IMEP #128 - Sulfur and Iron Bacteria Linked to Seafood Death

[BlueChip]

IMEP #128 - Sulfur and Iron Bacteria Linked to Seafood Death
Sulfur and Iron Bacteria Linked to Plant, Crab and Shellfish Kills
"Understanding Science Through History"
Iron and Sulfur Bacteria Key to Marine Soil Chemistry and Acid Conditions for Shellfish
The Anoxic and Oxic Zones as Possible Habitat Indicators for the Blue Crab
Early Shellfish and Soil Researchers Mention Bacterial Processes With or Without Oxygen
Viewpoint of Tim Visel – no other agency or organization
Tim Visel retired from The Sound School June 30, 2022

Along the waterfront is found the presence of steel sheet piles – they are connected by a long continuous couple or ball and socket joint.  They are commonly used in jetty and bulkhead construction on all our coasts.  They are long lasting – many times longer than wood.  Usually, the portion exposed to air (oxygen) has a red to brown coloration – rust.  The lower portion, especially below the low tideline, is often black.  These metal piles are subject to oxidation, which creates a weaker compound – an iron oxide when wet.  We know this weak flaky compound as the red-brown powder "rust" that forms on steel.  Since oxygen is available and active in moisture, we protect it with coatings to prevent oxygen from rusting iron.  This compound is noted as FeO.  The presence of water accelerated oxidation resulting in Fe2O3, iron oxide. (In chemistry notations Iron Fe (II) in oxygen and Fe(III) in oxygen-poor conditions).
In low-oxygen environments, iron (steel) is dissolved by sulfur acids.  In this case, iron (Fe) bonds with sulfur (S) to create a monosulfide or iron sulfide, FeS.  This metal sulfide is black.  Some people have seen green clays or green colors in eggs.  These are signs of high levels of iron sulfide in the presence of hydrogen sulfide (iron skillet and egg white) (yes, there can really be green eggs).

Some of the early examinations of Long Island Sound by Columbia University Grim et al., 1970, Geophysics, Vol. 81, pg. 649-666, March (1970), "Sub-bottom Studies of Long Island Sound" mentions this green coloration on page 658:
"On the basis of more than 200 cores (up to 6 feet in length) – describe the upper 6 ft. of the bottom sediments as a greenish-gray sandy to clayey.  Holocene silt, covered in most places by a few inches of black, gelatinous organic mud." 

The green color is attributed to the chemical reactions of iron.  Iron, sulfur compounds and organic matter share one attribute, they can all be "food" for bacteria.  Many people might remember the days of skin punctures of a rusty nail.  Such a puncture could often result in a quick trip to the doctor for a tetanus shot (personal experience noted).  What I don't recall was the why – rust could be the source of the bacteria Clostridium tetani or "lock jaw."  The truth here was the bacteria is found in soil, but is more prevalent in horse manure and I had a Welsh pony at the time (4-H Club).

But iron-oxidizing bacteria have also evolved to live off iron compounds, deriving energy from it chemically.  This is the reddish-brown slime that discolors streams and brooks – one once signaled the location of clumps of "bog iron" when iron clumps were smelted to form metal.
Many forms of iron oxide exist, and early foundries (called a bloomery) used limonite ferric hydroxide (FeHO2) to make iron.  Madison, CT had a bloomery and George R. Lange, III in a Wesleyan University 1998 Master of Arts research project (Hiking Trails of Madison Connecticut), describes this process on page 12:
"The Bog Iron Works Trail leads to the location of a small bloomery (iron processing plant), which produced bar iron from local bog ore in the late 17th century.  A charcoal-fired forge reached high temperature – and after being would bog iron ore was fed directly into the fire.  Small lumps of bog iron ore were reduced to a malleable iron.  Bog iron ore, which is a deposit of ferric hydroxide or limonite found in the bottoms of swamps or ponds, was used.  The deposits are formed during the process of decay by iron-fixing bacteria."

Location of iron clumps was frequently marked by an orange-red ooze at the peat (bog) surface.  This iron fixing occurs in a low-oxygen environment and the iron exposed to oxygen "rusts" and changes the color of the discharge.
Waters high in iron can stain household water fixtures brown (sinks, tubs, etc.).  These bacteria use the iron compounds for energy, allowing them to consume organic matter when oxygen is limited.

Other bacteria use sulfur compounds as an oxygen source when elemental oxygen is limited.  These are the sulfate-reducing bacteria when oxygen is low or absent, they break sulfate (SO4), a sulfur atom surrounded by four oxygen atoms, releasing hydrogen sulfide as a byproduct (H2S).  Waters high in sulfur can stain household water fixtures yellow, the color of sulfur.  They often have a "rotten egg" smell. 
The oxidation or reduction of iron and sulfur have dramatic consequences for marine soil chemistry.

It might be easier to think that the oxygen-sufficient soils are brown – noting that iron is "rusting" – oxygen-poor soils are often grey or black from the waste products of sulfate-reducing bacteria, hydrogen sulfide (H2S).  Sulfate-reducing bacteria obtain oxygen from the sulfate compounds dissolved in seawater.  Sulfate (SO4) is abundant in seawater.

These two elements are not "limiting" in our water – iron – from weathering of iron containing rock and sulfur from the seawater compound sulfate.  What is limiting in coastal water is elemental oxygen.  This has a temperature limitation; cold water can hold much more elemental oxygen but is without the plankton oxygen pump.  In warm water, plankton provides oxygen as a waste product from using carbon dioxide CO2.   In many areas, oxygen can undergo wide swings in respect to elemental oxygen.  For example, massive fish kills from oxygen deficits are rare in the colder months.  This is not the case during the summer, especially in shallow waters subject to the most daytime heating.  In our area, this is often the third week of August.  It is the coves and bays that are most susceptible to these wide swings in temperature and the bacteria that live in them.

One of these areas that could be investigated is the formation of jarosite, which is formed by the aerobic iron-oxidizing bacterium Thiobacillus ferrooxidans, which is frequently found in acid sulfate soils (See Appendix #4).  This compound, an iron mineral, has an unusual feature, it has a yellow color and was once an ancient paint.

Our shallow bay bottoms contain many strains of bacteria that alter soil chemistry.  One of the reasons that bay bottoms don't appear yellow when anaerobic (low or no oxygen) sulfate-reducing bacteria (Desulfovibrio) quickly reduce jarosite to FeS.
One possible link to habitat locations could be the blue crab.  Those that overwinter in oxic brackish habitats appear to have a brown stain (called rusty crabs) while those that winter (inhabit) estuarine waters have a pronounced yellow stain around the mouth (called yellow face) and those that have recently shed or live in saltwater have no stain and are termed "white belly crabs."

When a large migration of yellow face crabs moved east in 2012 (Report #9, The Search for Megalops, August 30, 2012, New England Crabbing Resources and Report #10, July 20, 2011) (See Appendix #9: CT Blue Crab Special Report, posted August 23, 2012, The Blue Crab ForumTM), crabbers noticed the sudden arrival of yellow face crabs.

The University of Connecticut Wracklines, Vol. 11, No. 1, Spring/Summer 2011, had a picture of a yellow face blue crab on its cover taken by Steve Joseph.  We really don't know the exact cause of blue crab staining (newly shed crabs have brilliant white and blue colors) except that rusty and yellow face crabs are usually hard shell crabs that have missed a shed or stopped shedding.  If they do not shed, chances of staining increase.  When large numbers of yellow face crabs appeared in Connecticut west to east, I wondered what could cause these stains.  In time, I have learned that changes in soil chemistry is the area linked to "winter kill."  This brings up the issue of studying our marine soils – it is only recently have they been considered a soil and the sulfur/iron chemistry of them as important or significant.

This lack of research in the impact of pore soil chemistry, especially with increased sewage solids (organic matter from nature and storm water) and increasing water temperatures, would reveal the transition of oxygen-requiring bacterial processes to favor those sulfate bacteria strains.  A deep marine compost would now allow more hydrogen sulfide to form.  Additional H2S hydrogen sulfide (the rotten egg smell of sulfide often mentioned just before a fish kill) increases the chance of sulfuric acid formation and a lower marine soil pH.  As habitats closer to land obtain more organic matter from bays and coves, these same areas show greater acidity.  Oak leaves, for example, have a pH of 4.7 and storm water has little opportunity for terrestrial buffering (acid rain) can have a pH of 5.0 or less.  Sulfur has a direct role in making rain water more acidic.  Acid soils have been linked to anaerobic sulfate bacteria and digestion of organic matter by bacteria in low oxygen conditions.  Acidic soils kill shellfish veligers (spat) in just a few seconds.  Those bays and coves that obtain large amounts of organic matter in hot low oxygen conditions can produce hydrogen sulfide kills of shellfish.  In a recent study of Wellfleet Harbor, Massachusetts, researchers found accumulations of "black mayonnaise" that were over 10 feet in depth.

These deposits, at times, were measured to contain organic composition greater than 21% (See Black Mayonnaise in Wellfleet Harbor: What Is It and Where Does It Come From?, Mittemayr et al., 2020, The Center for Coastal Studies, Providence, Massachusetts).  From page 13 is found this section – my comments (   ) Tim Visel:
"A first glance at the measure sulfur isotope signatures suggests sedimentary sources, including pyrite (iron disulfide FeS2, T. Visel) and sapropel.  Pyrite is generally considered to be the end product of sulfur diagenesis (think chemical composting, T. Visel) in anoxic marine sediment and sapropel describes anaerobic sediments that are rich in organic matter."

Anaerobic digesters have been suggested for bacteria composting of organic matter by methanogens', a bacterial like organism that produces CH4 methane.  The issue is that sulfur-reducing bacteria also exist and contaminate biogas with H2S hydrogen sulfide.  This is a concern because of the formation of sulfuric acids that damage metal pipes.  Research is underway to reduce AD biogas with the use of iron oxide.  Wastewater treatment facilities employ hydrogen gas sensors to reduce sulfuric acid damage to sewer (metal) lines (See Appendix #11).  Marine soils high in hydrogen sulfide have been reported to be toxic to submerged aquatic vegetation.  These high sulfide levels have been linked to sulfur bacteria.  The presence of sulfur-reducing bacteria in low-oxygen tanks has been studied for over a century.  This is shown in the development of marine sewage treatment plant use of living (biological) microbes in composting sewage organic matter.  Organic matter composting organisms in tanks quickly were killed as air supplies and source organic matter was stopped.  This was not always the case.  The forty-seventh Annual Report of the New Jersey State Agricultural Experiment Station and the thirty-ninth Annual Report of the New Jersey Agriculture College, June 30, 1926.

A report from James B. Lackey on page 517 has this comment:
"Tank 4 was shut off for a period of several months at one time.  Its protozoa quickly died out although 6 weeks after shutting off the tank, bacteria of a rod type were so numerous "as to give the impression of looking at a centrifuged mass of them."
And further –
"They may not have been if the proper kinds since it has been demonstrated that certain strains of bacteria apparently serve as food, whereas others are not used.  The continued presence of bacteria, however, and the fact that many of the tank protozoa are known saprozoic forms."
Further studies of sealed jars noticed an increase of H2S hydrogen sulfide gas (pg. 518).  Descriptions of sulfate-reducing bacteria include "gram negative and rod-like" and includes that of Desulfovibrio vulgaris and Vibrio vulnificus, a known human pathogen.

Iron in our marine soils considered not limiting.  In a 1978 study, Welsh et al. Estuarine Interactions Academic Press, Pg. 381-401, "The Effects of Reduced Wetlands and Storage Basins on the Stability of a Small Connecticut Estuary," Pg. 391-392, contains this segment pertaining to Alewife Cove in eastern Connecticut between the town of Waterford and the city of New London – my comments in (   ) Tim Visel:
"In addition to the salinity gradients, prevailing pH values in the pore waters (water in the voids between particles of aquatic sediments - soil, T. Visel) were extremely low for marine sediments (Bass, Becking et al., 1960).  During the summer months, a number of measurements dropped below the limits of the carbonate system (the ability to obtain buffering cation ions, T. Visel) pH 4.0 and may have resulted from the leaching or organic acids or iron from the large amount of allochthonous (from far away) material present (twigs and oak leaf fragments).  Dissolved materials in the surface water and the ground water intrusions could also contribute to low pH.  Iron commonly exceeds .3 ppm in local streams ... The decaying vegetation in the newly formed swamp upstream should certainly constitute a local source."

This low pH was thought to create lethal conditions for anything living in the upper 10 cm of sediment.  Welsh et al. comments pertaining to toxic pH conditions on page 393:
"At the lower pH values, even the microbial community would be limited to such forms as thiol bacteria and iron bacteria."
It is the abundance of iron that colors the organic compost of near shore areas.  This mineral is found on Connecticut's beaches and is noted as Fe3O4, iron oxide, and contains iron II and iron III.  It is black and easily collected with a magnet.  It is sometimes called iron sand.  In 1944, Charles Rufus Horte in Connecticut's Iron and Copper Part I 60th Annual Report of the Connecticut Society of Civil Engineers, 1944, mentions iron sand on page 132:
"About 1760 Jared Elliot of Killingworth, believing that since this material was picked up by a magnet, it must contain iron, began a series of experiments and eventually produced from 83 pounds of the sand 50 pounds, of as he said "very good if not the best iron." [He submitted a sample to the London Society for the Encouragement of Arts, Manufacture and Commerce for which he received a gold medal, the same year he would publish the first book on Improving Agriculture Practices, which included the use of bivalve shell and marine mud as a compost.]

I suspect the source of his iron sand was the beaches in Madison, which after strong storms, contain deep layers of garnet (red) and magnetite grey sands.  The large amount of this mineral is even mentioned as imparting local oysters their excellent taste.
In the 1887 US Fish Commission Bulletin, George Goode, editor Ernest Ingersoll quotes a letter from Elihu Kelsey of Overshores Madison, Pg. 222, the following (bed was near a small island called Overshores):
"We find the character of the soil to be of the greatest importance.  On our producing bed, the mineral ingredient of the soil is iron.  This renders the oysters healthy and of the finest flavor, so that our customers say they cannot be excelled."

The formation of hydrogen sulfide H2S can lead to sulfuric acids H2SO4 in low-oxygen marine soils.  These soils become so acidic, kill shellfish veliger's soon after contact.  It is thought that storms have a soil renewal process – adding oxygen and removing sulfides often termed "new sand" in the fisheries literature. 
These great clam sets often happen after coastal storms, washing shallow soils.  It was David Belding in his research on Massachusetts shellfish that introduced the concept of soil aging or soil succession.  He suggested that soils high in sulfide (brown to black) could be cultivated to improve soil conditions (deep ploughing).  In his report on the soft clam fishery (1930), he mentions the danger of soils increasing in organic matter content.  From Belding, The Soft-Shell Clam Fishery of Massachusetts:

"Organic material – Clams are usually absent from soils containing an abundance of organic material.  Organic acids corrode their shells and interfere with the shell forming of the mantle.  Such a soil indicates a lack of water circulation within the soil itself as indicated by the foul odor of the lower layers of soil, the presence of hydrogen sulfide."

In his study of the hard clam, he mentions soil importance under the "Methods of Operating a Quahog Farm."  From Belding (1912):
"(a) soil (2) Soils in which organic acids caused by the decay of plant life, are present, prove unsatisfactory for any catching of seed, interfere to a slight extent with the growth by destroying the shell.  The nature of the soil in some indirect way determines the appearance, the composition and the weight of the shell, as observations from various soils in nearby locations indicate."  (For an explanation of a soil CEC (cation exchange capacity), see IMEP #119: Marine Soils of the 1950's to 1980's, March, 2023.)

One of the signals of sulfide enrichment is the die off or dieback of submerged vegetation, especially eelgrass.  The open pore-increased soil circulation has been noticed by eelgrass researchers, such as Clarence Cottam and C. E. Addy in a paper presented at "The North American Wildlife Conference Transactions, Vol. 2 (1947)."  They raise an important issue – the nature of the soil.
Following is a segment from the Cottam and Addy's paper titled "Present Eelgrass Conditions and Problems on the Atlantic Coast of North America," commenting on the construction of the Cape Cod canal:

"This dike was built with sand pumped from the bottom of Buzzards Bay: work on this dike was beginning June 1936 and finished February 1937.  The dike thus consists of "new sand" that is sand which hasn't been in shallow water for many years, probably in historic times.  Yet on both the north and south sides of this dike are healthy stands of eelgrass, standing which have among the best when I saw during the entire summer Stevens, 1946."

This reference adds to the concept of rinsing soils of organic matter and reintroducing oxygen into them.  This has been mentioned as part of the shellfish habitat observations for over a century.  This is facilitated by strong coastal storms or man-induced cultivation (See Appendix #2, #3).  It is thought that the direct reduction of sulfide H2S, a respiratory disruptor of iron's ability to transport oxygen (this is the source of "iron poor blood" for us as hemoglobin red blood cells is now the circulatory system moves oxygen and accounts for its color red), is why oysters and clams stop feeding when high amounts of sulfide are present.  The shellfish industry has reported these thin or watery meats for over a century beginning in the 1900's.  Dr. Galtsoff in a 1947 study of the York River (Ecological and Physiological Studies of the Effect of Sulfate Pulp Mill Waste on Oysters in the York River Virginia, and A. E. Hopkins and Paul Galtsoff, The Effect of Sulphite Waste Liquor on the Oyster, 1931) proved a direct relationship to the presence of sulfide to oysters ceasing to filter water for food.  If long enough, the (presence of sulfide) oysters simply starved to death. 

Maine soft shell clammers have reported strong sulfide smells near or in clam graveyards.  One of the signals of a high sulfide event is that oyster meats appear thin and watery and seed shows no growth.  Clams are observed to "pop out" from the soil.  Other signs of sulfide to clams is slow growth and thin shells linked to CEC (also termed sulfide starvation – See IMEP #75: The Chemistry and Cultivation of Clam Soils, posted January 9, 2020, The Blue Crab ForumTM, Fishing, Eeling and Oystering thread).
(NOTE: In southern areas, a sulfide event happens in low oxygen as fish attempt to flee to more shallow water.  Easy catching of fish and crabs is described as a "jubilee.")

In a 1931 report by H.C. McMillen – Investigations of Oyster Mortality in Oakland Bay Washington – investigates thin watery oyster meats, no oyster growth and weakened clams.  These conditions were reported by oyster growers in 1926.  From page 168, Bulletin of the Bureau of Fisheries, is found this segment:
"Within the last three years, conditions have changed radically.  At the request of the oystermen, a preliminary survey of the beds was made in May 1929, which brought to light a few very definite facts.  Many of the oysters were dying, and a majority on the lower beds were already dead.  In the channels and on the un-diked ground, careful search revealed the presence of a few medium-sized oysters which were in the early stages of decomposition.  Clams were working out of the ground, and they were in such a weakened condition that one could pull the shell open with the fingers to examine the watery decomposing body within which still showed signs of life.  In the dikes of medium height, a few large oysters were alive, but they showed no signs of recent growth which could be expected.  When these oysters were shucked, they soon lost water from the body, and there remained a thin flabby piece of meat which had a decidedly bitter taste."

McMillen also reports on the mysterious die off of barnacles, mussels and hydroids at the same time.  This is the often mentioned "sulfide deadine" when excess organic matter (similar to wood paper pulp) is consumed by sulfur bacteria in low-oxygen conditions.  From McMillen pages 174-175 is found this section (part of the Hopkins Galtsoff 1931 study):
"Boats and scows used on the oyster beds have been cleaned and repainted at least once a year but in the last three years no growths have appeared, and the work of reconditioning has been found unnecessary.  Since these pieces of apparatus do not leave the oyster body, these facts indicate that abnormal conditions exist in the water over the oyster beds."

Oyster growers in Connecticut had experienced a suspected bacterial-sulfide oyster kill in 1890.  Oysters were killed, shells still "paired with rotting meats and horrible stench" (See IMEP #112, Parts 1 & 2, posted June 8, 2022, The Blue Crab ForumTM, Eeling, Oystering and Fishing thread).
The presence of hydrogen sulfide appeared at two times after a long cold winter and in heat with a pronounced thermocline (the bottom waters become stagnant).  Thurlow Nelson describes a sudden bottom kill in 1925.
From the New Jersey Agriculture Experiment Station Report of the Department of Biology, Thurlow C. Nelson, pg. 110, is the following:
   "The Death of the 1925 Oyster Set"

"Since the first time from the beginning, our work here in 1921, the oyster growers cooperated with us on a large scale in the planting of shells.  In every case, the shells were put out just prior to the time of expected set, the bulk of the shells being overboard by June 13.  As a result of this fortunate timing of shelling together with the very large sets which struck June 15 and 21 a heavy catch of spat without doubt the most extensive and valuable set which has occurred since 1921 was secured."

"Examination of commercial shells showed, however, that the great majority of spat failed to grow following attachment, the shells becoming snowy white in contrast to the deep reddish-purple color of the normal healthy spat.  Further examination proved that most of the spat had died within a few hours of attachment without making any new shell growth.  Samples of shells dredged up through July and August showed not over two or three living spat which had attached."

I suspect the high heat (reported also in the report) created a condition in which H2S hydrogen sulfide and sulfuric acid had killed these oysters.
Virginia Lee of the University of Rhode Island (1980) mentions the impact of hydrogen sulfide upon oysters in a salt pond.  Lee – "An Elusive Compromise Rhode Island Coastal Ponds and Their People" – contains this segment:

"After oxygen levels are depleted, toxic hydrogen sulfide is often produced, causing the rotten egg smell that lurks over mud flats and back coves.  For instance, in the summer of 1978, Fosters Cove in Charlestown Pond went anoxic for so long that the oyster spat growing on the aquaculture rafts died."
It was John Hammond on Cape Cod who pointed out this sulfide deadline, something that I noticed but did not connect to iron or sulfur bacteria, the growth of fouling organisms on mooring chain.  When organic matter is consumed by sulfate-reducing bacteria, they create hydrogen sulfide as a waste product.  In time of low oxygen (high summer temperatures) or in winter when the algae "oxygen pump" process is at a minimum, sulfide leaves the bottom and can be diffused into the water.  In many places, this is called black water as it interacts with iron, giving it the black color.  Sulfide is highly toxic and kills marine life as it leaves the bottom.  John Hammond pointed out that on moorings in salt ponds.  The first few feet will contain mussels, hydroids and barnacles but at a certain depth, this growth may stop, leaving a black chain.  This is the level that sulfide reached.  He also detailed finding dead fish in ice on salt ponds, fish were killed and floated and became part of the ice.  Massachusetts still puts out a notice to ice fishers – this is from "If You Find a Fish Kill" fact sheet, Division of Fisheries and Wildlife:

"Ice anglers may encounter signs of a low oxygen environment when they drill through the ice and notice the smell of rotten egg or observe sluggish or dying shiners.  The odor is hydrogen sulfide gas, which is a natural byproduct of low dissolved oxygen environments,  and is not likely the result of pollution.  Oxygen levels will return to normal shortly after the ice melts in the spring." 
One of the best descriptions of high temperature and low dissolved oxygen generation of sulfide I have found is in a 1983 Environmental Review Team Report on Frash Pond Stratford, Connecticut 1983, Kings Mark Environmental Review Team, Warren, CT 06754 (which can be found online at https://ctert.org/pdfs/Stratford_FrashPond_112.pdf).

Frash Pond, a relatively deep kettle pond similar to Cape Cod kettle, lies north of Great Meadows, a large salt marsh system and west of the Housatonic River.  Historic records indicated it was once a freshwater pond and is labeled as "fresh pond" on historic maps.  By 1886, it was totally connected to tidal estuarine waters in or near the Great Meadow system.  In the 1930's, areas near Frash Pond were ditched for mosquito control and several reconfigured channels during the next 50 years.  In the early 1980's, Frash Pond was associated with hydrogen sulfide odors and the "death of aquatic organisms" (not described).  A request was received from the Stratford Town Manager as the cause of hydrogen sulfide in the pond.  The study concluded that the origin of hydrogen sulfide was two-fold, a lack of elemental oxygen, especially in hot weather, and the bacterial breakdown of organic matter by sulfate metabolism.  Section VI of the report is found in Appendix #5.  For the full report, visit https://ctert.org/pdfs/Stratford_FrashPond_112.pdf.

It is not just the temperature (blue crab, for example, thrives in tropical waters) but the food (organic matter) that feeds sulfur bacteria and bacteria that release nitrogen.  Iron is plentiful and intersects the sulfur and nitrogen bacteria pathways.

We may have been given decades of advance warning on how deadly Vibrio strains in seawater could be.  In 1962, Butler Flowers Frank M. Flower and Sons of Oyster Bay, New York built a shellfish hatchery and by the late 1970's became a significant producer of oyster seed.  In 1980-81, the hatchery noticed a sudden onset of larvae mortality about 10 days after fertilization.  Samples of the culture water and larval organisms were sent to Carolyn Brown of the NOAA facility in Milford (referred to as the Milford Shellfish Lab) Connecticut, who was able to isolate two bacterial strains that resembled Vibrio anguillarum (See Appendix #10).  One strain (1) was not sensitive to penicillin but the second (2) was.  Strain 1 also produced nitrate (oxidizing ammonia) and one strain produced hydrogen sulfate (reducing sulfate).  In 2001, Vibrio splendidus was associated with a summer mortality of juvenile oysters (Crassostrea gigas) in France.  Vibrio disease has been linked to coral bleaching, winter flounder fin rot, lobster shell disease and usually associated with heat and low oxygen conditions.

While terrestrial farmers strive to replace organic matter in oxygen-bacteria soils, marine soils often have too much organic matter and cultivation might be the only way to reduce it – or oxygenate it so oxygen bacteria can live.  In closing, soil cultivation may reduce the chances of H2S hydrogen sulfide forming.
We don't pollute waters with hydrogen sulfide, bacteria does that and has been long associated with hot stagnant or poorly-flushed basins, large and small.  In a 2004 study of The Roles of Anoxia, H2S and Storm Events of Dead End Canals of Delaware Inland Bays Estuaries, Vol. 2, No. 3, pg. 551-560, Luther et al. mentions a bias in looking at the role of bacteria produced hydrogen sulfide - on page 551 is found this segment:
"Most studies concerning anoxia only document the loss or disappearance of O2 and do not document the presence or extent of H2S in the water column even though toxicity of H2S to many organisms is well known."

This frequently happens even though oxygen with H2S can form sulfuric acid, lowering the pH of bottom waters (my research observations, T. Visel).  Earlier research often noted this bacterial impact.  Claude E. ZoBell, in a paper written in 1938 (Scripps Institution of Oceanography New Series #72) titled "Occurrence And Activity of Bacteria In Marine Sediments" reprinted in Recent Marine Sediments by the National Research Council, 1955, pg. 424, has this section:
"Many types of bacteria influence the sulphur cycle in sediments.  It is not unusual to find from a few thousand to a few million bacteria per gram of mud that liberate H2S from sulfur-containing proteins, and those that reduce sulphates to H2S are widely distributed in marine sediments.  The H2S may combine with iron or other substances, it may be oxidized to and deposited as elementary sulfur or it may be oxidized to sulphates."

And comments on the role of bacteria on changing the pH in seawater – and its role in slowing shellfish shell formation – same page 424 – my comments, T. Visel (   ):
"Several bacterial processes may tend to increase the acidity of marine sediments and if these processes are over-balance the base forming reactions, calcium carbonate may be dissolved (shell erosion, T. Visel).  The liberation of phosphates or hydrogen sulfide from proteins, the oxidation of ammonium to nitrites or nitrates and the oxidation of Sulphur or its compounds to sulphuric acid, all contribute to acidity of the environment.  Whether base-forming (ammonia has a pH of 12, T. Visel) or acid-forming (sulphuric acid has a pH of 3.5, T. Visel), microbiological processes predominate depends upon the type and amount of organic matter present, the kinds and number of bacteria, the oxidation/reduction potential and other factors."

It is the kind of bacteria that determines habitat quality.  There is no free bacterial food when it comes to oxygen as some bacteria won't get a chance to consume it.  This is the bacterial war I mentioned in EC #7: Salt Marshes A Climate Change Bacterial Battlefield, posted September 10, 2015, The Blue Crab ForumTM.  It's just we rarely see or experience this bacterial conflict.  For example, we focus upon elemental oxygen O2 as the most critical measure of water quality and most benthic studies include it.  What is rarely mentioned is that active oxygen-requiring bacteria die off, another bacteria is ready to take their place at the dining table.  Nature will fill the void (i.e., empty chair).  For example, when O2 is low or depleted, some bacteria are able to use nitrate NO3 as an oxygen source.  If that becomes limiting (often in very hot weather), another group that can utilize iron oxide FE2O3 as an oxygen source steps in.  These bacterial strains are not as efficient as they need to expand energy to split off the oxygen but present a series of steps before all oxygen sources are eliminated.  Some types of bacteria can even utilize phosphate PO4 (anion) as an oxygen source.  When these alternate oxygen sources are depleted, these bacteria receive and allow bacteria that can utilize sulfate SO4 to increase.  Water that is warm naturally holds less oxygen and can accelerate the above process.

The growth of sulfate bacteria is intensified by coastal water warming.  That is why most fish kills happen in late summer as dissolved oxygen levels are lower.  When hydrogen sulfide builds, it kills oxygen-requiring aquatic life on the bottom.  Rapid sulfide oxidation generates heat and has a direct impact upon marine soils.  Hydrogen sulfide can "sterilize" bottoms, killing all but the most sulfide-tolerant worms.  This helps explain the increase of clam sets after winter storms, but we have little research into the bacterial responses of soils after natural cultivation. 
Although it is recognized that iron and sulfur bacteria have key roles in the production of iron sulfides, very little has been reported on them as they relate to marine soils and the man-made or natural cultivation of them.  This research could answer many seafood questions – my view, Tim Visel.

Appendix #1

The Soft Shell Clam Industry of Maine
Robert L. Dow Dana E. Wallace Paper
United States Fish and Wildlife Service
Circular 110
Washington, D.C. June 1961

Pg. 10: "In sand flats clam shells grow white, brittle and paper thin, in rocky flats the shells are dark and black, thick and with rounded and blunt edges.  Sometimes very slow growing clams will have overlapping layers of shell growth.

Pg. 11: "The growth rate of clams varies with environmental clams transplanted
From one type of sediment (soil, T. Visel) to another assume the growth characteristics
of the new area, showing that environment and not heredity is important in determining
growth."

Pg. 13: "A condition of watery-brownish or blackish-colored meats is referred to by diggers as "water belly." (This condition is recognized as glucose-sulfide starvation, T. Visel)

Pg. 18: "Though it has been definitely determined that chemical factors directly affect shellfish survival, high mortality rates and poor growth have occurred in growing areas where it was suspected that unfavorable chemical conditions were involved.  The presence of hydrogen sulfide in the water is reflected in a lowered pH – acid – alkaline factor.  The formation of hydrogen sulphide results from the decomposition of organic material on the surface, or mixed with the sediments, of the flats.  It would appear, then, that chemical factors may adversely affect clam survival and growth but these chemical factors depend upon the existence of unfavorable physical factors (clam flat soil, T. Visel), such as poor drainage and inadequate water circulation."

Pg. 20: "Erosion occurring above mean high water (from land or streets, T. Visel) may have more serious consequences than erosion within the intertidal zone (over the flats from storms, T. Visel) because a thin blanketing layer of clay, deposited on the surface of the flats (severely restricting water circulation within the soil, T. Visel) kills the clams.  Commercial diggers refer to these areas as "dead flats."

Other researchers on the east coast found a direct relationship between the growth of hard shell clams (quahog) and the percentage of soil clay fractions (Narragansett Bay has several studies that document this).  Perhaps the most direct and which made strong comments about the impact of clay was research from Alaska.

Appendix #2

Informational Leaflet No. 179, Cordova, Alaska
The Effects of An Experimental Hydraulic Harvester on Razor Clam Habitat by Richard Nickerson and Timothy S. Brown
State of Alaska
February, 1979

This report was published by the Alaska Department of Fish and Game, Cordova, Alaska.
Abstract:
"The effects of an experimental hydraulic harvester on clay levels in marginal and submarginal razor clam habitats was analyzed from two latin squares.  Post-treatment clay levels in the marginal habitat did not differ significantly from respective pre-treatment levels.  Clay levels increased significantly in control plots of the submerged habitat, but were significantly reduced in horizontally flushed plots."
Introduction (pg. 2):

"An adverse relationship was found to exist in the Cordova area between clay levels in razor clam bearing substrate and the presence of one-year old razor clams. That is, the density of one-year old clams decreased as levels of clay increased.  When clay levels reached 2.2% of the substrate composition, one-year old razor clams were not found."


Appendix #3
Email Exchange Soft Clam Habitat Spring 2008

I will send the Sandwich historical, but perhaps the entire Cape? I have an old US Fish Commission Report that gives a town-by-town fish/shellfish summary (1887), so I will send you a copy. The attached is just a draft of some research of three case histories for soft shell clams, but my focus is on marine soils porosity, pH and grain size. It's part of a Habitat Restoration Committee I'm on for the Long Island Study. My research indicates that soil does have a part in growth/setting. High organics, soft muck and low pH bottoms exhibit poor or slow growth. But loose sand almost clean and washed can set heavy. Have you noticed this on your Clam Plow" to cultivate soft shell clam-flats—published reports indicate increased sets. What I will present in an old argument here "man-made habitat creation" or natural made habitat creation." Both resulted in soil "cultivation" and some evidence of increased clam sets does exist.
I will send the historical report soon.  Tim Visel
Hi Tim,
Thanks a bunch! I have noticed much heavier clam sets in areas of my farm dominated by course substrate. I have rigged up an old 1950 Gravely L. series with a plow and am using it to plow my steamer nets out. The larval clams seem to like the netting and settle out under the net. With the plow, I can now get down deep enough to foil the most hardy predators.

My farm consists of muddy areas and sandy areas so I'm well aware of the phenomena you will prove. Again, let me know if I can assist.


Appendix #4

Geoderma
Volume 16, Issue 1, July 1976, Pages 1-7
Formation of mackinawite by the microbial reduction of jarosite and its application to tidal sediments
K.C. Ivarson, R.O. Hallberg
Abstract

A study was made of the microbial reduction of jarosite (K Fe3(SO4)2(OH)6), a mineral formed by the aerobic iron-oxidizing bacterium Thiobacillus ferrooxidans, and frequently found in acid sulfate soils developed on marine sediments containing pyrite (FeS2).

Under anaerobic conditions and in the presence of organic matter (lactate) and the sulfate-reducing bacterium Desulfovibrio desulfuricans, jarosite was soon reduced to a sulfide — a poorly crystalline form of mackinawite (FeS). Due to the presence of phosphate ions in the cultural medium, vivianite or its polymorph metavivianite (Fe3(PO4)2.8H2O) was formed also. Because aging transforms mackinawite into pyrite, it is suggested that in marshy areas where pyritic sediments and acid sulfate soils ('cat clays') occur, and where aerobic and anaerobic conditions exist, there is a generic relationship between pyrite and jarosite and the above two microbes help to maintain this relationship by cycling sulfur and iron between the two minerals. At the same time they perhaps aid in cycling phosphorus.

Appendix #5

KING'S MARK ENVIRONMENTAL REVIEW TEAM REPORT
FRASH POND
STRATFORD, CONNECTICUT
JANUARY 1983

King's Mark Resource Conservation & Development Area
Environmental Review Team
Sackett Hill Road
Warren, Connecticut 06754

VI.  THE ORIGIN OF HYDROGEN SULFIDE IN FRASH POND
Sulfur enters Frash Pond in the form of sulfate dissolved in seawater.  Saltwater being denser than freshwater remains at the bottom of the pond even when the surface water is fresh.  During the summer, solar heating at the surface further intensifies the density differences between the warm fresh water on top and the cold saltwater at the bottom.  The strong density gradient drastically reduces mixing in the pond, causing the saltwater at the bottom to become stagnant.  Isolated from the atmosphere, the bottom water rapidly gives up its dissolved oxygen to the respiration of organic matter in the pond sediments. 
Respiration consists of two linked processes.  First, organic matter gives up electrons as it is oxidized to carbon dioxide.  Second, an electron acceptor receives those electrons and becomes reduced.  In aerobic respiration, the electron acceptor is oxygen, which is reduced to water.  However, when ambient oxygen is exhausted, bacteria have the ability to continue oxidizing organic matter by using alternate electron acceptors.  Sulfur is an excellent alternate electron acceptor for anaerobic respiration by bacteria.  Initially sulfate can be reduced to elemental sulfur, and then elemental sulfur can be further reduced to sulfide.  Both sulfur products bear upon the situation at Frash Pond.
First, elemental sulfur is very insoluble, and quickly precipitates out of water.  This raises the possibility that the sediments of Frash Pond have been enriched in sulfur during various periods of its history.  Second, sulfides are very insoluble in the presence of metals, precipitating as black metallic sulfides, which give marine muds their inky, blue-black appearance.  Thus, the bottom of Frash Pond also may have been enriched in metals.  These include potentially toxic heavy metals introduced from nearby landfills.  Third, when sulfide is more abundant than the constituents with which it is insoluble, sulfide appears as soluble and volatile hydrogen sulfide.  The evolution of hydrogen sulfide gas which calls attention to Frash Pond each summer is but the tip of the sulfur-cycle "iceberg" which operates in the depths of the pond all year.

To summarize, there are four basic components to the problem with sulfur at Frash Pond:
1.   Source of sulfur:
Sulfate in seawater and, possibly, sulfur enriched sediments.
2.    Stratification:
Chemical and thermal density gradients in the water column.
3.   Organic matter:
The electron donor driving the reduction of sulfate to sulfide.
4.   Potential metal toxicity:
Possible pollution from nearby landfills.

Appendix #6
Online Resource Guide for Florida Shellfish Aquaculture
Sulfide Concentrations in Sediments and Water and Their Effects on Hard Clams

Purpose:
The hard clam Mercenaria mercenaria is a common inhabitant of estuarine sediments and an important aquaculture species in the U.S.  In coastal environments, high nutrient inputs lead to increased rates of phytoplankton production followed by increased organic material availability.  Some of this phytoplankton will die and sink to the bottom where it is consumed by bacteria.  As this organic material is decomposed, the increased bacterial respiration rates can translate to reduced oxygen availability, particularly in benthic waters and sediments. Sediments are especially prone to oxygen limitation because of reduced diffusion rates, and, as a result, deeper sediments often completely lack oxygen.  Under these conditions, other types of bacteria decompose the organic matter and, in doing so, produce hydrogen sulfide.  Sulfide is responsible for the black sediments found in many estuaries and is the source of the "rotten-egg" smell.  Therefore, although proximity to coastal sources of nutrient helps to ensure an abundant food source for hard clams, it also exposes them to hydrogen sulfide.  The combined effects of low oxygen concentrations and hydrogen sulfide are known to reduce the growth and survival of many bivalve species.

Results:
Hydrogen sulfide was found in sediment pore water near and within hard clam high density lease areas (HDLAs).  Porewater sampled from clam lease sites had mean sulfide levels up to 110 μmol/L.  The Derricks, Gulf Jackson, and Horseshoe Beach HDLAs were found to have significantly higher sediment pore water hydrogen sulfide concentrations than the Pine Island and Pelican Reef HDLAs. Non-lease sites had higher levels, with mean sulfide levels up to 300μmol/L.

In summary, hydrogen sulfide occurs in the sediments of Florida's west coast clam aquaculture areas at concentrations capable of reducing hard clam survivor.  The reduced survival of seed clams exposed to hydrogen sulfide in the laboratory and the beneficial effect of antibiotic suggests that hydrogen sulfide and accompanying bacterial growth play a role in hard clam mortality during the field growout process.

Appendix #7

Microbial Community Stability in Anoxic Sediments Under Conditions of Shifting Salinity, Oxygen and Sulfate
Libusha Kelly, MIT
Microbial Diversity, 2010

Abstract:

Microbial interactions in anoxic communities play a role in the construction and maintenance of many environments, including marshes, wastewater treatment plants, and the human mouth.  To explore the factors that cause community shifts and those that encourage stability in microbial communities, we enriched the anaerobic microbial populations from two anoxic sediments under conditions of high and low salinity.  After a preliminary enrichment phase, we further perturbed a subset of enrichments by amendment with sulfate and oxygen.  Physiological and genetic analysis indicated that the initial inocula from both sites contained a diverse community of bacteria and archae, including expected members like sulfate reducing bacteria, methanogens, and acetogens.  Our results suggest that oxygen had the most destabilizing effect of all perturbations on the methanogens and acetogens in the enrichments; sulfate amendment did not impact community composition and activity as severely.  Salinity appeared to have an affect on community composition and response; the saltwater incubations were consistently more affected under perturbation than the freshwater enrichments.  Finally, the sulfate reducing bacterial populations were more stable to perturbation generally than methanogen and actogen populations.

Appendix #8

CONTROLLING THE NITROGEN CYCLE WITH ANAMMOX BACTERIA, ENGINEERING ETHICS, AND A FRESHMAN ENGINEER'S INITIATIVE
By Ian Abrahamsen
University of Pittsburgh
Swanson School of Engineering


THE NITROGEN CYCLE: A BALANCE OF NUTRIENTS AND POLLUTANTS
In brief, the nitrogen cycle is the transformation of nitrogen gas in the air to other nitrogen compounds that, ultimately, turn back into nitrogen gas.  Unfortunately, man-made advancements have unbalanced the cycle with undesirable results, one of which is fixation, a problem that I believe can be solved by denitrification.

   Fixation occurs in one part of the nitrogen cycle when nitrogen gas, a diatomic molecule in the atmosphere, converts to other nitrogen compounds that plants must consume to survive [3].  This conversion, known as fixation, requires a large amount of energy, which can be achieved by the properties of chemical enzymes of small bacteria; these bacteria are called microbes and are found in soil [3].  Fixation has increased rapidly, as a result of the introduction of fertilizer, the mass farming of legumes, and the burning of fossil fuels [3].  All of the fertilizers used to make crops increase the rate of fixating microbes in the soil.  The roots of legumes increase this rate as well, and when fossil fuels burn, nitrogen compounds are released into the air as a byproduct.  The conversion of nitrogen molecules back to nitrogen gas, which is called denitrification, is currently not at the same pace as fixation; there is an abundance of nitrogen compounds in the air, causing an imperative challenge for engineers [3].  Fixed nitrogen compounds, including ammonia, nitrous oxide and nitric acid, are gaseous pollutants to our world and can cause acid rain, extra heat contained in the atmosphere (the greenhouse effect), thinning of the ozone layer, aquatic "dead zones", and water pollution [3].  Therefore, a balance needs to be attained between fixation and denitrification, by decreasing fixation, by increasing denitrification, or by a combination of both.

WATER DENITRIFICATION: AN ANSWER IN ANAMMOX

I have decided to focus on the solution of increasing denitrification, because in order to decrease fixation a decrease in fertilizer would be needed, which would decrease the amount of crops grown across the globe.  A possible option to improve denitrification is the use of anammox (Anaerobic, AMMonium, Oxidation) bacteria.  These microbes are able to break down fixed nitrogen into nitrogen gas without the requirement of oxygen, as most denitrifying microbes do [4].  Breaking down nitrogen without oxygen is a huge advantage because the oxygen-hungry microbes are costly to maintain in a laboratory setting, whereas anammox bacteria is much cheaper [5].  In fact, anammox bacteria plants have been estimated to save up to 90% more in comparison to standard sewage treatment plants [5].

   Since anammox bacteria prosper in marine environments, its advantages begin in the water.  Anammox bacteria naturally convert ammonia into nitrite, and then back into the nitrogen gas consumed by plants to complete the cycle [4].  Because these bacteria go through an organic process, anammox is an extremely functional answer to problems such as the sanitation of surface water, plus the cleaning of waste water [5].  In my opinion, anammox is an extraordinary engineering breakthrough, for it could also solve other worldwide problems.  For example, I could see this process not only benefitting the putrification of drinking water through removal of nitrogen pollutants found in waste, but also reducing the amount of nitric acid found in polluted bodies of water before the water is evaporated and condensed into precipitation.

ANAMMOX IN ACTION: THE APPLICATION OF THE BACTERIA

Anammox is a relatively recent solution to the challenge of the disturbed nitrogen cycle.  The first evidence of anammox bacteria was discovered in the early 1990's in Delft, the Netherlands [6].  This breakthrough was at a waste water facility, and although it took years to isolate the activity of these microbes in a laboratory, it was a promising beginning for denitrification [7].  Through these studies, and many others, anammox bacteria have been found in waste water and most habitually in ocean environments [4].  It has been estimated that anammox bacteria may account for nearly 50% of the nitrogen removal in marine environments [5].  Although this statistic is an impressive feat, it just shows what has been naturally occurring, and we now need a way to harness the different anammox bacteria and introduce them to environments with high levels of fixed nitrogen.

Appendix #9

The Search for Megalops
CT Blue Crab Special Report – Waves of crabs detected perhaps moving both east and west
NOTICE TO MEGALOPS EMAIL LIST = Attention Crabbers
Wednesday August 22, 2012


Information obtained on August 14-18 appears to contain reports that a series of large waves of adult blue crabs are moving along Connecticut's coast.
These crabs have a very different appearance and don't resemble the bright white/blue shells of spring and summer.  These crabs might be several years old perhaps the 2006 and 2009 Megalops set survivors.

Crabbing has soared in Branford and Guilford and now Milford.  This is a very good sign for western Connecticut crabbers.  The densest population this year based upon crab catch reports so far has been the New Haven estuary – Quinnipiac, Mill and West Rivers.  We do not know the source of these "new" crabs.

What to look for:

1.   These populations contain yellow face crabs– a piece of yellow around the mouth area.  Yellow face crabs have been observed in Clinton Harbor on August 18th. Yellow face crabs have not been observed in the CT River population as yet.

2.   A significant portion of the population has a second growth claw – a perfect claw that is about half the size of the regular claw – this is a crab that is several years old, it takes several years to re-grow a lost claw, perhaps 2 to 5 years.  Reports of any crabs with a second smaller claw would be helpful.

3.   The shells of these crabs show numerous injuries, sometimes shell damage that has healed, some may even be missing a point entirely.  I have seen two of these crabs in Clinton Harbor last Monday.  The shells show nicks, scratches etc.  It is obvious that they have been in these shells quite awhile.

4.   The underneath areas might have a brown growth, looks like an algal coating of some kind.  The claws may have dark brown patches or black streaks toward the tips.

5.   No female sponge crabs appear to be a part of these populations.
We may be able to track the movements of these crabs by the date and time they appeared in crabbing spots. (General location is fine).  I would appreciate any reports of these crabs or sightings.

Thanks, Tim Visel


Notice: WARNING – These crabs are very large averaging 7 to 7.5" point to point.  They have very strong claws and I have seen one at Clinton Harbor nearly cut a 5-inch crab in half.  They are what has been described as "rock hard" – hard shells; I banded one at the Indian River, Clinton over the weekend and it took two lobster bands in each knuckle to reduce fighting.  Please exercise caution etc.

Appendix #10

JSR Vol. 1, No. 1, Pgs. 83-87
1981
A Study of Two Shellfish – Pathogenic Vibrio Strains Isolated From a Long Island Hatchery During a Recent Outbreak of Disease
Carolyn Brown , National Oceanic and Atmospheric Administration
National Marine Fisheries Service
Northeast Fisheries Center Milford Laboratory, Milford, CT 06460

Abstract: "Two bacterial strains belonging to the genus Vibrio were implicated in a recent outbreak of disease in larvae of Crassostrea virginica at a Long Island shellfish hatchery.  Bacteriological observations made during the disease period suggested that the two bacterial pathogens represented an extremely small proportion of the total bacterial population in the seawater system of the hatchery.  This was further supported by the appearance of spontaneous disease only after the tenth day of larval development.  Although the two strains were morphologically distinct, their biochemical and physiological characteristics suggested that they were closely related to the Vibrio anguillarum.  The disease could be initiated in the laboratory when small members of the two pathogenic strains were added (2 cells/mL) after each change of larval culture water.  The two strains could be recovered from larval cultures 3 days after a single inoculum of less than 10 cells/mL of larval culture water, even though the water in the cultures was changed daily.  This carry-over of bacterial cells shows that extremely small numbers of pathogenic cells present in a seawater system can eventually lead to a disease situation.  Ultraviolet radiation was found to be an effective method of eliminating one of the two pathogens.  The other partially recovered from exposure within 24 hours."

Strain one was not sensitive to penicillin.  Strain two was and strain 1 produced nitrate while strain two produced hydrogen sulfide.

Appendix #11
Iron Sulfide Scale Management in High-H2S and -CO2 Carbonate Reservoirs
By:
G. Verri, K.S. Sorbie, M.A. Singleton, C. Hinrichsen, F.F. Chang and S. Ramachandran

Summary
Combined sulfide/carbonate-scale formation in wells producing from reservoirs with high carbon dioxide (CO2) and high hydrogen sulfide (H2S) represents a serious threat to production efficiency and system integrity.  Understanding of both the main source of iron that forms the iron sulfide (FeS) scale and the phase partitioning and effect of the acid gases (CO2 and H2S) is important in devising and implementing the correct sulfide-scale-control program.