The Bioremediation Discussion Group is the forum for all bioremediation related problems.
found the following interesting discussion:
question
I have a question relating to sulfide resistance mechanisms of sulfate reducing bacteria (SRB). Specifically I am interested in knowing what makes
SRB tolerate high concentrations of sulfide. Any references or pointers would be really appreciated.
Sridhar Viamajala
Research Associate
Washington State University
replies
Bruce Rittmann - John Evans Professor of Environmental Engineering - Northwestern University
While some bacteria might have specialized sulfide-resistance mechanisms, the widespread and widely used strategy is to make the
sulfide unavailable by precipitating it with ferrous iron or a number of other metals. This is a research theme in my group, and it is an
example of what we call bioprotection. In addition, bioprotection works to protect the community against metal toxicity.
So, it is a two-way street.
Valerie Anne Edwards - President - Alken-Murray Corporation - http://www.alken-murray.com
I tackle this problem differently, by introducing selected bacteria with
sulfide oxidizing capabilities. Specifically, I prefer Thiobacillus
denitrificans and Paracoccus pantotrophus, which can utilize sodium nitrate
as nitrogen source and as alternate electron acceptor, creating sulfate or
organic sulfur instead of sulfuric acid, which is the end result when some
other strains of Thiobacillus. We also include sodium nitrate and
dolomitic limestone to further buffer pH in the system. When organic carbon
sources are limited, these strains can use carbon dioxide as their carbon
source.
In an oxygenated system, Starkeya novella can oxidize sulfide and
mercaptans. All of these strains can use carbon dioxide as their carbon
source when introduced to a system lacking organic carbon sources, but
Thiobacillus denitrificans is unable to use organic carbon sources at all,
a totally chemotrophic species.
I also use three strains of a new species of Bacillus I isolated from a
Georgia humified soil deposit, with cationic polymer activity which
inhibits all gram-negative strains, thus creating an environment conducive
to development of unique Bacillus. My new species of Bacillus acts a lot
like the Paracoccus pantotrophus, facultatively heterotrophic, utilizing a
nice array of organic carbon compounds or alternatively utilizing carbon
dioxide while oxidizing hydrogen sulfide and denitrifying any leftover
sodium nitrate, creating sodium sulfate and nitrogen gas. Dr. Frederick
Cohan of Wesleyan University did the DNA hybridization work for these
strains to prove that they are NOT Bacillus mojavensis, as initially
identified by Accugenix using 16S rRNA. According to Dr.Cohan, they are
actually closer to B.subtilis spizizinii and B.valismortis than to
B.mojavensis. I have been using all of these strains commercially since
March of 2003, with great success.
Another treatment option for sulfide oxidizing strains that can denitrify
and use carbon dioxide as their carbon source is the treatment of WWT
effluent with excess nitrate and sulfide and no remaining organic carbon.
In the past, an organic carbon source, such as methanol or acetic acid
would be added and an attempt at creating an anaerobic environment would
have been necessary to force Bacillus or Pseudomonas aeruginosa to
denitrify. T.denitrificans, Paracoccus pantotrophus and my new Bacillus
will all denitrify even when oxygen is present as another option, avoiding
the addition of another pollutant in order to eliminate one that is present.
Hello Valerie-Anne Edwards,
My name is Casey Hubert and I'm a PhD student at the University of
Calgary. I recently read your BioGroup posting about sulfide tolerance
where you took more or a sulfide oxidation angle. I have done some
experiments on sulfide removal with nitrate and molecular analysis of my
experimental system shows some
present. I suspect they are probably heterotrophic nitrate reducers
(organics are also present in the system), and I was intrigued by the
description of your
posting - by calling them facultative heterotrophs, so you mean they are
also capable of oxidizing inorganics (ie sulfide)?. Can you refer me to
any papers in which you have discussed or characterized these
Such information, if it is available, might be useful to me as I try to
understand my own results.
Dear Casey,
Yes, my four sulfide oxidizing Bacillus can grow on two selective media i
used to detect their talents and compare with Thiobacillus denitrificans.
The primary media was a thiosulfate based media designed to support
Thiobacillus denitrificans in a BBL GasPak jar with GasPak Plus envelope
to assure no presence of free oxygen and plenty of carbon dioxide. This
was my primary selection media used to screen soil isolates fairly early.
Those that showed good strong growth on that media and also on TSA (not
on Nutrient since they absolutely require 0.5% NaCl, tolerate up to 10%
NaCl), i continued to test their talents and enzymes produced. Since I
wanted sulfide oxidation, and not necessarily thiosulfate oxidation, I
created another selective media, this time with sodium sulfide and sodium
or potassium nitrate in it under the same anaerobic conditions and they
jumped up and said "hey look at me - I love this stuff better than the
thiosulfate media". My final media used acetic acid to adjust pH instead
of hydrochloric acid to see if a tiny bit of an alternate carbon source
would accelerate them and two of the strains that test positive on acetic
acid media with no other carbon source were even more thrilled to go
after the sulfide. The media was so stinky it had to be poured in a fume
hood and it was immediately evident which strains worked by odor
dissipating in two days and color change from green to blue, actually
increased because of use of nitrate instead of acid produced in the
thiosulfate media without nitrate present. They are also capable of a
nice assortment of carbon digestion, as long as at least 0.5% NaCl is
present. Another strain from the same soil showed unusual talents with
nitrogen compounds, first degrading citrate to ammonia changing green to
blue and then 12 hours later back to green, which one of the sulfide
babies did also. I then tried them both in a media used to verify our
ammonia oxidizing nitrifiers are working. See the procedure at
http://www.alken-murray.com/QC2.pdf for details. I ended up
discovering that only two of the strains, the new genus between Kurthia
and Bacillus (5.6% or more from each) and one of the sulfide babies
showed positive results on this media, taking around a week to change the
color to a very pale yellow-orange. I have Chemetrics kits that will
reveal presence of nitrite and nitrate, so i made the STP media without
any color to interfere and used it to check whether nitrite or nitrate
were created and nitrite was plentiful, some ammonia still remained and
no nitrate was created by either strain. I also like the two Paracoccus
pantotrophus I use for comparable behavior in a gram-negative with the
advantage of slime production by the gram-negatives to protect them from
toxic chemicals in the environment and also protect their friends in the
same consortia. The Paracoccus, Starkeya novella and Thiobacillus
denitrificans work well in consortia with my Bacillus babies. Only the
Bacillus can be used in a liquid formula containing 15 to 30% sodium
nitrate, non-toxic micro-nutrients that accelerate performance and
germination of spores once the preservative is diluted enough to allow
them to awaken and go to work, enhanced by a chemical formula to make
them work even better. In liquid form, i usually mix in some FOG
degraders since H2S is often present in collection systems that have a
high buildup of FOG and odors from fatty acids, thus fatty acid degraders
also included. Nitrate alone cannot accomplish sulfide remediation, it
only catalyzes the result if the right bugs are present to be able to use
it. By combining both, I can be absolutely sure of my end result. My
only problem with the sulfide Bacillus has been commercializing them.
They do not grow really well in high numbers unless sodium sulfide and
potassium nitrate and 0.5% NaCl are added to the media. They will not
grow well in typical media used for large scale fermentations. Once we
got over the odor issue and gave them what they wanted, they grew just
like champs with final freeze-dried culture count of 4.2 to 5.85 E11
CFU/g , but without I was lucky to get a result over 1.0 E 10 CFU/g, not
so good for commercial value. My main goal when I began playing with that
specific soil was to find the sulfide oxidizing strains and I was stunned
when nothing I could isolate was gram-negative despite good thiosulfate
growth. Apparently a high-molecular substance in the soil is deadly to
gram-negatives, opening the door for Bacillus to develop new traits.
Accugenix initially identified them as Bacillus mojavensis, so I wrote to
Dr. Cohan of Wesleyan University, who named Bacillus mojavensis and a
number of other Bacillus closely related and we swapped strains. I tested
his and he tested mine and only one of his grew very faintly on the
Thiobacillus media and none on the sulfide-nitrate media, even with a
tiny bit of acetic acid added as an accelerant. Only two of my sulfide
strains can use acetic acid at all and those two also use lactic acid as
sole food source. Dr. Cohan sent me a program and bootstrap showing that
they are actually further away from B. mojavensis and closer to
B.subtilis, spizizinii and B. valismortis, which I found fascinating. Dr.
Cohan conjectures that bacteria pick up a trait and then the DNA changes
to mirror that change. Nobody knows what makes B. mojavensis different
except DNA, so perhaps it has some hidden talent as my babies would have
if I had not been on a mission to find sulfide oxidizing strains and they
turned out to be a new species of Bacillus, the highlight of my work with
bugs. Having screened for salt tolerance for over four years now, I have
assembled an interesting group of strains that i can formulate with, as
needed and I plan to patent 17 of them in a specific formula to solve
something I have been advised not to discuss prior to applying for the
patent. I sent that group of strains to ARS culture collection today,
liking their pricing for patent deposit better than ATCC.
Louis B. Fournier, Ph.D. - STAR Environmental, Inc.
I'm not sure I recall what the exact question is/was. However, suffice it to say that if sulfide in a waste stream is being treated, it might be easier to simply add an appropriate concentration of hydrogen peroxide. Depending upon pH, peroxide reacts with sulfide to form either sulfate ion or colloidial sulfur. This is commonly used to treat sewage effluents in order to eliminate sulfidic odors.
Fred Heyrich - Bio-SURGE, Inc./Neotech.
I use a quadra-peroxide and a non-ionic surfactant to effect conversion to
sulfate from sulfide. Very prompt. I would be willing to send to you
samples for a lab-scale or pilot study. If intersted please contact me by
reply. The technique is rather basic but depends on a quadra-peroxide (300 ppm diluting
an 18% basic solution. All dilutions are rated non-haz.
