hrs, 20 min, 4 ml was collected into two epi tubes for bottles 1-15 (epi tube #s 130-159). All blanks sampled as above (epi tube #s 160-177).

At t5 = 25.5 hrs (tubes 1-7) and 26 hrs (tubes 8-21), 14 ml of liquid was collected into 15 ml Falcon Tubes and frozen at -80°C. For the rocks in bottles 1-15, contents of each assay were collected in a 50 ml Falcon tube, any remaining liquid poured off, and frozen at -80°C.

A Few Notes:

• The liquids collected definitely had particulates in them. we should consider filtering to avoid any fluorescence from particles. On the other hand, i’m not sure how post assay filtering affects MUF fluorescence, so it’s open for discussion. Since you have worked with sediments, you have a better idea than me.
• The rocks in bottles 13-15 were finer than the rest because by the time I reached those bottles, the good rock chip pieces were gone. this is likely not a huge problem, but worth noting here so we are both aware.

J2-481, Lohiau, Marker 2 Rock Enzyme Assays, 12-13 october 2009

One basalt was collected from Marker 2 at Lohiau site:

not much rock, so only enough to fill five bottles. i chose to use Leu-AMC as the substrate since it can be used as a measure of microbial activity in general, as opposed to MUF-P, which is more indicative of nutrient stress (Gordon Taylor MEPS paper).

these is the same rock used in a 13C bicarbonate uptake experiment done by Beth! Orcutt and myself, started on 12oct.

Table 2- Amount of substrate added to each bottle. Bottles 27-28 contained no rocks (experimental blanks). All bottles contained 30 ml Trisma buffer.

bottles from J2-481 Expt

bottles were prepared the same as above, but there was a little less rock per bottle (7.5-9 cm3) per bottle. 30 ml Trisma buffer was added to each. started experiment at 16.35h local time (t0) and took 1.5 ml x2 epi tubes for each blank (bottles 27 and 28). as above, all bottles incubated in the refrigerator at 4°C and at each sampling time, all samples frozen at -80°C.

t1- 18.00h, 1.5 hours- 1.5 ml of each bottle with rocks sampled into 2 epi tubes, frozen. blanks not collected.

t2- 19.40h, 3 hours 5 minutes- 1.5 ml x2 epis samples for each bottle, including blanks, and then frozen.

t3- 00.10, 13oct, 7 hours, 35 min- sampled same as t2

t4- 05.45h, 13oct, 13 hrs, 10 min- sampled same as t2

t5- 18.00h, 13oct, 25.5 hrs- 15 ml of liquid from each bottle was poured into a 15 ml Falcon tube labeled with the bottle #. the r ocks were collected in 50 ml Falcon tubes and excess liquid poured off. all samples frozen at -80°C.

J2-482, between Marker 57 and 38, Enzyme Assays 14-15 october

J2-482 rocks R1 and R2 collected during transit between Markers 57 and 38. brought on board and processed afternoon of 13 october. rock chips were collected into a clean amber glass bottle and the bottle filled with sterile seawater (Sigma) and put at 4°C until morning of 14 october, when the experiment was set up.

Images of rock collection by ROV Jason:

This experiment was set up identically to the one from J2-477, with 15 experimental bottles and 6 blanks. Refer to table 1 above for molarities of substrate added per bottle/blank.

Experiment started at 07.15 local time 15oct09. 2 ml each from bottles 16-21 were sampled into epi tubes 232-237, respectively (bottle 16=232, 18=234, etc.)

t1- 09.05h, 1 hr 50 min, 1700 ml into two epi tubes per bottle for all bottles, stored immediately at -80°C

t2- 11:15h, 4 hrs, sampled as t1

t3- 16:45h, 8.5 hrs, sampled as above

t4- 20.25h, 13 hrs, 10 min, sampled as above

t5- 10.15h, oct15, 27 hrs. 14 ml of liquid sampled in 15 ml Falcon tubes labeled with the dive number and bottle number (and “FeMO ’09 enzymes). Bottles 16-21 sampled this way. Rocks collected into 50 ml Falcon tubes and frozen at -80°C. These tubes labeled the same way as the liquids, but do not say t5 on them.

For this experiment, the first half of the bottles have larger chunk pieces of rocks than the latter half, rock volume per bottle seems to vary widely. it will be important to weight the bottles with the rocks in them versus an empty bottle (so not to contaminate the rocks if we do later work like DNA extractions, etc) to get the weight of rocks per bottle for normalization purposes.

Picture of the bottles incubating in the refrigerator (4°C):