Wild Yeast Harvesting at Lake Merritt

Make Yeast Collection Wort Vials
For collecting the yeast I made a fresh wort and put 10 mL in 15 mL tubes using a sterilized burette pipette. I wanted the 5 mL of head space to allow space for any krausen formation and so that when I mixed the wort I could move a fair amount of oxygen into the liquid to get the yeast going.

   Collection Wort Recipe (~1.04 gravity):
   20 g DME (Dried Malt Extract)
   180 mL Distilled water

boil for 1 hour and then transfer to tubes and allow them to cool before using. Label each 15 mL tube with the date of creation.

In this mock up I made vials that I added 100 ul of Vodka (40% by volume ethanol) to 10 mL of wort to see if a very low amount of alcohol (0.4%) could help select for yeast over other organisms and denoted these vials with a B.

Collection Tool Kit

1) 70% rubbing alcohol in a spray bottle - used for cleaning hands and tools. I would use 70% ethanol but its harder for me to get.

2) Knife with scissors - used for cutting sections of fruit etc to get in vial. Sterilized every time before use

3) Ziplock bag with napkins - used for cleaning tools with alcohol

4) Yeast Collection Wort Vials - always visually check them before heading out, just in case of contamination. The fresher the batch you can make the better, but vials should be able to be kept for 1-2 months at 4C.

Field collection kit

Field Collection of Yeast

I went to Lake Merritt's gardens in downtown Oakland to hunt for wild yeast. I looked for fruit and flowers since they would be potential sources of sugars and complex carbohydrates that might attract yeast. Before each sampling I would spray my hands, the top of the vials and the knife with alcohol. For fruit and flowers that were small enough I would use the alcohol treated knife/scissors to deposit fruit into the Yeast Collection Wort Vials. I would then screw on the top and then shake vigorously for 30 seconds in order to try and get any yeast present into solution and also aerate the wort. I immediately labeled the vial with the date of collection, a unique identifying number, and a comment about the collection.

Controls - upon getting home I used 1 ul of German Hefeweizen yeast from White labs as a control to make sure that the system was a viable condition for yeast. Since I wanted to see if the addition of alcohol (vials labeled B) had any selectivity I added 1ul of the White Labs yeast and 1ul of Lactobacillus from yogurt to a few vials.

Vials ready to start fermentation

Fermentation 

Ideally I would have an incubator that I could control the temperature of the collected materials, but my apartment does not afford me this luxury, so I simply placed the vials in a jar in relatively dark space and let them incubate. I checked them about every other day to see if there were obvious signs of fermentation (krausen, bubbles, etc). It took almost three days before I saw bubbles coming from the bottom of the tubes and krausen formation in the controls. The photos below are all from three days after sampling.

Controls made with Hefeweizen Yeast. Notice the krausen and bubbles

a) Hefe yeast - krausen present b) Grape - krausen present c) Catctus fruit - mold present, no krausen

I was expecting the fermentation to take about 2 weeks, but I was surprised to see some still going at that time so let them go a full 3 weeks before moving onto the next step - sampling and plating.

Plating and Sampling

Part of the reason for choosing a 10 mL ferment for the collection vials is that there is enough liquid to taste multiple times and still plenty to either start a new fermentation with or streak out plates. Notes on each sample are in the summary table at the end of this blog and I will just highlight the few that I think are noteworthy here. The grape was just as expected. It was boozy in flavor and had a grappa like nose to it. Microscopy showed plenty of health budding yeast cells. I really was hoping the burgmansia would come through, but it was lack luster, not much taste compared to the wort control and the scent was reminiscent of the flower but not as intoxicating. The orange flower was the one that caught me off guard. Upon opening the vial it had much more pressure then the hefeweizen yeast controls and the scent, oh the scent! its smelled just like the flower. The flavor was like a warm fermented saison and microscopy showed a healthy number of budding yeasts. After getting a feel for each ferment I then decided to streak out the material on DME Agar plates.

   DME Agar Plates
   30g DME
     5 g Agar
     1g Fermax Yeast Nutrient
   264 mL distilled water

boil for 10 minutes in an attempt to completely dissolve the agar. Pour into 5.5 cm diameter petri dishes. This made approximately 15 plates. I tried to make a relatively sterile place to pour by cleaning my bathroom floor with 70% isopropanol alcohol and then putting down a small sheet of aluminum foil to work on and cleaning my hands and all tools with isopropanol. I let the plates set up for 1 hour and then place in the oven at the lowest setting to help the plates dry out. All plates were labeled with a pour date. These plates should be able to keep 1-2 months at 4C. It might be helpful to add ampicillin to these plates if you are just looking for yeast, but I was curious to see everything that grew from these collections. I believe it would also be helpful to boil the material for ~30 minutes to one hour before pouring as I think some contamination I saw on control plates was due to spores in the DME, agar, or yeast nutrient.

To some of the plates I added 50ul of the fermentation culture and then used 6mm sterilized (boiling water follow by isopropanol) to spread the plates. 50ul turned out to be way to much and 1 ul would probably be a better spreading concentration.

Orange flower yeast plated and under the microscope

Juniper Berry yeast - looks like a Brettanomyces due to the pseudohypha

Left to right: Budding yeast from Orange flower, Bacteria clusters from Burgmansia, Fungal hyphae from mold in cactus fruit ferment. All 400X with inverted scope.

Next step is to get single colonies from these cultures and then to scale up fermentation from these single colonies by transferring krausen to select for faster fermentinng yeast...or at leas that is the theory! I will start with 500 mL or 1 L ferementers so that they are small

Sample	Test	Fermentation notes	Scent notes	flavor notes	microscopy
Control	2017.09.24 A -ctrl	no noticeable change	similar to wort	wort	NA
Control	2017.09.24 B -ctrl	no noticeable change	similar to wort	wort	NA
AK Juniper	2017.09.24 1 A	mold at surface after a week	wort + musty	wort like	NA
Burgmansia	2017.09.24 2 A	small bubbles 3 days	slight scent of flower	wort with delicate sweetness	granular cells ~15 um
Burgmansia	2017.09.24 2 B	small bubbles 3 days	slight scent of flower	wort with delicate sweetness	granular cells ~15 um
Orange Flower	2017.09.24 3 A	krausen in 3 days, high pressure at 3 weeks	strong orange scent + cloves	tastes like orange saison	budding yeasts dominant
Cactus Fruit	2017.09.24 4 A	mold in 3 days, chunky cloud at 3 weeks	musty	wort like	long hyphae (mold)
Grape	2017.09.24 5 A	krausen in 3 days, bubbling still at 3 weeks	boozy scent like grappa	boozy, sharp	budding yeasts dominant
Grape	2017.09.24 5 B	krausen in 3 days, bubbling still at 3 weeks	boozy scent like grappa	boozy, sharp	budding yeasts dominant
Fig	2017.09.24 6 A	no obvious signs of fermentation	wort + lite must	wort	NA
Hefeweizen Yeast	2017.09.24 7 A	krausen in 3 days, low action at 3 weeks	clove / banana	young hefe, banana	budding yeasts dominant
Hefeweizen Yeast	2017.09.24 7 B	krausen in 3 days, low action at 3 weeks	clove / banana	young hefe, banana	budding yeasts dominant
Hefe + Lacto	2017.09.24 8 A	krausen in 3 days, low action at 3 weeks	clove / banana	young hefe, banana	budding yeasts dominant
Hefe + Lacto	2017.09.24 8 B	krausen in 3 days, low action at 3 weeks	clove / banana	young hefe, banana	budding yeasts dominant

A = regular wort
B = wort + 0.4% ethanol

Isolation of single yeast cell (colony) 

From the streaked plate a loop was used to get just a small amount of yeast and then used quadrant streaking technique (use the loop first in one quadrant, turn it or clean it, run it through first quadrant into next, repeat until 4 quadrants touched) in the hope of getting a single colony that could be picked and propagated.

single colonies of Orange Flower Yeast

The most isolated yeast colony on this plate (top) was then taken and used to make another plate of single colonies. From this next plate mini-stabs were made by pouring DME agar into 1.5 mL tubes and collecting colonies into them. These tubes can be held at 4C for extended periods of time and sampled to restart the colony. One of these stabs was given to Yann on 2017.10.27 to try to propagate back home with Nuevo Mundo Cervecería.

1.5 mL tubes with DME Agar. Orange Yeast inoculated "mini stab"

Currently working on making stabs of the other yeast isolates from grape and juniper and then the big task: figuring out how to domesticate these yeast. Here is the best paper I've read on domestication of yeasts and what happens to their genome over the process of domestication.

http://www.cell.com/cell/fulltext/S0092-8674(16)31071-6?_returnURL=http%3A%2F%2Flinkinghub.elsevier.com%2Fretrieve%2Fpii%2FS0092867416310716%3Fshowall%3Dtrue

The biggest take homes are domestication involves increasing the ability of yeast to metabolize maltose (Mal1 locus) and for most yeast strains, excluding German Hefeweizen and Saison, it also means losing the pathways that cause phenolic flavor (production of 4-vinyl guaiacol ). Yeasts at the far end of domestication also show a decrease in sexual reproduction.

How to Sequencing a Genome using the Sanger Sequencing Method

Just found this website that someone built of the FLASH animations I made way back in 2006 while working at the the Joint Genome Institute. The nice thing is that it makes the animations easier to get to and click through. Check it out here:
http://heejong.com/mccauley/DNA_Sequencing_Animations/player.html

2015.11
The Move

On a precipice
I could see the long trail
behind
ahead
Green tunnels

Upon that height

I knew I wanted
to walk on the crest
of a trail
with many precipices
clear views
rocky shoes

Poem: 2013.12.11 ZZ walking

In step
We walked
Talked
talentedly about the world

The city installed
new lights
That fragmented the shadows.
Alone they gave me pause
in the night,
how suddenly something stable
had come undone

And upon returning home
to your letter
I found the rest of world
too had unwound

A noticeable difference
in our gaits multiplied by years
had walked us over a chasm.
You and an unconceived Child
to one side and I to the other

I knew you had stopped
And only with the scare
of nausea in your steel stomach
And the doctor's denial
did that crack in the sand
vacate the space between us
to where we could not stand side by side

In step
We shall walk
Talk
apart

USA; now like visiting a 3rd world country

I've spent many of extra nights and incalculable hours not getting somewhere due to political problems in others countries and always considered these situations symptoms of places that still have large political instability. This past week a visit from a German friend made me apply this same diagnosis to the US. Nancy wanted to go to Lassen, but we were unable to plan it because of the federal shutdown, so we agreed to go to Tahoe as we could still have some time in the mountains, but another political hiccup interfered as BART went on strike Friday. Nancy had flown into SFO, so what should have been about an hour or so worth of transportation to get to the east bay turned out to be closer to 3 hours. As a country we need to stop being so greedy and think about how tantrums effect other people. Some how in the last twenty years the word compromise seems to have escaped the national lexicon. I'd like to see it come back.

At least the mountains still know how to compromise with the changing weather

Poem: ZZ departing

The burden upon you

With the weight of your parents expectations

Becomes that of finite lite

in your flight

For I remember a fortnight

Where upon the bed you turned many a times away and inward

Till turning turned upon itself

And in the sheets, a thing that gave me retreat.

A blackhole covered in white linen

So strong that even your voice could not escape.

Under the sun

I know now never to stay for a fight

Because in your search for one

I was undone

The razor in hand that runs through your life

Divides the world such that

There is no picture to hang on the wall

To stand back from and say

"Ah there it is, I'm sure, it is all"

But in the mountains upon

The ridge of our relations

A view of all directions

But a trail didn't not emerge

The wind carries no weight yet knows its path

And

Absence becomes the heaviest thing of all

Poem: Partial Prime Design

“Partial Prime Design” 2011.10.29 (11, 7, 5, 3, 2 and 53, 41, 23, 7, 7)

Beneath the bright banner
Of sierra skies
The milky way stretches without resolve
And into our world
Flecks of the universe dissolve
Over head and under covers
We try to make sense
Of all the innumerable bodies
That strike us
Why some tilt our axis
And the others accumulate only as dust

From the dusty trail we depart
To wrap ourselves around a mountain
And to swim in a cold blue alpine body
Upon whose shores
We are wrapped in late summer’s heat
And swim beneath your shawl in the humid warmth of each other’s breath
While Goethe calls from peak to peak

In the meadows far below
Prime petals punctuate
The serene green of life
Where a river of glass
Cuts the land in two

Cloud fire
Leads back
To our Origin

Next to Goethe
We watch stars fall

Occupy Oakland Protest, October 25th 2011, Downtown Oakland, California

Photos from Occupy Oakland tonight.

Protest was peaceful in Snow Park. The police did not chase the crowd to Snow Park, but remained in downtown around city hall. I don't know why the crowed decided to go back to city hall in order to confront the police, because by that time a lot of the shit-starters had showed up and began lighting trashcans etc on fire and that triggered the tear gas.











I am thankfull that the protesters that came in front of my building (1502 Alice St shown here) did not cause vandalism like the Oscar Grant riots.

Book Review: Outliers

Outliers: The Story of Success by Malcolm Gladwell

My rating: 4 of 5 stars


Outliers is very much in the same vein or at least style as Malcom Gladwell's preceeding titles. Quirky bits of information that when rallied around the same arguement come up with something interesting. I feel that with this book Gladwell actually takes on a larger arguement than his other books, its ultimately the Nature vs Nurture arguement but using extremely successful people as the case studies. Gladwell argues for nurture being the largest component of success and uses everything from the Canadian Hockey Team to Bill Gates to why Asian kids are good at math to form his arguement.



Some nick-nack info highlights



Canadian Hockey Team - Most players are born in January, February and March. Their birth month line them up to be the biggest kids in their early teams. 10-12 month differences when you are 5 and 6 can mean you are dramatically bigger. The bigger and better players get special attention, more training, and ultimately more hours of practice, so by the time they are in their late teens they are significantly better.



10,000 hour rule - People with 10,000 hours of practice at one thing tend to be experts and if you get this 10,000 hours in before say the age of 20, you have a much greater chance to go really far.



Bill Gates - had access to one of the first computers at a super young age and would spend hours upon hours on it, so by the time everyone else is starting to hear about personal computers, Gates has already logged in 10,000+ hours of programming time. Similar stories for Bill Joy and Steve Jobs.



Math - kids that can focus do good in math. Your ability to focus on one subject for a period of time has a direct correlation to your math abilities. Asian kids do better in math because they come from a culture of extreme focus that grew out of needing to tend to rice paddies. Western societies grew wheat and other less intensively cultivated crops, so their focus ability is culturaly less. This part of the book gets a bit dodgy, but the chinese proverbs vs western proverbs are good:



"If a man works hard, the land will not be lazy" - Chinese



"If God does not bring it, the earth will not give it" - Russian



You can change your nurture -

1) as seen by Korea Airlines turning its horrible air record around, by changing the nurture/culture of its pilots and co-pilots to be not Korean.

2) As seen in the KIPP school in the Bronx, where kids are sort of reconditioned to be in a different culture than that which is around them and the students do amazingly well.



The basic underlying statement is that nurture plays a huge role in success. You have to be in the right environment at the right time to be wildly successful, but you can choose to change your environment and to some degree your nurture. I think the real question that is dodged all the way through is what is success?



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Poem: Today

Today

Copper caressed skies propped up
The sunset, floating Mt Tam
Against the edge of another day

And across it- beyond it
In that same sky
Across and beyond the ocean

You wander over copper colored landscapes
Yet no matter how far you wander
Colors never change

Some items in life distill beyond context
And when we ask what is next?
We already know in our hearts the eternal concepts.

Poem: Bar 355 July 24-ish

In the night is sight,
When downtown Oakland has changed,
When I can walk unastranged into a local
wateringhole
where the music doesn't stop,
The bottom doesn't drop,
out of the place because I'm young and white
buth this is also the what bothers me about tonight
where are the people that lived here when I was here at the start?
they now live at the afar extensions of the BART
SYStem
The music is the same but the relation has changed.
MJ is not the same to the displaced as he is to those that replaced.
A song - a world is read differently by
every-fraction-of-people-in-time(s)
And what does this tell us of the world?
Should we read in frame, out of frame, which games - is everything
THE SAME?
or ultimately different?
Depeche mode draws the line
in this bar of contemporary time.

Desert Solitaire by Edward Abbey

My rating: 4 of 5 stars


There is a lot in the this book that resonates with someone that has spent enough time in the outdoors in one place with themselves to make grand conclusions about one's own insignificance.


Some of my favorite quotes from the book:
Language makes a mighty loose net with which to go fishing for simple facts, when facts are infinite.

The beauty of Delicate Arch explains nothing, for each thing in its way, when true to its own character, is equally beautiful (p45).

A weird, lovely, fantastic object out of nature like Delicate Arch has the curious ability to remind us- like rock and sunlight and wind and wilderness- that out there is a different world, older and greater and deeper by far than ours, a world which surrounds and sustains the little world of men as sea and sky surround and sustain a ship (p45).

Industrial Tourism is a threat to the national parks. But the chief victims of the system are the motorized tourists. They are being robbed and robbing themselves. So long as they are unwilling to crawl out of their cars they will not discover the treasures of the national parks and will never escape the stress and turmoil of the urban-suburban complexes which they had hoped, presumably, to leave behind for a while (p64).

We have agreed not to drive our automobiles into cathedrals, concert halls, art museums, legislative assemblies, private bedrooms and other sanctums of our culture; we should treat our national parks with the same deference, for they, too, are holy places (p65).

A ventursome minority will always be eager to set off on their own, and no obstacles should be placed in their path; let them take risks, for godsake, let them get lost, sunburnt, stranded, drowned, eaten by bears, buried alive under avalanches - that is the right and privilege of any free American (p69)

A man could be a lover and defender of the wilderness without ever in the his lifetime leaving the boundaries of asphalt, powerlines, and right-angled surfaces. We need wilderness whether not we ever set foot in it (p162).

Grand Canyon, Big Bend, Yellowstone and the High Sierras may be required to function as bases for guerrilla warfare against tyranny (p163).

In any case, when a man must be afraid to drink freely from his country's rivers and streams that country is no longer fit to live in (p202).

But the love of wilderness is more than a hunger for what is always beyond reach; it is also an expression of loyalty to the earth, the earth which bore us and sustains us, the only home we shall ever know, the only paradise we ever need - if only we had the eyes to see (p208).

No, wilderness is not a luxury but a necessity of the human spirit, and as vital to our lives as water and good bread. A civilization which destroys what little remains of the wild, the spare, the original, is cutting itself off from its origins and betraying the principle of civilizations itself (p211).

Henry Mountains, Utah

Its a great country; you can say whatever you like so long as its strictly true- nobody will ever take you seriously (p293).

And thus through language create a whole world, corresponding to the other world out there. Or we trust that it corresponds. Or perhaps like a German poet, we cease to care, becoming more concerned with the naming then with the things named; the former becomes more real than the later (p322).





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Philippines and Taiwan Photos (2010.01)

27 day stint in Taiwan and the Philippines. Here are a few tempting photos. It was amazing!!

Philippines:
More photos of the Philippines at:
http://www.flickr.com/photos/damon_tighe/sets/72157623344784827/




















Taiwan:
see more photos of taiwan:
http://www.flickr.com/photos/damon_tighe/sets/72157623455846184/

Book Review: "House of Glass" by Pramoedy Ananta Toer (2009.07.08)

House of Glass by Pramoedya Ananta Toer


  rating: 5 of 5 stars


View all my reviews.

The final piece of the Buru Quartet. Excellent change in perspective in this novel compared to the first three of the Quartet. Not only a shift in Narrator, but an almost meta-textual analysis of the first 3 books. Pramoedya is a great crafter of not stories not only in content, but in structure.

Poem: from Santa Fe and some photos (2009.05.28)

12:30am 2009.05.28 Santa Fe, NM. Fusion club

the air breathes
soft and high
and I am alive
ethanol courses
actions recourse
and the stars shine
ambivalent
in the black blanket
punctuated with bolts
New Mexico
Old and New
falters forever
in capitalism
in Santa Fe
in error and
perfect existence

12:40am 2009.05.28
Turquoise cuts adobe
in mid day light
and at night
they-
grey, stand in contrast
to the shadows
to the shallows
we don't dare illuminate
for darkness penetrates
this place
in the bowls and souls
deep seats and retreats
it bleeds and seeds
the next generation -
infinite penetration
of half dead frustration
Santa Fe

12:50am 2009.05.28
sell, sold, soul
so you remember
the past-
the commoditized
capitalized product of this town,
where Heroes fought
Savage Indians,
but scratch that hatch
it ain't sellable
brine it
shine it
design it
legitimize it (the past)
-build it (the past) and they will come
but once they've cum
its all undone.

1:05am 2009.05.28
Rockies rock bottom
Santa Fe by day
find a way to masks the resource pull
that in time shall levy its toll
in drops absent
water limited
this town fate scripted in
Chaco and Bandelier and Other(s)
places far and near
How small our imagination is
for the teachings of history

1:20am 2009.05.28
Coral colony of clouds
coral the Jemez on the horizon
in the high dry sky of New Mexico
the germinating seed of Huxley's anti-Utopia
The land of life in protest


Santa Fe contrasting Adobe


Iris and cliff in the Jemez


The Buckets swimming hole in Jemez


Clouds over ABQ


Porch adornment in Santa Fe

My Nerd Job: biofuels and shipworms (2009.05.15)

So we just got samples today of shipworms for single cell micromanipulation. Why in the world would anyone be interested in shipworms and their symobiotic bacteria? These little worms are extremely good at breaking down wood and the reason they are good at breaking down wood is the bacteria that associate with their gill structure produce cellulases that degrade cellulose, a major component of wood. If you can degrade wood into into component sugar you can ferment it and turn it into a stiff drink or a biofuel. Possibly enzymes from these bacteria could lead us away from using corn and other food stocks as feed stocks for a biofuel infrastructure in the US, not to mention that most of plant biomass is cellulose so having access to it would greatly increase the effeciency that we could take a crop and turn it into fuel.

Lore has it that Columbus got an extra year long stay in the Bahamas because shipworms ate up his boat so bad that he could not sail back to Europe until all the wood had been repaired.

So here is the process (Hands are Dan our collaborator):

1) Capture a worm from a piece of wood. The worm is right at the tip of dental tool


2)Dissect the worm to get out its gill where the bacteria associate



3)Pulverise the gill to free up the bacteria. Below is the gill aparatus:



4) Look at the bacteria under the microscope:



5) Capture singe bacteria:



6) Lyse open the cell to get the DNA and amplify it.
7) Sequence the DNA and find gene for cellulases

Down the Road:
8) industrialize the cellulases for use at biofuel plants
9) Drive down US consumption of foreign oil. Create a fuel that cycles carbon instead of digging out of the ground and putting it in the air.