American Society of Agricultural Engineers
2950 Niles Road 
St. Joseph
Michigan 49085 9659 USA
Voice: 616/429 - 0300
FAX: 616/429 - 3852

Paper No. 944068

AN ASAE MEETING

PRESENTATION

REATMENT OF CATTLE WASTE FOR RECOVERY OF SINGLE CELL PROTEIN 1

by

A. W. Gebriel
Graduate Research Assistant
Civil Engineering
Texas Tech University
Lubbock, TX 79409-1023

N. C. Parker3
National Biological Survey
Texas Coop Fish and Wildlife Research Unit
Texas Tech University
Lubbock, TX 79409-2125

C. B. Fedler2
Associate Professor
Civil Engineering
Texas Tech University
Lubbock, TX 79409-1023

Written for presentation at the 1994 International Summer Meeting
sponsored by
THE AMERICAN SOCIETY OF AGRICULTURAL ENGINEERS
Hyatt Regency Crown Center
Kansas City, MO
19-22 June 1994


SUMMARY:

Purple sulfur bacteria grow in anaerobic lagoons in the presence of light and use H2S as an electron donor for photosynthesis. Purple sulfur bacteria reduce odor levels from anaerobic lagoons and cell protein of the bacteria is a useful by-product that can be included in the diet for poultry and fish. Experiments were conducted to determine the effect of reactor depth and surface area on purple sulfur bacteria production. The did change of bacterial concentration in response to the changing sulfide concentration and light intensity were studied by measuring changes in baacriochlorophyU~ content.

KEYWORDS:

Anaerobic reactor, Lagoon, Feedlot, Bacteria

This Is an original presentation of the author(s) who alone are responsible for its contents.

The Society Is not responsible for statements or opinions advanced in reports or expressed al Its meetings Reports are not subject to the formal poor review process by ASAE editorial committees, therefore, are not lo be represented as refereed pubticatlons.

Reports of presentations made al ASAE meetings are considered to be the property of the Society Ouotation from this work should state that It is from a presentation made by (the authors) at the (listed) ASAE meeting.

1 Research support was provided by the Water Resources Center, Texas Tech University and the Texas Higher Education Coordinating Board Advanced Technology Program Project No. 003641064.

2 Corresponding author.

3 Jointly sponsored by Texas Tech University, Texas Parks and Wildlife Department, the National Biological Survey, and the Wildlife Management Institute.


INTRODUCTION

Wastes from agricultural related activities (from both crop and animal production) are consistency identified as the most pervasive non-point pollution source in the United States. Agricultural waste is one of the leading causes of surface water quality problems and ground water contamination (Jensen, 1991; Sweeten and Melvin, 1985). The most common method of treating agricultural waste is with lagoons. Lagoons have been used for treating a variety of wastes, ranging from agricultural, municipal to industrial activities. They are very effective especially for treating animal waste. Red, pink, or purple pigmentation of lagoon water has been frequently observed. This pigmentation is attributed to the presence of purple sulfur bacteria that grow in anaerobic lagoons. The presence of these bacteria has been associated with reduced odor from lagoons (Lotringen and Gerrish, 1978). In addition to odor reduction, purple sulfur bacteria have been considered as a supplementary feed for animals, especially fishes and poultry (Kobayashi and Tchan, 1973).

Purple sulfur bacteria are found in nearly all aquatic environments. They are present where light reaches anaerobic, sulfide-containing zones in lakes (Takahashi and Ichimura, 1968; Guerrero et al., 1985; Overmann et al., 1991), in lagoons treating a variety of wastes (Sletten and Singer, 1971; Hart and Turner, 1965; Holm and Vennes, 1970), and in sewage treatment plants (Siefert et al., 1978). They grow anaerobically in the presence of light and use H 2S as an electron donor for photosynthesis. By utilizing H 2S (the main cause of lagoon odor) purple sulfur bacteria reduce the odor level from anaerobic lagoons. In addition, cell protein of the purple sulfur bacteria is a useful by-product that can be included in the diet for poultry and fish. Effective treatment of animal waste and production of bacterial biomass requires knowledge of environmental conditions that favor growth of purple sulfur bacteria. Light intensity, anaerobic conditions, sulfide concentration, temperature, and pH have been reported to affect the growth of purple sulfur bacteria (Pfenning and Truper, 1989) with light intensity and sulfide concentrations the most important factors. However, light penetration and sulfide concentration are influenced by depth of the lagoon and the concentration of the waste. Studies in the past have focused on monitoring the presence of purple sulfur bacteria and the environmental conditions that existed where they were found. Most of these studies were done in lakes where the sulfide concentration and other nutrients are low and extent of light penetration is deeper than in waste treatment lagoons. The main emphasis of this research is to determine the effect of depth and surface area of a reactor on growth of purple sulfur bacteria using cattle waste as a substrate.

P121FIG1.jpg (32161 bytes)
Figure 1: Schematic representation of the overall system for the production and utilization of purple sulfur bacteria.

OBJECTIVES 
To utilize purple sulfur bacteria as a supplementary feed for animals, sufficient quantities of bacterial cells are needed. The production of bacterial cells is a result of anaerobic waste treatment. Figure 1 presents of the system under consideration, in a broader perspective.

The physical parameter of the reactor plays a very important role in modifying the abiotic factors that affect growth of purple sulfur bacteria. Depth of a reactor sets a limit for light reaching sulfide containing zones. Depth affects the quality and intensity of light reaching the sulfide containing laya and the thickness of the aerobic zone that may develop in the reactor. When the reactor is open to the atmosphere, oxygen will diffuse into the reactor creating an aerobic zone at the top of the reactor. Depth also affects the sulfide gradient that may develop in the reactor. Surface area of a reactor affects the total amount of light reaching the surface and the amount of oxygen that may diffuse into the reactor, the surface water that would get most of the light may become aerobic. Surface area is also important in suspended growth systems since this factor sets a limit to the number of bacteria that can grow at a particular depth. Previous studies do not provide sufficient information on factors that affect the growth of purple sulfur bacteria in a reactor that simulates a lagoon environment. The main objectives of this study were to fill information gaps and to investigate the possibility of producing purple sulfur bacteria in sufficient quantities for use as a feed supplement. The specific objectives were:
1. To determine the effect of reactor depth and surface area on purple sulfur bacteria growth.
2. To characterize the diel change of bacterial concentration in response to the changing sulfide concentration and light intensity, and
3. To develop a sulfate-sulfide balance model.

SULFUR BACTERIA

Bacteria that oxidize or reduce significant amounts of organic sulfur compounds exhibit a wide diversity of morphological and biochemical characteristics. One group, the sulfatereducing bacteria grow anaerobically and reduce sulfate, SO42-, to hydrogen sulfide, H2S. A second group, the photosynthetic green and purple sulfur bacteria, grow anaerobically in the presence of light and uses H2S as a electron donor for photosynthesis. The sulfurr is oxidized to sulfate or elemental sulfur. A third group, the aerobic sulfur-oxidizers, oxidizes reduced sulfur compounds aerobically to obtain energy for chemoautotrophic growth (Clesceri et al., 1989). The sulfate-reducing and the photosynthetic green and purple sulfur bacteria are the most important in anaerobic environment.

The purple sulfur bacteria grow anaerobically and can fix carbon by anoxygenic photosynthesis. Common to all species is the presence of bacteriochlorophyll-a and carotenoid pigments; only one species (Thiocapsa pfennigii) contains bacteriochlorophyll-b (Pfenning and Truper, 1989). The most commonly identified species of purple sulfur bacteria isolated from waste treatment lagoons are classified under the family Chromatiaceae and belong to the genus Chromatium, Thiocapsa, Thiospirillum, and Thiopedia. The most common species are Chromatium vinosum, Thiocapsa roseopersicina, and Thiopedia rosea.

Purple sulfur bacteria have been observed in lagoons 0.9 m to 4 m deep treating animal waste (McFarlance and Melcer, 1977), in a lagoon 0.9 to 2.5 m deep used for treating cattle waste (Wenke and Vogt, 1981), and in lagoons treating poultry waste with a depth range from 0.4 to 1.3 m (Dornbush and Andersen, 1964). McFarlane and Melcer (1977) reported that in a 5 m deep lagoon treating waste from a meat packing plant, no purple sulfur bacteria were evident, but subsequent incubation of a sludge sample in the light resulted in the growth of purple sulfur bacteria.

In wastes containing sulfate, the sulfate-reducing bacteria anaerobically reduce sulfate and other sulfur compounds to hydrogen sulfide, H 2S. At depths where light can penetrate the purple sulfur bacteria grow anaerobically and use hydrogen sulfide, H 2S, as an electron donor for photosynthesis, and the sulfide is oxidized to sulfur or sulfate. Thus, purple sulfur bacteria interact with many other species of bacteria and by closing the sulfur cycle, purple sulfur bacteria and sulfur reducing bacteria will stimulate each other's growth. It has been reported that light, oxygen concentration, and sulfide concentration control the presence and proliferation of purple sulfur bacteria. These abiotic factors are disscussed briefly.

Light

In natural water, light is both absorbed and scattered. In clear oceans, light penetrates up to about 100 m (Jerlov, 1976) but in most lakes, where dissolved and particulate organic matter are abundant, light penetrates to only the upper 10 m (Wetzel, 1975). In lagoons treating wastewater where the amount of dissolved and suspended solids are usually high, light penetrates even less. Interestingly, for the purple sulfur bacteria to thrive, not only the presence of light but also the intensity and the quality of light are important. For example, most Chromatiaceae tolerate light intensities up to about 500 UE m -2 s-1 (Eichler and Pfenning, 1991; Eichler and Pfenning, 1988), Thiopedia were usually observed in natural environment at light intensities of about 0.5 to 2 UE m -2 s-1 (Steenbergen and Formals, 1982). From a laboratory experiment using a tungsten lamp, Eichler and Pfenning (1991) reported that optimal growth of Thiopedia rosea occurred at light intensities of 100 UE m -2 s-1 and intensities above 150 UE m -2 s-1 inhibited growth completely.

Purple sulfur bacteria primarily use the visible component of the light spectrum (40~ 760 am wavelength). They can also use part of the infrared region, 800-900 nm wavelength, (Takahashi and Ichimura, 1970). The infrared region is strongly absorbed by water in Me tap few centimeters (0-50 cm); hence, it is significant only in shallow ponds and esturine environments. From laboratory experiments, Eichler and Pfenning (1991) reported a light transmission between 440 and 500 nmfor best growth of Thiopedia rosea.

Oven, Sulfide Concentration, pH, and Temperature

In addition to light, sulfide concentration and anaerobic condition are the necessary requirements for the presence and growth of purple sulfur bacteria. While most purple sulfurr bacteria are not tolerant to aerobic conditions, Thiopedia has been found to be abundant in oxygen concentration ranges from zero to 2.5 mg/L (Schegg, 1971; Genovese, 1963; Caldwell and Tiedje, 1975). Different species have a different level of tolerance to oxygen and sulfide concentrations. For instance, Thiopedia has some tolerance to aerobic conditions, whereas Chromatium species has no tolerance. On the other hand, Thiopedia is sensitive to high sulfide concentration, whereas Chromatium has a better tolerance to high sulfide levels. Depending on the concentration of sulfide and oxygen, Caldwell and Tiedje (1975) reported a stratification of different species of purple sulfur bacteria (oxygen tolerant species at the top and sulfide tolerance species at the lower part of the lake).

P121FIG2.gif (150315 bytes)
Figure 2. Layout and flow diagram of the experimental setup. (a) plan view,
(1)different surface area rea reactors (2) different  depth reactors (3) culture tank (b) side view of reactor.
Thiopedia is observed in sulfide concentration of less than 2 mg/L (Caldwell and Tiedje, 1975). Caldwell and Tiedje (1975) also reported a decline in the number of Thiopedia where the sulfide concentration was 4 to 8 mg/L. Photoautotrophic growth of lh~oped~a is inhibited by a sulfide concentration above 16.0 mg/L (Eichler and Pfenning, 1991). Takahashi and Ichimura (1970) reported that the presence of purple sulfur bacteria in a reservoir with a sulfide concentration of 40 mg/L and Holm and Vennes (1970) reported an optimum sulfide concentration of 45 to 60 mg/L for the purple sulfur bacteria in the lagoon environment. Moreover, on the basis of enrichement culture results, different species of purple sulfur bacteria have different sulfide tolerance levels ranging from (13-16 mg/L) for low sulfide tolerant species to (128-257 mg/L) for high sulfide tolerant species (Pfenning, 1978).

The purple sulfur bacteria are found in nature at temperatures ranging from 8 to 80 0C and a pH range of 6 to 9.0. Most species, however, exhibit an optimum at a temperature range between 18 and 30 OC and a pH range 7.0 to 8.5 (Pfenning, 1978).

MATERIALS AND METHODS

To conduct the experiment under natural light conditions, a physical setup consisting of twenty reactors was constructed beginning Fall 1992 and continuing through early 1993. Reactors were constructed from polyvinyl chloride (PVC) pipes. The experiment was divided into two groups. The first group consisted of different depth reactors with a constant surface area and the second group contained different surface area reactors at a constant depth. The first group of reactors consisted of depths of 0.9 m (3 ft), 1.8 m (6 ft), 2.7 m (9 ft), 3.7 m (12 ft) and 4.6 m (15 ft) with a constant diameter of 30 cm (12 in), each reactor was replicated twice. Duplicate reactors in the second group consisted of PVC pipes with inside diameters of 20 cm (8 in), 30 cm (12 in), 46 cm (18 in), and 61 cm (24 in) with a constant depth of 3 m (10 ft). Sampling ports were provided at various depths on the outer surface of all reactors to withdraw samples. A completely randomized design with two replications was used and the reactors are housed in two insulated buildings to reduced the diel temperature fluctuation. The process flow diagram for the design is shown in Figure 2. Two 1.8 m (6 ft) wide, 0.76 m (2.5 ft) deep circular tanks were placed to mix the raw feedlot waste and keep the purple sulfur bacteria culture used to inoculate the reactors. The experimental setup was fitted with the necessary valves, pipes, pumps, and mixers for unloading and loading the waste.

Reactor Loading and Inoculation

Cattle waste used to load the reactors was obtained from the Lubbock Feedlot located south east of Lubbock, TX. To determine the constituents of the cattle waste, six samples were collected for chemical analysis for solids, and major and minor nutrients (Table 1).

TABLE 1: Chemical composition of cattle waste used to load the reactors

Component Concentration, mg/L CV %a
Total solids 25,878 10
Total volatile solids 20,013 9
Total volatile solids, % 2.0  
TKN 329 12
Ammonia, NH3-N 264 3
Nitrate, NO3-N < 0.03 0
Sulfate,  SO4 640 7
Phosphate,  PO3 -P 231 12
Chloride, C1 693 6
Potassium, K 909 10
Calcium, Ca 682 8
Magnesium, Mg 208 7
Sodium, Na 33 1
Iron, Fe 16 12
Zinc, Zn 4 21

a CV(Coefficient of variation of six samples)

A loading rate, 2% volatile solids, with a theoretical 21 days hydraulic detention time was used. The different surface area reactors were first loaded on February 20, 1993, and the different depth reactors were first loaded on March 27, 1993. All the reactors were inoculated with a culture of purple sulfur bacteria obtained from the feedlot lagoon with a ratio of 1:2 (one part culture to two part waste by volume). The bacteriochlorophyll-a concentration of the inoculate culture was 1,800 mg/L. After the initial inoculation, a pre-determined amount of digested waste was drained from the bottom of the reactors and fresh waste was added at the top every week. The volume drained from the reactors was determined to achieve a 21 day hydraulic retention time. The following definitions were applied for hydraulic retention time and loading rate. Hydraulic retention time equals reactor volume divided by the volume of influent loaded. Loading rate equals influent volatile solids concentration times volume added divided by the reactor volume (Fischer, 1983).

Data Collection and Analysis

Temperature, light intensity, and dissolved oxygen concentration were measured at various depths. Liquid samples were collected for determinations of sulfide concentradon, bacienochlorophyll-a (Bchl a) content, pH, and other nutrients. Terrestrial and underwater photosynthetically active radiation (400 700 rim wavelength) were measured using LI- COR light sensors. Temperature measurements were done at various depths (surface, 0.3 m, 0.9 m, 1.8 m, and 2.7 m) using Copper/Constantan thermocouple wire. The light sensors and the thermocouple wires were connected to a Cambell Scientific 21-X Micrologger for automatic data recording and storage. The sulfide concentration of each sample taken from the reactors was measured using an ORION model 94-16 silver-sulfide ion electrode (ORION, 1989; ORION, 1992) within a 24-hour period. Oxygen concentration was measured bi-weekly using a portable YSI-5739 probe fitted with a 3 m (10 ft) long cable. Growth of purple sulfur bacteria were determined by measuring changes in bacterioch10rophyll-a concentrations (Austin, 1988). The samples for bacteriochlorophyll-a determination were frozen until they could be analyzed.

P121fig3.gif (43631 bytes)
Figure 3: Percent of the terrestrial photosynthetically active radiation reaching a given depth. The percentage is calculated from simultaneous measurements taken under water and above the water surface. Reactor (PSB): purple sulfur bacteria culture in the reactor, Reactor(Tap water): reactor filled with tap water, and Lagoon(PSB): in the lagoon purple sulfur bacteria culture.

RESULTS AND DISCUSSIONS

Light Intensity

The light intensity reaching below 2.5 cm (1 in) from the surface was less than 2% of the terrestrial radiation measured at the same instance. Measurements taken at the feedlot lagoon also indicated the same condition, Figure 3. Most of the radiation is rapidly absorbed by the water itself and dissolved and particulate matter contained in the water. A small portion is also absorbed by the bacterial cells on the surface. For the bacteria culture below 8 cm (3 in) from the surface, a light intensity less than 2 mE m~2 s-l was detected.

Photosynthetic bacteria are adapted to low light intensities and exist in environments where light intensity is less than 0.1% of the incident light (Parkin and Broclc, 1980; Mas et al., 1990). Where Thiopedia are found in natural environments, a light intensity of 0.5 to 2 m E m~2 s-l were reported (Stcecubergen and Korthals, 1982; Folt et al., 1989). On the other hand, from laboratory experiments using a tungsten lamp, Erichle and Pfenning (1991) reported optimal growth of Thiopedia occurred at light intensities of 100 mE m~2 s-l. In natural environments where purple sulfur bacteria are found, light intensities as high as 100 mE ~2 s-l could exist only in the top few millimeters, especially in highly turbid lagoon water.

Temperature

The mean- monthly temperature inside the 61 cm (24 in) wide reactor from April to December 1993 range from 24 C in July to 4 C in December and there was no observable temperature stratification inside the reactor during the experimental period. The daily temperature records indicated that the temperature fluctuation inside the reactor is negligible except at the surface. For example, the difference between the maximum and minimum temperature recorded on July 15, August 15, and September 15, 1993 was 4, 7, and 9 C, respectively, at the surface and less than 2 C for depths below the surface (Figure 4).

P121FIG4.jpg (265131 bytes)
Figure 4: Sample profile of daily temperature records. The bars indicate the temperature range, the difference between the maximum an minimum readings.

Dissolved Oxygen Concentration

During the experimental period the oxygen concentration inside the reactors was less than 2 mg/L close to the surface and not detectable at deeper depths. An oxygen concentration up to 4 mg/L was measured after heavy rain events. This made the surface layer (less that a few centimeters) relatively aerobic creating a micro-lake ecology.

In lakes where purple sulfur bacteria are found, the top layer is usually aerobic, but as the depth increases the layer becomes anaerobic. Purple sulfur bacteria are usually found between the interface of the anaerobic and aerobic layers (Fort et al., 1989; van Gemerden et al., 1985). Since the reactors are open to the atmosphere, the rain that has a relatively higher concentration of dissolved oxygen as compared with the water inside the reactor diluted the top few centimeters and increased the oxygen concentration and reduced the sulfide level. The purple sulfur bacteria migrated downwards to the anaerobic layer and completely disappeared from the surface layer. The increase in dissolved oxygen level was especially apparent in the culture tank, 1.8 m (6 ft) wide and 0.8 m (2.5 It) deep, used to inoculate the reactors with the purple sulfur bacteria which was not full at the time. The overall effect of the rain in the reactors was very minimum since the reactors were full and fresh waste was added from the top that mixed the surface layer with the layer beneath it.

Depth and Surface Area Effect

Depth and surface area of a reactor are thought to have an important effect in modifying environmental conditions that develop inside the reactor. To determine their effects liquid samples were taken from the venous ports and analyzed for bacteriochlorophyll-a. Analysis of variance on the treatment means of bacteriochlorophyll-a data was carried out and the treatments mean bacteriochlorophyll-a values from the five depth treatment were significantly different from each other at (a=0.025). However, the LSD analysis unveiled that the mean bacteriochlorophyll-a concentration from the 0.9 m (3 ft) deep reactor was significantly greater from the mean bacteriochlorophyll-a values of all the other reactors. And there is no significant difference between the values from any of the other treatments, i.e., the mean values from 1.8 m (6 ft), 2.7 m (9 ft), 3.7 m (12 ft), and 4.6 m (15 ft) deep reactors are not significantly different from each other, Figure 5. This suggests that the 0.9 m (3 ft) deep reactor was the best to grow the purple sulfur bacteria under natural conditions in the operating conditions discussed in this paper. To compare the bacterial concentration in the top 0.9 m (3 ft) depth of the different depth reactors, average bacteriochIorophyll-a values taken from ports located in the top 0.9 m (3 ft) depth of each reactor was analyzed and the mean  bactermchlorophyll-a values were significantly different from each other (a=0.05).

P121FIG5.jpg (157349 bytes)
Figure 5: Bacteriochlorophyll-a and pH values for the different depth reactors. The values are average of the samples taken from all the ports on November 21, 1993. Values marked by the same letters are not significantly different( a=0.1).
The result of this analysis reinforces the importance of depth of a reactor. The high bacterial concentration in the 0.9 m (3 ft) reactor was not simply because the top layer was more productive than the deeper layer, but the bacterial growth was also affected by rate of nutrient supply from the sludge layer. The difference between purple sulfur bacteria concentration in the reactors was also conspicuous from visual observation. Throughout the experimental period the 0.9 m (3 ft) deep reactors appeared deep pink while the purple sulfur bacteria in the other reactors were only lightly pigmented.

The treatments mean bacteriochlorophyll-a values from the different surface area reactors were not significantly different from each other. Within the range considered in this experiment, surface area had no significant effect on the growth of purple sulfur bacteria, Figure 6. The other difference apparent from Figures 4 and 5 is a positive correlation between pH and the corresponding bacteriochlorophyll-a concentration. In all cases, the higher bactermchlorophyll-a concentration corresponds to a higher pH value. Other studies done in feedlot lagoons indicated a similar positive correlation (Holm and Vennes, 1970; Wenke and Vogt, 1981). The pH values are also within the range reported by Holm and Vennes (1970) and Wenke and Vogt (1981). Since changes in pH in an anaerobic environment is a result of microbial activity, a slightly alkaline pH where purple sulfur bacteria grow may be a good indication that a suitable condition has been established.

Sulfate-Sulfide Cycle in a Sealed Reactor

The sulfur cycle in an anaerobic environment is entirely different from carbon decomposition under anaerobic conditions. The decomposition of organic matter into different carbon forms takes place in different stages. Different microorganisms in series are involved in decomposing the complex organic matter into simpler forms. The sulfur cycle in anaerobic environment mainly involves two major bacteria. The bacteria that reduces sulfate into hydrogen sulfide and the bacteria that utilize hydrogen sulfide for photosynthesis activity.

P121FIG6.jpg (24042 bytes)

Figure 6: BActeriochlorophyll-a and pH values for the different diameter reactors.The values are average of the samples taken from all the ports on November 21, 1993.

The photosynthetic activity of purple sulfur bacteria is also different from algae in that purple sulfur bacteria do not utilize their photosynthesis end product when there is no photosynthetic activity. Algae use carbon dioxide for photosynthetic activity and give off oxygen during the day time and at night oxygen is used for respiration and carbon dioxide is given off. In purple sulfur bacteria photosynthesis, the two sulfur bacteria cycle sulfur compounds where one uses the end product of the other. Sulfate is reduced by the sulfate reducing bacteria into hydrogen sulfide and the sulfide is utilized by purple sulfur bacteria as an electron donor for photosynthetic activity. Purple sulfur bacteria give off sulfate or elemental sulfur as a by-product of photosynthesis. Thus, sulfate reduction and hydrogen sulfide utilization takes place simultaneously by the two microorganisms. A schematic representation of the major sulfur cycle and the interaction between the sulfate reducing and purple sulfur bacteria is presented in Figure P121FIG7.jpg (37889 bytes)
Figure 7: Schematic representation of the major sulfur cycle in anaerobic environment.

Referring to Figure 7, if a reactor is sealed the amount of sulfide leaving the system and sulfide oxidized by oxygen are zero. In a stabilized system, sulfide precipitation by heavy metals is negligible and can be taken as zero. Neglecting the amount of sulfur stored in the cell and used for cell build up, the following mass balance is applicable for sulfide.

Rate of  Change of  Sulfide Concentration

=====

Rate of Sulfide  Production by  Sulfate Reducers       ____ Rate of Sulfide Utilization by
Purple Sulfur Bacteria

Similarly, the following mass balance is applicable for sulfate.

Rate of  Change of  Sulfide Concentration

=====

Rate of Sulfide  Production by  Purple Sulfur Bacteria       ____ Rate of Sulfide Utilization by
Sulfate Reducers
P121TIME.jpg (32369 bytes)
Figure 8:Diel variation of sulfate-fulfide (a) and bacteriochlorophyll-a (b) concentractions in a sealed 0.9m(3 ft) deep reactor. The data points are averages of the samples taken from the two reactors.
From these two mass balance equations, the concentrations of sulfide and sulfate are dependent on He activities of both the sulfur reducing bacteria and purple sulfur bacteria. It is difficult to measure the activity of the two bacteria independently in a natural environment. However, changes in the concentrations of sulfide and sulfate could be used to indicate the combined activity of the two types of bacteria.

To demonstrate the sulfate-sulfide cycle, the data taken from the two 0.9 m (3 R) deep sealed reactors on February 7, 1993 was examined. Water samples were collected from four ports of the two reactors at a bi-hourly interval and analyzed for sulfate, .sulfide and bactermchlophyll-a concentrations, Figure 8. The differences in the concentration of sulfide and sulfate of the samples taken from the different ports of each reactor are small.

After sunrise, between 0600 and 0800 hr. photosynthetic activity of purple sulfur bacteria resumes and the sulfide is used up and decreased linearly until 1400 hr and after 1400 hr the sulfide concentration in the reactors increased until it reached a steady state. The sulfate concentration generally decreased, however at a slower rate, Figure 8(a). Between 600 and 1400 hr the sulfide was utilized much faster by the purple sulfur bacteria than it was supplied by He sulfate reducing bacteria through sulfate reduction. The empirical model that describes the diet change of sulfide concentration is:

    y = b - ax  for  x 1400 hr

al *(1-Exp(-a2 *(x-8 ) ) )     for x > 1400 hr

Where x is hours since photosynthesis begins, hr y is sulfide concentration, mg/L, and

a= 23. mg/L hr. b=226 mg/L, al = 153mg/L, and a2=0.673/hr.

Between 600 hr and 1400 hr. the sulfide concentration was reduced from 232 mg/L to 33 mg/L. This reduction is about 86% and the average rate of change of sulfide concentration was 25 mg L-1 hr~1. Multiplying by the volume (67 L) of the reactor, 1675 mg of sulfide per hour was utilized between 600 hr and 1400 hr. Since sulfide is also produced through sulfate reduction, the amount

P121FIG8.jpg (35204 bytes)
Figure 9: Relationship between sulfide concentration and bacteriochlorophyll-a content. From samples taken from the surface only.
of sulfide utilized by the purple sulfur bacteria was greater than 1.7 g/hr. After sunrise the bacteriochlorophyll-a concentration started increasing and reached a maximum around 1400 hr. The time at which the highest concentration of bacteriochlorophyll-a occurred corresponds to the lowest sulfide concentration (Figure 8) and the maximum daily solar radiation. The diel change in bacteriochlorophyll-a concentration follows the diurnal change in solar radiation. Even though, there was sunlight after 1400 hr for photosynthetic activity, the bacteriochlorophyll-a concentration started declining and sulfide concentration started increasing. Between 600 and 1400 hr the bacteriochlorophyll-a concentration increased from 2140 mg/L to 2722 mg/L this difference is about 600 mg/L. Assuming one gram of bacteriochlorophyll-a yields one gram of dry cell mass, if the bacterial cells were harvested from the entire reactor at 1400 hr about 182 gm could be recovered at one harvest. The rate of change of bacteriochlorophyll-a content, between 600 and 1400 hr. was 75 mg L-1 hr~1. The change in bacteriochlorophyll-a content in the whole reactor was 5025 m g/hr. For every milligram of sulfide used about 3.0 mg of bacteriochlorophyll-a was produced.

Sulfide Concentration

In almost all studies dealing with purple sulfur bacteria, either in the field or under laboratory conditions, sulfide concentration and bacteriochlorophyll-a content were measured. Some investigators have indicated that sulfide concentration inhibits growth of the bacteria if it is above a certain concentration. However, the levels reported varies from author to author. From the experimental setup used in this research, attempts were made to establish a relationship between sulfide concentration and bacteriochlorophyll-a content from the samples taken from the surface only the open reactors at various dates between 1 100 hr and 1200 hr. Sulfide concentration could either limit or inhibit the growth of purple sulfur bacteria. Below and above about 7 to 8 mg/L of sulfide concentration, bacteriochlorophyll-a content was lower having a peak at 7 to 8 mg/L, Figure 9. However, from the sealed reactor data, a sulfide concentration as high as 230 mg/L was recorded at 0600 hr and 90 mg/L at 1200 hr. The corresponding bacteriochlorophyll-a concentration were 2100 ~g/L at 600 hr and 2630 mg/L at 1200 hr. These bacteriochlorophyll-a concentrations are three times higher than the values obtained either from the different surface area reactors or from the reactors deeper than 0.9 m (3 ft). If, in fact, sulfide concentration is inhibiting, it should be reflected in the values of bacteriochlorophyll-a content. The tolerance to high sulfide levels measured in the sealed reactors may be explained as follows.

The purple sulfur bacteria culture in the reactors is composed of different species and e sulfide concentration increases with depth, Figure 10. The species that have higher tolerance to high sulfide concentrations and no tolerance to oxygen concentration grow close to e sludge layer andvise versa (Caldwell and Tiedje, 1975). When the reactors were sealed, the purple sulfur bacteria species that tolerate high sulfide concentrations and no tolerance to oxygen concentration may have dominated the system.

P121FIG9.jpg (32690 bytes)
Figure 10: Sulfide variation with depth in the sealed reactors.

SUMMARY AND CONCLUSION

The most common method of treating feedlot waste is with lagoons. Purple sulfur bacteria grow photosynthetically using hydrogen sulfide as an electron donor in anaerobic lagoons. Utilization of hydrogen sulfide by purple sulfur bacteria reduces lagoon odor levels. Purple sulfur bacteria also create a slightly alkaline environment that favors the dissociation of hydrogen sulfide into bi-sulfide ion (HS-). Since the HS- is not a gas, it stays in solution and this contributes to reduction of odor from hydrogen sulfide.

Besides odor reduction from lagoons, purple sulfur bacteria have been considered as a supplementary feed for animals, especially for fish and poultry. Effective treatment of animal waste and production of bacterial biomass requires knowledge of environmental conditions that favor the growth of purple sulfur bacteria. Among the factors that affect the growth of purple sulfur bacteria, sulfide concentration and light intensity are the most important factors.

A field experiment that consisted of circular reactors with five different depths and four different surface areas was conducted under natural light conditions. Growth of purple sulfur bacteria were determined by measuring changes in bacteriochlorophyll-a concentrations.

Most of the sunlight, 98%, was absorbed at the surface, hence effect of light intensity was only significant in the top few millimeters of the bacterial culture. Sulfide concentration could either limit or inhibit the growth purple sulfur bacteria. The bacteriochlorophyll-a  concentration in the shallowest reactor, 0.9 m (3 ft) was 2 to 3 times greater than the other reactors that went to a depth of 4.6 m (15 ft). The treatment mean bacteriochlorophyll-a values from the different depth reactors were significantly different (a=0.025), but the mean bactenochlorophyll-a values from the various surface area reactors were not significantly different. Thus, depth but not surface area of reactors influenced the growth of purple sulfur bacteria.

In sealed reactor, the sulfide concentration decreased linearly by 86% between 0600 hr and 1400 hr. During the same period, the bacteriochlorophyll-a concentration increased by 27 %. For every milligram of sulfide used, about 3 mg of bacteriochlorophyll-a was produced. From the reactor (volume of 67 L), a maximum of 182 gm bacterial cell could have been recovered during a single harvest if the bacterial cells were harvested when bacteriochlorophyll-a was maximum (around 1400 hr).

REFERENCES

Austin, B. 1988. Methods in aquatic bacteriology. John Wiley and Sons, New York, N. Y.

Caldwell, D. E., and J. M., Tiedje. 1975. The structure of anaerobic bacterial communities in the hypolimnia of several Michigan lakes. Can J. Microbiol 21:377-385.

Clesceri L. S., A. E. Greenberg and R. R. Trussell. 1989. Sulfur bacteria. Standard methods for the examination of water and waste water, 17th edition. American Public Health

Association and American Water Works Association, Washington; D.C.

Dornbush, J. N., and J. R. Andersen. 1964. Lagooning of livestock waste in South Dakota. Proceeding of the 19th Industrial Waste Conference, Purdue University. 317-325.

Eichler, B., and N. Pfenning. 1988. A new purple sulfur bacterium from stratified fresh water lakes, Amoebobacter purpureus sp. nov. Arch. Microbiol 149:395-400.

Eichler, B., and N. Pfenning. 1991. Isolation and characteristics of Thiopedia rosea (neotype). Arch. Microbiol. 155:210-216.

Fisher, J. R. 1983. Methane production from livestock waste. Proceedings of the 1983 livestock waste management conference. University of Illinois at Urbana-Champaign.

Folt, C. L., M. J. Wevers, M.. P. Yoder-Williams and R. P.Howmiller. 1989. Field study comparing growth and viability of a population of phototrophictacteria App1. Environ. Microbio. 55:78-85.

Genovese, S. 1963. The distribution of H2S in the Lake of Faro (Messina) with particular regard to the presence of "Red Water" p.. 194-204. In: C. H. Oppenheimer (ed.) Marine

microbiology. Charles C. Thomas, Publisher, Springfield, IL. (ed.)

Guerrero, R., E. Montesinos, C. Pedros-Alio, I. Esteve, J. Mas, H. v. Gemerden, P. A. G. Hofman, and J. F. Bakker. 1985. Phototrophic sulfur bacteria in two Spanish lakes: Vertical distribution and limiting factors. Limnol. Oceanogr. 30:919-931.

Hart, S. A. and M. E. Turner. 1965. Lagoons for livestock manure. J. Water Pollut. Control Fed. 37:1578-1596.

Holm, H. W. and J. W. Vennes. 1970. Occurrence of purple sulfur bacteria in a sewage treatment lagoon. Appl. Microbiol. 19:988-996.

Jensen, R. 1991. Agricultural role in nonpoint source pollution. Texas Water Resources Institute, College Station, TX. 17(1): 1 -6.

Jerlov, N. G. 1976. Marine optics. Elsevier, Amsterdam.

Kobayashi, M. and Y. T. Tchan. 1973. Treatment of industrial waste solutions and production of  useful by-products using a photosynthetic bacterial method. Australia. Water Res. 7: 1219-1224.

Lotringen, J. M. V. and J. B. Gerrish. 1978. H2S removal by purple sulfur bacteria in swine waste lagoons. Proceeding of the 32th Industrial Waste Conference: Purdue University, 440-448.

Mas, J., C. Pedros-Alio, and R. Guerrero. 1990. In situ specific loss and growth rate of purple sulfur bacteria in Lake Ciso. FEMS Microbial Ecology. 73:271-281.

McFarlane, P. N., and H. Melcer. 1977. The occurrence of purple sulfur bacteria in anaerobic lagoons-Theory and application. Proceeding of the 32th Industrial.Waste Conference,

Purdue University. 497-506.

ORION. 1989. Silver-sulfide electrode Model 94-16 user manual, Boston, MA.

ORION. 1992. Ion selective electrode catalog and guide to ion analysis, Boston, MA.

Overmann, J., J. T. Beatty, N. Pfenning and T. G. Northcote. 1991. Characterization of a dense, purple sulfur bacterial layer in a meromictic salt lake. Limnol. Oceanogr. 36:846-859.

Parkin, T. B. and T. D. Brock. 1980. Photosynthetic bacterial production in lakes: The effect of light intensity. Limnol. Oceanogr. 25:711-718.

Pfenning, N. 1978. General physiology and ecology of photosynthetic bacteria. In: R. K. Clayton, and W. R. Sistrom (ed.) p 3-18. The photosynthetic bacteria. Plenum Press,

New York, NY.

Pfenning, N., and H. G. Truper. 1989. Anoxygenic phototropic bacteria. In: J. T.Staley, M. P. Bryant, and J. G. Holt (eds.) p 1635-1708. Berg's manual of systematic bacteriology vol. 3. Williams and Wilkins. Baltimore, MD.

Schegg, E. 1971. Production and destruction in the trophogenic. Schweiz. Z. Hydrol. 33:425- , 532.

Siefert, E., R. L. Irgens and N. Pfenning. 1978. Phototrophic purple and green bacteria in a sewage treatment plant. Appl. Environ. Microbio. 35: 38-44.

Sletten, O. and R. H. Singer. 1971. Sulfur bacteria in red lagoons. J. Water Pollut. Control Fed. 43:2118-2122.

Steenbergen, C. L. M., and H. J. Korthals. 1982. Distribution of Phototrophic microorganisms In the anaerobic and microaerophilic strata of the lake Vechten (The Netherlands): Pigment

analysis and role in primary production. Limnol. Oceanogr. 27:883-895.

Sweeten, J. and S. Melvin,. 1985. Controlling water pollution from nonpoint source pollution. EPA, Washington, D.C.

Takahashi, M. and S. Ichimura. 1968. Vertical distribution and organic matter production of photosynthetic sulfur bacteria in Japanese lakes. Limnol. Oceanogr. 13:644-655.

Takahashi, M. and S. Ichimura. 1970. Photosynthetic properties and growth of photosynthetic sulfur bacteria in lakes. Limnol. Oceanogr. 15:929-944.

van Gemerden, H., E. Montesinos, J. Mas and R. Guerrero. 1985. Diel cycle of metabolism of phototrophic purple sulfur bacteria in lake Ciso(Spain). Limnol. Occanogr..30:932943.

Wenke, T. L. and J. C. Vogt. 1981. Temporal changes in a pink feedlot lagoon. Appl. Environ. Microbio.41:381 -385.

Wetzel, R. G. 1975. Limnology. Saunders, Philadelphia, PA.