Abstract
Manufactured soil, also known as Fabricated Soil (FS), is a natural mixture of decaying substrates rich in aluminosilicate, carbon, nitrogen, phosphorus and potassium sources (complete composition and quantity cannot be disclosed at this time). This substrate is usually used for landscape rehabilitation, and is an excellent source and example of environmental remediation. Fabricated soils could be a solution to the problem of soil erosion.  This became a serious issue as all over the world millions of acres of soil are damaged. This study examines the use and application of FS in Western Pennsylvanian soils that were previously degraded by acid mining drainage. We are still trying to determine if fabricated soils are a long-term or short-term solution to the problem. After the exposure of FS into the natural habitat for the duration of three years, we evaluated bacterial activity in soil, as this is an important indicator of soil health.

Introduction

Soil mineral content, presence and effects of plant population, and microbial activity in general are important soil attributes that the authors have been studying in Fabricated Soils (FS) for the past three years [11, 14-16, 18]. It was stated by several researchers that at this time, still little information is available concerning the occurrence of natural transformation of bacteria in soil.   This is because few bacteria are known to possess the genes required to develop competence and because the tested bacteria are unable to reach this physiological state in situ [6].

Bacterial functions and activity in soils are important for many reasons [2, 5]. One example is clearly illustrated by the presence of Actinomycetes that although they are abundant throughout the soil profile, they tend to be most abundant in, or adjacent to plant roots [5]. Our preliminary observations suggest that Actinomycetes are found in colonies that form thread-like filaments in the soil. They are particularly effective at breaking down tough substances like cellulose (which makes up the cell walls of plants) and chitin (which makes up the cell walls of fungi) even under harsh conditions, such as high soil pH [22].

Another one deals with the applications of “Humic Coverage Index” that define the availability of contaminant-degrading bacteria in soils [6]. They are responsible for major bioavailability and biodegradability of contaminants in soil. Certain bacterial strains are particularly important in nitrogen cycling [9, 10, 19]. Free-living bacteria fix atmospheric nitrogen, adding it to the soil nitrogen pool. Other nitrogen-fixing bacteria form associations with the roots of leguminous plants such as lupine, clover, alfalfa, and others [19].

Needless to say legumes, clover in particular, were used purposefully to grow on the fabricated soil plots in our experiment. Actinomycetes are also capable of forming associations with some non-leguminous plants and fix nitrogen, which is then available to both the host and other plants in their vicinity [1, 3, 4, 19, 20].

Materials and Methods

In our research, seven soil samples were obtained from four different locations named by the presence of certain trees grown on these specific plots such as Poplar Plot or Willow Plot. The samples were collected in July 2005 and stored on ice for a day during transportation to U.S. Micro-Solutions where bacterial and fungal analyses occurred. All samples were assayed at the same time by the following method. A portion of each sample was weighed on an analytical balance and then placed in a measured amount of sterile deionized water. These were allowed to stand at room temperature for thirty minutes, vortexed vigorously, and dilutions were streaked to trypic soy agar (TSA) and inhibitory mold agar (IMA) plates. All TSA and IMA plates were incubated at 27°C for up to ten days. The bacterial colonies were enumerated, identified and the number of colony-forming units (CFU)/gram of material calculated. The authors acknowledge the assistance of U.S. Micro-Solutions, Inc. (Greensburg, PA) in determining the bacterial activity and identification of microorganisms in fabricated soil samples.

Results and Discussion
Most of the samples tested had different Bacillus spp., meaning more than one type was recovered from the samples plated on TSA and IMA agar.  Nine types, not individual species, of Bacillus were identified. Their morphological description is shown below in Table 1.

Table 1. Bacillus spp. Types.

Bacillus represents a genus of Gram-positive bacterium, which is ubiquitous in nature (soil, water, and airborne dust). Bacillus produces large, spreading, gray-white colonies with irregular margins when grown on blood agar. This bacterium has a unique characteristic in that it has the ability to produce endospores when environmental conditions are stressful. The only other known spore-producing bacterium is Clostridium. Although most species of Bacillus are harmless saprophytes, two species are considered medically significant: B. anthracis and B. cereus [3-5, 7, 9, 10, 21, 22].

B. cereus. Unlike B. anthracis, B. cereus is a motile bacterium which can cause toxin-mediated food poisoning. It is known to inhabit many kinds of food including stew, cereal, milk, and most recently it has been found in fried rice. The two toxins released by the bacterium lead to vomiting and diarrhea, symptoms similar to those of Staphylococcus food poisoning [3-5, 7, 9, 10, 21, 22]. The pie charts below represent the details on bacterial presence in different types of soils, giving a comparison of bacterial species and their percentage in soil samples tested in 2004 and 2005 (Fig. 1-9).

Bacterial variety changed within one year in mining soil bringing non-fermenting gram-negative bacillus to a 100% rate, and causing Bacillus spp. to disappear (Fig. 1 and 2). At this point we do not have an explanation why this took place but assume that climatic conditions of the area and allelopathic interactions within bacterial community itself or flora grown in vicinity could affect the change in species presence on the mining soil plot. This plot was chosen as a control sample for comparison results obtained from other areas.

Bacterial composition changed within one year in top soil as well by bringing the number of colony morphology types down from eight to five. Fermenting gram-negative bacillus were no longer present in 2005 testing, and were substituted by non-fermenting gram-negative bacillus at a 47% rate.  Bacillus spp. 9 and 1 disappeared from the sample but Bacillus spp. 2 and 3 were till present. (Fig. 3 and 4).  

At this point again, we do not have an explanation why this took place but assume that climatic conditions of the area and allelopathic interactions within the bacterial community itself or flora grown in the vicinity could affect the change in species presence on the top soil plot. This plot also was chosen as a control sample for comparison results obtained from other areas.

So, top soil was a double-control in the experiment, soil biota presence and composition changed within the year by bringing again in play non-fermenting gram-negative bacillus type 1 and 2 and changing Bacillus spp. composition from 1-4,9 to only Bacillus 2 and 3. The variety of species was lost and the percentage was distributed in a way that Bacillus spp. presence significantly dropped from 70% to 10%.

Original fabricated soil tested in 2004 had eight different bacterial species, including Bacillus spp. 1-4 and 6, gram-positive bacillus and cocci, and unidentified Actinomycetes (Fig 5). In a fabricated soil tested in 2005 six species were present mostly non-fermenting gram-negative bacillus of types 1 and 2, unidentified Actinomycetes and three Bacillus spp. 2, 3 and 9 (Fig 6).

Fig. 7 shows ten different species (between the different colony morphology Bacillus spp. types and other listed microorganisms) and representatives of bacterial flora found in the Poplar Plot sample: Bacillus spp. 1, 2, 4 and 9, Micrococcus, fermenting-and-non-fermenting microorganisms and more.

Table 2 shows the presence of bacterial flora in Poplar plot tested in 2005. Due to the overgrowth of bacterial flora, there was no percentage calculation in the sample.
 

Table 2. Fabricated Soil. Poplar Plot. Tested 2005.

Interesting enough Willow plot was the only one that maintained presence of most of the bacterial from 2004 (Fig. 8 and 9). Bacillus spp. 1, 2, 6 and 9 were present in 2004 testing, and Bacillus spp. 1, 2, 3 and 9 were in current year analysis.

Non-fermenting Bacillus maintained their presence as well as Coryneform gram-positive Bacillus. Though unidentified Actinomycetes disappeared mostly from each sample tested.

Table 3 summarizes the presence of bacterial microbiota in soil samples tested both in 2004 and 2005. It is obvious that a variety of species diminished for the past year, however, most of bacterial Bacillus spp. were there still playing an important role in maintaining soil fertility.

Bacteria are becoming increasingly important in bioremediation, meaning that we can use them to help in the reclamation of contaminated lands [2, 8]. Bacteria are capable of filtering and degrading a large variety of human-made pollutants in the soil and groundwater so that they are no longer toxic. The list of materials they can detoxify, just to name a few includes herbicides, heavy metals, and petroleum products [9, 10]. By monitoring soil organism dynamics, we can detect detrimental ecosystem changes and possibly prevent further degradation [21]. The response of each group of soil organisms, i.e., soil saprophytic bacteria, symbiotic bacteria, saprophytic fungi, mycorrhizal fungi, protozoa, and nematodes, can be used to indicate the effects of contaminants on soil health [10].

During the exposure of fabricated soil into the natural environment, the activity of bacterial communities changed the growth of trees, whereas the green mass (all plants) increased remarkably, these trees developed deep root systems, and the level of nutrients such as potassium, nitrogen and phosphorus was also on the rise. Thus, fabricated soil became an enrichment substrate for plant growth and propagation, and for microbial activity, as the interrelations between all living components of the soil constitute successful strategies in fighting against soil degradation and erosion. Fabricated or so-called ‘artificial soils’ are high in fertility and can be used in the areas where erosion and loss of topsoil is evident. Examples of possible applications could include areas of former coal mining and stripping activities, or areas of steel production, which unfortunately determined heavy environmental impacts in Western Pennsylvania.

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