DIEGO CAIVANO

The phenomenon known as Black Plug Layer (BPL) has become a major problem for golf courses. Traditionally, research has centered on the "menaces of the greens": weeds, fungi, infestation of insects, algae in ponds and death of the greens. Within the last ten years, the focus of investigative efforts has shifted toward solving the microbial problems that many courses are plagued with. This paper will focus on a process that is more of a nuisance to golfers, but can seriously jeopardize microbial environments inside and outside of golf courses. The phenomenon is known as Black Plug Layer (BPL), it is produced by a small consortium of bacteria converging to form an environmentally toxic biofilm. This paper will address several issues: how and why the BPL is formed, attempt to clarify what causes BPL, where this phenomenon is occurring and how some possible treatments might alleviate the dilemma.

Among ecologists, golf courses do not rank significantly high. To the everyday laymen, the lushness and beauty maintained within these parks, is admirable. However, the truth be told, most scientists would not disagree that golf courses, save the ones built on landfills, are ecological disasters. For the most part, the elegant courses that can uphold their "natural beauty" can be directly attributed to the extreme amounts of chemicals their habitats are subjected to. In 1990, the Environmental Protection Bureau of the New York Attorney General’s Office conducted a survey of 52 Long Island golf courses. They found that these golf courses applied 21 different herbicides, 20 fungicides and 8 insecticides annually - a total of 50,000 pounds of active ingredients - to maintain high playing standards (6). This study also revealed that the average amount of pesticide per acre treated per year was seven times the average amount of pesticide applied on horticultural land. The actual numbers were astonishing, an average of 18 pounds of pesticide per acre per year are used, compared to the already high 2.7 pounds used on agricultural land (6). Compound this with the golf course’s blatant abuse of natural resources and it can become an explosive combination.

In some dry ridden areas, like New Mexico, golf courses may use up to a million gallons of water in a day (6). Not only are ecologists worried about how this misuse of water may damage areas with already low water resources, but this water eventually ends up as silt and pesticide loaded runoff in habitats not accustomed to high levels of water. Due to maintenance practices, such as the one mentioned above, golf courses have recently become extremely controversial. Plainly stated, in order to uphold a pleasant playing field for members, the microenvironments within a country club are subjected to various harsh regiments of environmentally toxic chemicals. More importantly, the current maintenance habits on golf courses directly affect the surrounding environments.

The trend of golf course maintenance personnel, until recently, has been to treat their problems with what is causing many of their problems - chemicals (1). Fortunately, the United States Golf Association, in the early 1990’s created the USGA Turfgrass and Environmental Research Committee. This committee is responsible for establishing guidelines for reducing the environmentally harmful chemical practices which golf course superintendents have been conducting for years. The recent trend in research universities, including Cornell, has been an organic approach (6). Some courses have begun using a new method, which employs composted turkey manure to help alleviate a common fungal problem known as pythium-root (1). In doing so, Bob Feindt the superintendent of the Country Club of Rochester has cut their fungicide use by more than 60 percent. Research has also been geared towards combining the instincts of the knowledgeable course superintendents with computer technology. This and other, perhaps, "too high-tech" approaches to golf course maintenance are beginning to be seen on the market. The reason for a pessimistic review of technology is the full-costs and practicality of these new innovations. The opportunistic side to this is that a lack of funds from golf courses is not an imminent problem and most all superintendents are committed to keeping up with technology. For instance, in 1993, there were 14,375 existing golf courses - an area that if combined together would be roughly the size of Delaware and Rhode Island (6). To add to this size, the National Golf Association projects that about three new courses a week will open until at least the year 2000. Since these Country Clubs make their customers pay outrages prices for memberships and golfing privileges, money shouldn’t be hard to collect compared to the high price of completely destroying the environment. However, a few obvious points should be noted: even if country clubs have the budgets to spend on expensive maintenance equipment, this does not mean they will spend it on new technology; secondly, the age and the standard of golf courses are important in how they are maintained. A public course, which is not as expensive and may not have the same quality of courses as private ones, may chose to use the less expensive chemical methods of maintaining its greens. Chemical practices are effective not only in cost but in the range of problems solved for the greens. In general it is simple to see why over fertilizing and use of pesticides could begin to cause complications. One could list numerous difficulties which most people are aware of to the over-administration of chemicals. However, these same practices are at the root of causing the problem, which has been labeled as Black Plug Layer.

One of the many problems greenskeepers encounter is what has been unscientifically named as the Black Plug Layer (BPL). The problem usually occurs, but is not limited to, the specific area of the golf hole known as the green. From the perspective of the greenskeeper the disease’s symptoms expressed by the turfgrass include, chlorosis, browning, wilting, and the increased susceptibility of the grass to certain molds and viruses, along with a characteristic smell and black sludge (7). Occurring between zero to ten centimeters, or more, below the surface in depth (3). The black film also appears to create a surface that is impermeable, and this is why greenskeepers have often noticed the increased time in water absorption on the affected greens. Many theories have been composed as to the cause of BPL. The extent of these hypotheses has ranged from algal by-products and sulfur reducing bacteria to biofilms.

Most of the early research done on BPL concentrated on finding the source: biological, chemical and/or physical agents that produced the biofouling of greens. There were several studies that posed different hypothesis, of which the three most relevant hypotheses will be examined by this paper. At that time each of these papers were considered to contain important information pointing towards what could be the cause of the black plug layer. The earliest literature on the subject dates back as far as mid-1987 and studies are still currently being conducted. Since these experiments were all completed in close proximity to each other we will analyze them in chronological order, for sake of maintaining clarity.

The first study conducted on the biofouling of greens explained the black layer as primarily being caused by sulfur-reducing bacteria (1). The experiments entailed adding sulfur to two anaerobic columns, each containing different genera of sulfur-reducing bacteria: Desulfovibrio and Desulfotomatuclum. The experiments lead to the production of black sludge and to the characteristic hydrogen sulfide gas as is seen on courses. This group indicated that their research did not show algae as being the primary cause of the black plug layer. Their set of experiments only indicated that algal growth occurred after the formation of the black plug layer and only in the presence of sulfur. Their proposed mechanism for the production of the toxic, hydrogen sulfide gas was via the sulfur-reducing bacteria (2). They suggested that the anaerobic, sulfate-reducers were metabolizing soil organic matter as a source of food, the by-product was electrons which got released into the environment during respiration (1). The sulfate in the soil, having a strong affinity for electrons, would accept them and thereby get reduced, hence the name sulfur-reducing bacteria. As for the black color to the soil, they indicated that the hydrogen sulfide, when available, would react to form black metal sulfides. This is due to the fact that the gas is highly reactive with metals, such as iron or copper. The argument posed by this group was that BPL was due to anaerobic microbial respiration (1). On the exterior the claims maid by the authors seemed reasonable, but the fact that the materials or method section was not included, made the literature seem inconclusive and textbook-like; as if what had been published was "matter of fact". The next paper, however, took an alternative approach in explaining the mysterious black layer. Instead of concentrating on the chemical or biological explanations for the problem they went a step further by attempting to explain the physical reasons allowing the development of the chemical and/or biological factors, involved with BPL.

The second article made an effort to address the physical properties of this problem, which was not regarded in any of the other studies. They proposed a reason for how the physical environment of golf greens could develop, permitting the proliferation of the bacteria causing the black layer. Furthermore, they postulated that the origins of the phenomena, the physical cause of BPL, could be traced to the movement and accumulation of silt and clay particles into distinct strata (3). The stratification would result in poor water infiltration, which could lead to waterlogged conditions causing low oxygen content. The researchers suggested that these conditions could prove an excellent environment for growth of the bacteria and algae, which had been mentioned in other research efforts. The proposal of an alternative hypothesis was going to be researched in the summer of 1987. The project was designed to determine if BPL was associated with soil strata containing high concentrations of silt and clay particles. The three objectives of this project were going to be: to identify the physical properties of black layer, to compare physical properties of BPL with physical properties of soil strata and to attempt to correlate the formation of BPL with the concentration of silt and clay in turfgrass (3). As interesting as this effort sounded, the funding for the research was not attained. Furthermore, the data compiled at the time this project would have started was showing that BPL was probably due to chemical and biological reasons (5).

An entirely separate hypothesis than the first two, laid partial blame of the BPL phenomena on mucilage produced by blue-green algae. The thought behind this theory was that the algal by-products would cause impedance in the water percolation into the sand and initiate the process of causing BPL (7). The algal mucilage was considered as a good source for producing two conditions: the sulfur odors and the necessary anaerobic environment, which would then permit bacteria to develop a black layer. However, the article states that all of these factors failed to explain the formation of the black layer, until an unspecified consortium of bacteria was introduced (7). Nonetheless, all of the above factors, once combined gave the characteristic symptoms of black plug layer (9). The problem with this theory was with its scientific reasoning, the characteristic black layer only developed when bacteria had been added to a sand column colonized by blue-green algae (8). This varies greatly from the natural setting of a golf course, were bacteria do not mysteriously appear once an algal colony is established and create a black plug layer.

The studies mentioned above seemed to follow non-traditional mechanisms in research. The scientific reasoning was flawed, in that there was no preliminary investigative effort to conclude what the possible sources for the turfgrass BPL could be attributed to. Normally, for industry related research, the group interested would go through a series of logical steps until coming to a conclusion, without making any unreasonable assumptions. An example of the thought process could be to establish a problem, isolate the condition, reproduce it, find a possible mechanism to reduce the effects of the given dilemma. In the cases above the mechanisms had been decided before the BPL had been isolated. Not only were these experiments considered to be important, at the time, most people in the field accepted the findings without question. These theories were adopted very easily because they were somewhat simple for the golf course superintendents and its community to understand. Recently, microbiological research has turned to the much more complex and novel arena of biofilm systems.

Biofilms have recently become a major topic of research. The reason being the extent of applications and consequences they may contribute to science. The microbiology community is now realizing that most of the microbial activity in an open ecosystem takes place in and around biofilms (13). In fact, researchers know believe that bacterial biofilms predominate numerically and metabolically in virtually all nutrient-sufficient ecosystems. Over time, the extensive bacterial genotype has been able to respond phenotypically to the pressures of environmental stimuli (4). This type of evolutionary plasticity that bacteria continue to show can explain the fact that bacteria constitute the most successful form of life on earth. The reaches of biofilms can vary greatly; these sessile organisms predominate in most of the environmental, industrial and medical problems being currently researched (13). Ironically, for a hundred and fifty years microbiologists have concentrated on the free-floating or planktonic phase of bacteria, instead of the biofilm growth phase and the ultramicrobacterial phase (13).

A Biofilm is defined as a matrix-enclosed bacterial population adherent to each other and/or to surfaces (4). The biofilm phase appears to be one of the two growth phases bacteria have. A third phase called the ultramicrobacteria phase, is a phase in which bacteria have been observed as existing in a wide variety of oligotrophic environments predominantly dormant awaiting for dissemination (4). These two phases are very different from the planktonic growth phase discovered by Louis Pasteur. One can view these two growth stages as a strategy. Bacteria have adapted them, to enable themselves to thrive in a wide variety of environments. Currently the view is that a set of reactions to environmental conditions is codified in patterns of gene expression, which is triggered by specific environmental stimuli. One of the benefits bacteria in biofilms posses is the incredible resistance to antibacterial agents - at least 500 times more resistant than free-floating bacteria (4). In 1990, researchers were able to observe that biofilm bacteria were morphologically and metabolically different from the same species of free-floating bacteria. Figure 1, on the following page, shows a model of a single species biofilm colony at different stages of development.

When free-floating bacteria encounter a hard surface several changes occur. Once a hard surface is encountered and adhered to, the bacterial cell

undergoes a phenotypic change. Reporter genes were used to find that algC and algD were unregulated at the time of adhesion. These genes control the production of enzymes in the alginate synthesis pathway, which are necessary for the post-adhesion formation of biofilms (4). The other key observation in the formation of biofilms was the necessary expression of polysaccharides to establish the biofilm colony. The adhesion process has also been found to trigger the expression of an important genetic marker known as the sigma factor. Its role is to depress a large number of genes, making the biofilm phenotypically distinct from the planktonic bacteria (4).

The complex microbial community that is established as a biofilm is considered to hold some other unique properties. For instance, bacterial biofilms can form with several species of bacteria in one colony. Biofilm bacteria form communities that are metabolically active, constructing microniches for themselves (13). As a whole these communities create a primitive homeostatic and circulatory system, which is provided by the biofilm matrix. The bacteria in biofilms bind together and to surfaces by secreting a mesh of polysaccharide fibers. The clumps of polysaccharides are what create the water passageways in which nutrients are delivered and wastes are removed; much the same as our circulatory system does. Through these waterways, the colony creates and maintains a homeostatic environment for itself (4). The difference between the physiology of planktonic and biofilm bacteria is pronounced. For example, proteins in the form of enzymes are usually found in random mixtures of free-floating bacteria. Within a biofilm colony however, bacteria developed an efficient system of floating enzyme mixtures. These mixtures aid in the maintenance of physiological cooperativity within this community. In these ways biofilms, in structure and function, resemble the tissue formed by eukaryotic cells (4). These findings on biofilms create a much better interpretation for many problems that had unexplained mechanisms. One of these being the black plug layer phenomenon, which previous theories had left many unanswered questions.

Interesting results were obtained from research investigating BPL under conditions similar to those on golf greens. In 1989, the first paper to associate biofilms with black plug layer was published. Although biofilms had been studied since 1978, this was the first time the two ideas were correlated. The study was concerned with determining whether a consortium of bacteria was involved in the formation of a biofilm causing a black layer. As well as if there were conditions, which were more prone to be associated with the bacterial generation of a black plug layer. What was found was that consortia of bacteria were found to form synthetic BPL, more so than individual strains did (10). Another important observation in this study was that columns, which did not have sulfur reducing bacteria, did create biofilms but did not have the characteristic blackening and gassing of the sand columns (10). At this point in time, however, it was still unclear how the biofilm contributed to the black plug layer.

In 1990, a study was conducted on a golf course in Canada, which revealed two important observations. The most important being the careful diagramming of the black plug layer. The diagramming of a section of a BPL led to the realization that the processes, which protruded out of the colony, actually were associated with each other biologically. This was of importance because it enabled the researchers to make the connection between BPL and bacterial biofilms. In order to accomplish this the turfgrass was removed along with the three millimeter sand green layer. What were exposed were the uppermost edges of the existing black plug layer, which was mapped onto transparency sheets. The BPL was removed in 1mm layers until it had been mostly removed, the result was a three dimensional diagram of a section of a BPL infested golf green (5). The study was primarily concerned with describing the physical structure of the BPL and its inter-relatedness to the roots of the associated turfgrass. The result of the reconstruction of the mass can be seen in Figure 2.

Figure 2. The pen and ink drawing of the black plug layer by Cullimore et. al., shows four observed structures. Noted in this drawing are the major columnar process (MJCP), the minor columnar process (MNCP), the interconnective lateral plates (ILP), and the peninsulate lateral plates (PLP) (5).

If one compares the conceptual biofilm model and the diagramed BPL one can see that they are not that different in shape. This was also very important in correlating the two ideas. The other important correlation was the consortium of bacteria causing the BPL. This is because in a natural environment many species are available and it seemed less likely than not, to blame such a wide-ranging problem on a single species of bacteria.

Recent research has gone in another direction than what had been looked at before. The concepts and ideas are basically the same, but the context of how they are interrelated has changed. Current thought is that the environmental stress created by the management of the greens combined with the construction of golf greens enables the formation of biofilm colonies (11). Another surprising difference in thinking from the past is how biofilm bacteria in BPL are characterized. Together the variations on how BPL works have given a more complete idea on what BPL is.

It has been established that the construction of turfgrass on golf greens creates a difficult media to work with. They are generally constructed to meet the US Professional Golf Association’s specifications. These specifications are made up of different sizes of sand supplemented with peat moss. The top 30 cm of putting green soil are made up of a mixture containing 85% sand and 15% peat moss. Below 30 cm the soil is made up of larger grains of sand and pea gravel that serve as a base for the upper layer of finer sand and grass. This environment would seem to be ideal for normal bacterial growth, which occurs in soil (11). However, these greens are an extremely stressed environment. First of all, as mentioned earlier, large quantities and frequent doses of pesticides, soil conditioners and synthetic fertilizers are applied. Physically, stress is applied to greens in several different ways. This ranges from the amount of compaction created by foot traffic to temperature and light intensity to the intense watering schedules need for maintenance. Due to the high porosity inherent to the greens’ soil and the daily watering schedule, most of the dissolved organic carbon in the surface soil is flushed. To compound the problem the frequent chemical treatments significantly reduces the species diversity in the soil. The combination of chemical and physical stresses on turfgrass soils set up the development of the BPL (11). The next step in creating a better understanding of black plug layer was to better describe the distinct bacterial community memberships of the BPL in different golf greens.

The objective in identifying bacteria isolated from distinct golf course BPL problem areas was for several reasons. Primarily, the study was designed to determine if a common species was the cause of the biofilm colonies in geographically separate greens. From the 3 different golf greens in Southern California there were 41 species isolated and there were six isolates from a study completed at a site in Canada (11). Preliminary studies showed that there was only one common species between the four sites. However, the common species was a bacteria found in rabbits, common inhabitants of golf courses. The bacteria from the California study were then tested in Postgate’s medium. This medium is for isolating sulfate reducing bacteria and approximately half of the isolates tested positive. These results were quite different from those put forth by earlier research efforts. As was discussed earlier, previous conclusions had deemed sulfur-reducing bacteria as the cause of the toxic hydrogen sulfide gasses. The mechanism proposed at the time was as a by-product of the respiration by sulfur-reducing bacteria. The organic sulfur was used as a source of energy, as a product of respiration electrons were given off and accepted by the electron hungry sulfur in the environment (1). The results from the Postgate’s medium test are contrary to the theory that BPL is made up of sulfur-reducing bacteria. An alternative pathway for the hydrogen sulfide production was suggested. Desulfuration is a process by which an enzymatic degradation of sulfur containing amino acids takes place (11). The theory behind this pathway is by selective pressures. In other words, the stress of limited carbon sources may induce the genes responsible for synthesis of specific enzymes that break down sulfur, such as cysteine desulfhydrase. This reasoning seems to be more appropriate than earlier explanations for several reasons. To begin with, most sulfur-reducing bacteria show limited tolerance to oxygen, with few exceptions, therefore in an aerobic environment of a golf green they would not prosper (11). Another reason why this line of thought may be jaded is that BPL is an anaerobic environment, for the electrons to be given off respiration has to occur. It is impossible for respiration to occur in an environment that lacks oxygen. Although there was bacteria which could breakdown sulfur bonds to initiate the production of gas, there was no common link between the BPL from the various sites. Important data was attained from the cluster analysis, they showed that the bacteria could be grouped into clusters as a functions of substrate utilization (11). This meant that the bacteria that form BPL do not have to be from a single species, but that they do need to be able to adapt to over-stressed environments. This is also why the concept of biofilms can be applied to BPL. The research in biofilms has showed that any bacteria can take on that type of growth pattern, explaining why different types of bacteria were isolated in different golf courses except for the bacteria found in all the greens, which is carried by rabbits. The biofilm concept also explains why the BLP is in the form of the mucoid globular mass found on the field. Innovating research involving black plug layer and biofilms is being conducted to better understand the theories involved.

Currently a project underway is concerned with counter-attacking BPL in golf greens without having to employ harsh chemical methods or remove the entire green. These were the methods previously used to rid a BPL infestation. With chemicals the green was not playable for months until the bacterial problem was resolved and the turfgrass returned to playing conditions. Other drawbacks with this solution are obvious; the environment for one, the health of golfers which may be exposed to and the cost can range from $1,000 to $10,000 (6). When the BPL was severe enough the whole green needed to be removed. This entails excavating nearly three feet worth of existing green and repacking layers upon layers of sand and peat moss according to USPGA specifications. This could cost depending on the size of the green anywhere from $5,000 to $20,000 (11). Our research project involves the clean up of the black layer biofilm using biotechnology. The product is named WT-2000 and the composition of this solution is of surfactants and enzymes. This product has been used in the bioremediation of wastewater where the BOD (biological Oxygen Demand), COD (Chemical Oxygen Demand) and TSS (Total Suspended Solids) are beyond the acceptable defined parameters (12). Examples of treated industrial sites ranges from municipal wastewaters to dairy, soy bean and cured meats processing plants. Since all of these issues resolved by WT-2000 involved bacterial, it can be assumed that the increase of oxygen in the anaerobic system will better the circumstances of the golf greens. The idea behind the project is that the solution will catalyze the breakdown of the biofilm polysaccharides. Once this occurs, individual bacteria will begin to decompose the remaining biofilm in conjunction with the surfactants and enzymes. The draining of the clogged water will occur after the bacteria further down in the soil digest some of the biofilm. The end goal in the project is to be able to completely restore the golf green without having to be invasive nor chemically damaging to the rest of the environment. The incentive for the golf course to use this method will be economical because the cost of this product is roughly $.50 -$2.00 per 1000 gallons (12). An experiment has been designed to test the product against the biofilm for results on its degradation.

The design of the experiment is rather simple. Nine sterilized acrylic columns were packed to scale with USPGA soil materials, so as to replicate the turfgrass environment. Flow rates were established by flushing the columns with several liters of sterilized creek water that receives the nutrient run-off from an Irvine golf course. The columns were then inoculated with a mixture of black plug layer bacteria grown in the lab from a BPL infested golf course. The flushing with a nutrient source and FeCl3 (as an indicator for hydrogen sulfide and black metal sulfide production) was continued until all the columns had established BPL and dropped there flow rates significantly (indicating clogging). Three columns were set aside for controls, and the other two sets of three columns were given different treatment doses in their daily flushings of sterile creek water (11). The project has been underway for three weeks and for the next two weeks the data will be collected on the flow rates for later analysis. The relevance of this data will be two-fold. Primarily, the data compiled will show if the intentions of the product as a biodegrading agent will reach its predicted goal of restoring normal conditions to golf greens. The other concept this data will solidify is the support for the existing theory of the relationship between biofilms and black plug layer.

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6. Grossman J. 1993. How green are these fairways. Audubon. September/October 1993.
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8. Hodges CF. 1987. Blue-green algae and black layer Part II. Landscape Management. November 1987.
9. Hodges CF. 1989. Another look at black layer. Golf Course Management. March 1989.
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11. Lyon S. 1997. Multivariate analysis of black plug layer microcommunities in turfgrass soils. Applied Microbiology and Biotechnique. (Currently with publisher)
12. Podella C. and Baldridge J. 1997. Advanced BioCatalytics Corporation Brochure
13. Potera C. 1996. Biofilms invade microbiology. Science. 1996, 273, 1795-97

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