:

Time and Temperature in Kombucha Ferment Kombucha Mushroom Tea is a Raw Active Continuous 
Process. The higher the temperature the faster the ferment and the shorter the time to ferment. All things
are in a constant state of flux. Like a chicken egg that hatches because the time and temperatures are in
their proper relationship.
Wine is made with yeasts. Vinegar is made with bacteria. Separate process's.  Kombucha Tea is made with
both yeasts and bacteria in a synergic process. Once this process reaches the precise stage we seek, we
then bottle to maintain that stage—or serve a refreshing elixir as is.

All the available research on Kombucha Tea has identified Kombucha Tea as a process of fermentation by
yeasts and bacteria, sugar and black tea (camellia sinnesis, oxidized / fermented). There is a finite number
of yeasts and bacteria combinations that would produce kombucha, however the possible combinations as
well as synergy of various strains of kombucha tea possible have lead one major kombucha researcher
(Michael Roussin) to conclude that  Gluconic Acid, Acetic Aid, and fructose are what is in All
Kombucha Tea. This combination is what makes Kombucha Tea distinct from other health tonics such as
Apple Cider Vinegar or lacto-ferment health beverages like Ginger Beer or those produced by Water Crystals
(Japanese Water Crystals, Water Kefir Crystals, etc), and commercial fermented probiotics beverages like
B.E. Wholegrain Liquid.

The taste and character of the Kombucha Tea is only partly formed by the type of tea used in the ferment.
Tea contributes nitrogen (maybe more) that both the yeasts and the bacteria rely upon to create Kombucha
Tea.  The composition, character and preparation of the tea affect the peculiarities of the Kombucha Tea. 

 We know there is fructose in our ferment because we did put sugar in. How fast the sugar is metabolized
into glucose plays a large role in the overall time of the ferment. Regardless of how long we ferment, some
fructose will always be left behind. That percent not converted to glucose remains behind largely as fructose
and various nutrients. Some contribute to taste. Fructose is sweeter than glucose.  Minerals creating a
slightly bitter taste.  Other components of the peculiar sugar we use affect taste as well as the formation,
creation and destruction of acids. 
Home Brewed Kombucha Tea Basic 2 Step Dance

1. Conversion of sugar to alcohol

2. Conversion of alcohol to beneficial acids and nutrients

Generally, the conversion of sugar to alcohol is complete within 7-10 days when fermented at  21C- 27C 
(74F - 84F). A “hot fermentation” for instance in some red wines, may be at higher temperatures 27C- 30C
(80-85F). note: these two temperature ranges spread the often suggested temperature range of making
Kombucha Tea, i.e., 70-85F. Why choose one over the other or what is happening at what temperatures is
our quest. 

In the pursuit of Kombucha Tea, we are neither making wine – which almost exclusively rely upon yeasts,
nor vinegar, which rely almost exclusively upon bacteria. We are making Kombucha Tea, which is a
Serendipitous Collective adventure Of Bacteria and Yeasts. The tea that we drink is but a Kodak
Moment.

Yeast work fastest at about 1-2% sugar concentration. High sugar levels actually retard the growth of
yeast; Initial concentrations above 25% are problems. To make the strongest possible elixir increase the
sugar gradually. For example adding 1/3 on Day 1. 1/3 on Day 2, and the final 1/3 on Day 3. If you use a 
Hydrometer, add sugar at 1.040, bringing ferment up to 1.060-65, let it go until 1.040 add sugar again up
to 1.060 and so on until you've added the sugar you want.

Acetic acid taste like vinegar. We can easily test the pH of our ferment.  The pH does not give us the actual
acetic acid level, but the whole acid level including the crucial Gluconic Acid. However it may also include
such acids as; Lactic acid, Citric acid, Oxalic acid, Itaconic acid, which are more commonly found, and some
more random acids like; Benzoic acid, Butanoic acid, Decanoic acid, Hexanoic acid, Nicotinic acid,
Pantothenic acid, Propionic acid, Octanoic acid, d-Saccharic acid, Glucaric acid, Succinic acid,  and  almost
always the  ~Keto-gluconic acid, (2-Keto-gluconic acid, 5-Keto-gluconic acid, 2-Keto-3-deoxy-gluconic acid)
which may have been confused for what was reported as Glucuronic Acid in Kombucha Tea). For properly
fermented and anti-microbial  Kombucha Tea should be fermented to a pH in the range of 2.5 – 3.0 +/-
with around 33 g/L total acids.  (according to research by Cornell University).

Kombucha Tea is made by yeast that is acid tolerant, The principle role of the yeast is to convert the sugar
into ethanol (alcohol) for the bacteria to feed upon. To a lesser extent to produce carbon dioxide to make
our Kombucha Tea sparkling and effervescent – without suffocating the bacteria which require oxygen.
Yeasts provide a portion of the taste of Kombucha Tea.  Some species of yeasts can convert sugar into both
alcohol and acetic acid, but not produce gluconic acid by itself.  One species of yeast isolated from
Kombucha Tea has been identified as Zygosaccharomyces kombuchaensis sp. n. (type strain NRRL YB-
4811, CBS 8849) this species seems unique to Kombucha Tea.

The growth rate of yeast is normally related to temperature. While most yeast will survive cold
temperatures, growth rate is very slow and species dependent.

In general optimum yeast temperature range is 16C – 30 C [60-85F]. Lower temperatures are difficult to
handle and can lead to increased levels of S02, volatile acid and hydrogen sulphide and some bad smelling
stuff. Additionally at low temperatures, the beginning kombucha ferment is very susceptible to pathogens
and foreign or domestic yeasts that may attempt a takeover over the chosen yeasts that has yet to
establish itself, or has gone dormant because of the cold allowing another species better suited to cold
temperatures to take command.

Temperatures above 35C [95F] will usually stick [stop] most fermentation. At 41C [105F] most yeast begin
to die off and at 140F yeast dies within a few minutes.

My research indicates that the most common range is  23C - 29C [74F - 84F] and is fairly easy to maintain
in the average household. Kombucha heating units or seedling trays are encouraged by kombucha authors
and researchers like Harald Tietze and Guenther Frank and myself. A regulator can be added to help
maintain ideal temperatures.

A kombucha ferment that does not produce a mushroom, or a very quick acetic tasting ferment (faster than
its normal production), or a normal ferment with very thin and opaque mushroom is probably due to the
demise of the kombucha bacteria.

This may be because the yeasts have produced an intolerable amount of alcohol for the bacteria to survive
in, and /or an intolerable amount of carbon dioxide and the bacteria have suffocated. The bacteria need
oxygen as do the yeasts in part of their ferment. Boiling water or distilled water or very hot temperatures
all reduce the available oxygen. Stirring the water well before starting helps increase oxygen levels. Stirring
during the active ferment will increase oxygen as well as disturb the growth of the cellulose (mushroom). In
this case stirring/disturbing the cellulose does not adversely affect the formation of the acetic or gluconic
acids, you’ll just have an ugly or thin mushroom. Another cause may be due to the rise of dormant specie
within the kombucha or to outside influences - wild and airborne bacteria or yeasts, soaps, harsh sunlight,
smoke, paint fumes, or neglect of sorts. Not all species of Acetobacteraccae produce a mushroom but all
will produce acetic acid.  

Our other major player in our serendipitous adventure is the bacterium. Its role is to produce the beneficial
acids and to reduce the sugar and alcohol content. To avoid an over-bearing straight vinegar drink we need
to maintain a higher ratio of gluconic acid to acetic acid. This produces a pleasant semi-sweet, semi-sour
elixir known as Kombucha Tea.

Step 2 Conversion of alcohol to beneficial acids and nutrients

In a basic home ferment the starter tea contains both the yeasts and the bacteria (from a precious batch)
and a kombucha mushroom (the DNA). When the feeding solution is added (sugar and camellia sinnesis,)
the yeasts begin an active ferment. Depending upon he type of sugar selected, the bacteria  compete with
the yeast for available glucose. Usually the bacteria have to wait for the enzymes that the yeasts produce to
break down the sugar into glucose and fructose. As the yeasts commence producing alcohol the bacteria
begin feeding on the alcohol converting it into beneficial acids and nutrients. The bacteria also begin the
cellulose production (mushroom) in an attempt to seal off  "its" food supply.

As you'll see in the charts below, too much alcohol will destroy the bacteria. And too much carbon dioxide
(also produced by the yeasts) will suffocate the bacteria who require oxygen.

In a commercial venture these two steps can be done completely separate. Our Home-Brew however is
really a dance, as one partner moves, the other partner glides. You are the conductor of the great
symphony that the yeasts and bacteria dance to thus creating a mystical elixir and a magical moment.

All research indicates that specific strains of Acetobacteraceae are responsible for both the acetic acid and
the gluconic acid that create Kombucha Tea.  

A few members of the family Acetobacteraceae are cellulose-producers, while only six members fix
nitrogen, a component found in tea as well as other herbs. (see herbs for fermenting kombucha)  One strain
isolated in Kombucha Tea has been identified as Gluconacetobacter kombuchae sp. nov. (RG3T (=LMG
23726T=MTCC 6913T). RG3T does both as well as produce cellulose (kombucha mushroom).  According to
Michael Roussin the mushroom contains the “DNA” of the ferment, the instructions and expectations to be
followed to maintain the strain. Although this seems far-fetched we’ll present some research below that
indicate that certain behaviors are developed specific to this life-force known as Kombucha Tea. The state of
health of the bacteria and thus the health of the Kombucha ferment is reflected in the size and texture of
the mushroom. 

One strain of bacteria Lactobacillus camelliae sp. nov. (lactic acid bacteria isolated from fermented tea
leaves) is heterofermentative: produces 50% lactic acid and considerable amounts of ethanol, acetic acid
and carbon dioxide but not gluconic acid.

The yeasts in Kombucha Tea secrete an enzyme which cleave the sugar into glucose and fructose and the
yeast then produce ethanol and carbon dioxide. The  acetic acid bacteria create alcohol dehydrogenase and
aldehyde dehydrogenase. If the bounds of aldehyde dehydrogenase are over stepped we get some pretty
foul smelling Kombucha Tea. If everything is as it should be we obtain acetic acid, gluconic acid, some
fructose and a very tasting, pleasant healthy elixir – the Tea of Immortality as the Chinese referred to it.

Part II  Temperatures and the Production of Acetic & Gluconic Acid.

Acetic acid bacteria are classified into two genera,

Acetobacter and Gluconobacte.

1. Acetobacter prefers to oxidize ethanol more strongly than glucose 

2. Gluconobacter prefers glucose (sugar) more than ethanol (alcohol),

Among the Acetobacter species, Acet. aceti, Acet. pasteurianus, Acet. polyoxogenes and Acet. europaneus
are the most popular strains for making acetic acid as they prefer ethanol over glucose. And if they run out
off ethohol or sugar they do not cannabalise acetic acid later (turing it into carbon dioxide and your vinegar
– or kombucha tea to fizzy water). These  mesophilic strains have an optimum temperature for growth at
about 30 C. [86°F]. Three Acetobacter strains have the ability to produce acetic acid at 38–40 C  [100 F –
104F ] from vinegar mash. Saeki et  al. (1997

Isolation of acetic acid-producing bacteria

Eighty-six samples including 35 from fresh fruits, 24 from flowers, 14 from rotten fruits, six from soils, five
from wine with dregs and two from the wood of fruit trees were used to isolate acetic acid-producing
bacteria. Sixty-nine strains of acetic acid-producing bacteria were obtained, 21 from fresh fruits, 11 from
flowers, 28 from rotten fruits, one from soil, three from wine with dregs and five from the wood of fruit
trees. The abilities of the tested isolates to oxidize ethanol to acetic acid in a medium containing 5% ethanol
are listed in Table 1.

Acetobacter aceti IFO 3283, an excellent bacterium for vinegar production (Saeki 1993), Six of the isolated
strains produced more acetic acid than all the reference strains, Seven strains had abilities to oxidize
ethanol similar to Acet. aceti One strain, isolate I14–2, with the highest acetic acid productivity was twice
that of Acet. aceti IFO 3283. Isolate I14–2 was isolated from spoiled banana collected at Taichung, Taiwan.
[what this shows is that specific strains are developed from the foods they eat, in a sense  you are what you
eat!]

Acetic acid-producing bacteria, isolated from vinegar

used as reference strains in the Charts below.

Acet. aceti IFO 3283, Acet. aceti DSM 2002, Acet. xylinum ATCC 11142, Acet. pasteurianus ATCC 9432,
Acet. pasteurianus ATCC 6438 Acetobacter sp. CCRC 12326,

Effects of initial acetic acid, and ethanol concentrations and temperature, on acetic acid
production

Optimum temperature of acetic acid production was determined by incubating the isolate at various
temperatures and the residual activity of acetic acid production in each sample was compared with the
control sample which was cultured at 30 C. [86F].

Time course of acetic acid production from ethanol by Acetobacter sp. I14–2

In the time course study of acetic acid production from ethanol by isolate I14–2, it was found that acetic
acid accumulation paralleled cell growth (Fig. 1).

Ethanol was consumed steadily until cell growth reached stationary phase after 3·5 d of cultivation,
1
whereupon acetic acid accumulation reached a maximum level of about 50 g l .

It was found that gluconic acid formation occurred during the log phase along with acetic acid accumulation,
and no further production was observed during the stationary phase.

Effect of initial acetic acid concentration on acetic acid production

…. Gluconic acid production by these three strains was inhibited steadily by increasing initial acetic acid
concentration. This indicates that gluconic acid formation is repressed either by acetic acid added initially
and produced during fermentation, or by low pH caused by acetic acid….

Effect of initial ethanol concentration on acetic acid production

…. the amount of acetic acid produced by the isolate increased in proportion to the initial ethanol
concentration, while the amount of gluconic acid produced decreased with the initial ethanol concentration.

The optimum ethanol concentration for acetic acid production was 5% …The production of gluconic acid,…
reached the highest level in a medium without ethanol addition. …. Acetobacter would oxidize glucose to
gluconic acid when ethanol is absent, but would prefer to use ethanol rather than glucose when ethanol is
present.

Effect of temperature on acetic acid production

… over a temperature range from 20 to 37 °C  (68F – 98.6F). with an optimum at 30 C. [86F] (Fig. 4a).
….The effects of temperature on gluconic acid production and cell growth were coincident in trend with
acetic acid formation. In this study, Acet. aceti IFO 3283 and Acetobacter sp. CCRC 12326 did not grow or
produce acetic acid when they were incubated at 35 C. They had an optimum temperature for acetic acid
production at 30 and 25 °C, respectively (86 - 77 F) (Fig. 4b,c). When the temperature was raised, a delay
in ethanol oxidation was observed. The lag period for acetic acid production from ethanol by Acetobacter sp.
I14–2 was prolonged to about 24 h at 35 and 37 C (Fig. 5). However, ethanol was completely exhausted in
3–4 d and acetic acid production reached almost the same maximum level when incubated at both 30 and
35 °C. Although the consumption of ethanol at 37 °C was obviously delayed, isolate I14–2 could still
1
produce 41 g l of acetic acid. The decrease in amount of acetic acid produced might be caused by
ethanol volatilization at a higher temperature.

Acetobacter sp. I14–2 showed a higher acetic acid productivity than either Acet. aceti or Acetobacter sp.,
and could oxidize ethanol to acetic acid at temperatures above 30 °C. [86F]. When incubated as a static
culture, this isolate did not form a pellicle [mushroom] on the liquid surface, but generated aggregates
suspended in the culture. [a typical Mother of Vinegar] Table 2.  [And that's why it’s hard to tell the
difference between say apple cider vinegar (an acetic acid ferment/ Though more often the MOV (mother of
vinegar) will be floating deeper and less gelatinous

Industrial vinegar production by acetic acid bacteria is carried out at about 30 C. [86F], either by traditional
processes or by continuous submerged culture. Most strains used in vinegar production are mesophilic.
These strains could not grow at temperatures above 30 C. [86F] or produce acetic acid when they were
cultured at temperatures higher than 32C  [89.6 F]

….. Acetobacter sp still actively produced acetic acid when it was cultured at 35 C [95F]. and 37C  [98.6 ]
for 3d, and retained about 97 and 68%, respectively, of the activity compared with that at 30 C. [86F]. The
same result was observed for ethanol consumption by Acetobacter sp. the lag time was prolonged when the
temperature was elevated to 35  [95F]. and 37C  [98.6 F] (Fig. 5). The yield of acetic acid from ethanol by
Acetobacter sp. was 95% after incubation at 30 C. [86F] for 3·5 d. In comparison, the usual yield of acetic
acid by acetic acid bacteria ranges from 60 to 98% (Bar et  al. 1987). The yield of acetic acid by Acet.
altoacetigenes reached 92% after 5 d of fermentation (Entani et  al. 1987). Acetobacter aceti and
Gluconobacter oxydans subsp. sphaericus exhibited 80 and 92% yields, respectively (Bar et  al. 1987; Saeki
1993). Acetobacter sp. which could grow well at a wide temperature range of 30 C. [86F] - 37 C  [98.6F],
showed a yield of 95% (Lotong et  al. 1989). The yields of three thermophilic strains, were about 87·5% at
30 C. [86F] (Ohmori et  al. 1980). However, the yield of strain no. 554 was very low at 37 C, and strain S-
30 had lost its ability to produce acetic acid at 35 C.  Acetobacter sp. expressed an outstanding
thermotolerance with a yield of 85 and 82% when it was cultured at 35 and 37°C, respectively, for 6 d. The
yield at 37 °C by Acetobacter sp. I14–2 is comparable with that of Acet. lovaniensis SKU 1108, a
thermophilic bacteria (Saeki et  al. 1997). Acetobacter sp. I14–2 generated acetic acid at a rate of 0·61 and
1 1
0·59 g l h at 30 and 35 °C, respectively., the acetic acid production rate of Acetobacter sp.
I14–2 at 30 C. [86F] was comparable with those of some thermoplilic strains such as Acetobacter sp.
no.550, 554 and S-23 (Ohmori et  al. 1980). In addition, the production rate at 35 or 37 °C was higher than
that of a protoplast fusant strain, no. 116, which possessed a high resistance to acetic acid and ability to
grow at high temperatures (Fukaya et  al. 1989). The reasons for thermostability of thermophilic strains are
still uncertain. Ohmori et  al. (1980) suggested that the increase in tolerance to acetic acid or ethanol might
account for their thermophilic properties. Saeki et  al. (1997) studied the stability of aldehyde
dehydrogenase and alcohol dehydrogenase, but they did not find any significant differences between
thermophilic and mesophilic strains. We considered that tolerance to acetic acid or ethanol did not
contribute to the thermotolerance of Acetobacter sp. I14–2 as there was no remarkable increase in
tolerance to these compounds. Further studies are needed to elucidate the increase in stability of enzymes,
or the change in membrane structure in Acetobacter sp. I14–2, which might account for its thermostability.

Some authors have reported that several thermophilic strains lose their acetic acid resistance and ethanol
oxidation capability in the stationary phase (Ohmori et  al. 1982; Takemura et  al. 1991), but it was not
observed in Acetobacter sp. I14–2. With the advantages of thermotolerance, resistance to ethanol, high
acetic acid productivity, and easy preservation by lyophilization, isolate I14–2 is suitable for vinegar
making.

Changes in major components of tea fungus metabolites during prolonged fermentation.

Chen C, Liu BY
J Appl Microbiol  Nov 2000 (Vol. 89, Issue 5, Pages 834-9)

Changes in major components and microbes in tea fungus broth (or kombucha; teakwass) prepared from
nine different sources during a prolonged fermentation of up to 60 days were investigated. Cell
concentrations of both yeasts and acetic acid bacteria in broth were generally higher than those in the
cellulosic pellicles. The residual sucrose concentration decreased linearly with time, although the rate fell
after the first month. Metabolic fates of glucose and fructose produced as a result of the hydrolysis of
sucrose were different. Glucose was not produced in parallel with fructose (0.085 g 100 ml(-1) d(-1)) but
was produced with a lower initial rate (0.041 g 100 ml(-1) d(-1)). Both titratable acidity and gluconic acid
increased steadily with time for all samples, although gluconic acid was not generated for 6 days until the
fermentation had begun. Acetic acid increased slowly to a maximum value of 1.1 g 100 ml(-1) after 30
days; thereafter, it decreased gradually. Gluconic acid contributed to the titratable acidity and thus, the
taste of tea fungus broth, during the final stage of fermentation. It is concluded that the desired quality or
composition of kombucha can be obtained through the proper control of fermentation time.

Summary (work in progress August 2007)

Both MOV and kombucha use Acetobacteraccae (bacteria) and yeast to ferment. Not all Acetobacteraccae
produce a mushroom (cellulose), but they all produce acetic acid. It is more typical for Vinegar to be
produced with one of those non-cellulose producers and therefore MOV is less formed (aggregates
suspended in the culture)  more ball like thing floating submerged. Commercial production is after the
acetic acid, not the gluconic acid or cellulose which detract from acetic acid production. Normal vinegar 
production is 5% or higher acetic acid, while kombucha ferments rarely goes above 1% acetic acid, maybe
a rare 3%, but then declines over longer time. It may be the sugars are different (ACV = apples = fructose,
kombucha = glucose) where kombucha with fructose produces almost all acetic acid and little, if any
gluconic acid (Guenther Franks “Kombucha its Correct Preparation and Use…).

More and more commercial vinegars are not fermented as in days of old, (Four Thieves, Balsamic, Braggs
Apple Cider Vinegar) and rely only on the second stage (bacteria) feeding on added alcohol (plus
flavors ...). The studies (as above)  are for the the maximum acetic acid potential. However, our interests
are in the full fermentation. The yeasts play a significant role - even though they are not absolutely
mandatory (one could use watered down gin). However the studies are important to the  of zymurgy 
kombucha tea. From these studies we better understand the Time and Temperatures and Influences of our
home  Kombucha Tea ferment our SCOBY.

Within the family Acetobacteraccae, of the select few who produce cellulose only six members fix nitrogen,
a component found in tea (camellia sinnesis). Thus you have the makings of kombucha: a cellulose paddy
(mushroom), tea (not apples),  acetic acid, and gluconic acid and fructose - Kombucha Tea.
Wine brewers want high alcohol content.Vinegar brewers want high acetic acid content
Kombucha brewers want high Gluconic acid, decent acetic acid and as complete reduction of alcohol as
possible.
Hopefully this presentation will help home brewers chart their own course.

Table 1 Acetic acid production from ethanol by isolated strains + ref strains

Fig. 1 Time courses of acetic acid and gluconic acid production in relation to cell growth of Acetobacter sp.
I14–2. Seed culture (1%) was inoculated into a main culture containing 5% (v/v) ethanol and 2 g l1 acetic
acid, and cultivation was conducted with shaking at 50 rev min1 and 30C (86F) The amounts of ethanol,
acetic acid and gluconic acid of the fermentation broth were determined as described in the text. Ethanol
(■); acetic acid (&U25CF;); gluconic acid (○); O.D.600 (t)
Fig. 2 Effect of initial acetic acid concentration on acetic acid production. Various concentrations of acetic
acid and 5% (v/v) ethanol were added to the main culture medium before the test strain was inoculated.
Ethanol, acetic acid, gluconic acid and cell growth were determined after Acetobacter sp. I14–2 (a), Acet.
aceti IFO 3283 (b), or Acetobacter sp. CCRC 12326 (c) was inoculated into the main culture medium and
incubated at 30C (86F) with shaking at 50 rev min1 for 3 d. Residual ethanol (■); acetic acid produced
(&U25CF;); gluconic acid (○); O.D.600 (t)
Fig. 3 Effect of initial ethanol concentration on acetic acid production. Main culture media supplemented
with 2 g l1 acetic acid and various amounts of ethanol were inoculated with Acetobacter sp. I14–2 (a), Acet.
aceti IFO 3283 (b), or Acetobacter sp. CCRC 12326 (c) and incubated at 30C (86F)with shaking at 50 rev
min1 for 3 d. Residual ethanol (■); acetic acid produced (&U25CF;); gluconic acid (○); O.D.600 (t)
Fig. 4 Effect of temperature on acetic acid production. Main culture media supplemented with 2 g l1 acetic acid and 5% (v/v) ethanol
were inoculated with Acetobacter sp. I14–2 (a), Acet. aceti IFO 3283 (b), or Acetobacter sp. CCRC 12326 (c) and incubated at various
temperatures with shaking at 50 rev min1 for 3d. Residual ethanol (■); acetic acid produced (&U25CF;); gluconic acid (○); O.D.600 (t)
Fig. 5 Time course of acetic acid production from ethanol by Acetobacter sp. I14–2
at various temperatures: 30C  (86F)  (&U25CF;); 35C (95F)   (□); 37C (R) (98F)
from   Journal of Applied Microbiology 86 (1), 55–62.