139
An initial examination of the contents of commercial aquatic
microbial algae suppressing formulations
Paul H. Lord
0
INTRODUCTION
At least five commercial products are currently marketed with the claim that they
inhibit algae growth in fresh water ponds by introducing bacteria. These products are:
1. Pond Saver® by Plant Health Care, Incorporated® (1997);
2. Cygnet Bio Blend® by Aquatic Nuisance Plant Control and Cygnet Enterprises,
Incorporated® (1998);
3. Bacta-Pur Klear Bacteria® by Aquatic Eco-systems, IncorporatecfV (undated);
4. Pond Clarifier® by Organica, Incorporated® (1999); and
5. LakePak™ WSp® by Becker-Underwood, Incorporated® (1998).
These products appear to be based on research initiated in the 1970s (Shilo, 1970;
Granhall and Berg, 1972; Burnham, 1973; 1975; 1977; Daft, et aI., 1973; Reim et al.,
1974; Martin, 1976; Burnham et aI., 1976; Darveau and Lynch, 1977; Delucca and
McCracken, 1977; Mayfield and Inniss, 1978; Berger, 1979) after bacterial associations
with algae cultures were noted in the 1960s (Safferman and Morris, 1962; Mayer et aI.,
1964; Blasco, 1965; Shilo, 1967). Some ofthe 1970s research documented the
competition for usable nitrogen compounds between aquatic (photosynthetic) algae and
aquatic (nonphotosynthetic) bacteria. Follow-up research in the early 1980s continued
(Yamakana and Glazer, 1980; Gromov and Mamkaeva, 1980; Burnham et al., 1981,
1984; Burnham, 1981 a; 1981 b; 1984; Cole, 1982; Ha1emejko, 1984). However, research
in the later 1980s and 1990s, into this potential algae control mechanism, is sparse with
only a few known related papers published (Coder and Goff, 1986; Peterson et aI., 1993).
By way of contrast, there are a larger number of papers published in the last two decades
discussing the use of bacteria as inhibitors of various aquatic macrophytes (e.g., Gunner,
1982; 1983; 1984; Galbraith, 1984a; 1984b; Pennington, 1984; 1985; Underwood, 1991;
Shearer, 1997). Recent texts devoted to the algae (Vymazal, 1995; Stevenson, et aI.,
1996) devote less than a half page each to adverse bacterial interactions with them. A
review of the literature identified 17 bacteria taxa likely to be used in such formulations.
These are identified in Appendix A.
Despite manufacturers' marketing claims, local experience has shown that only
rarely do bacterial formulations make a noticeable improvement in water quality
(Harman, 1998). During 1998 the use of Pond Saver in application on a small private
pond was supervised by Albright (1999). The bacteria provided no noticeable difference
o SUNY Oneonta biology graduate student enrolled Graduate Microbiology (taught by Professor L.
Bukovsan). Present address: 100 Sunset Ridge, Cooperstown, N.Y. 13326; E-mait [email protected];
telephone: (607) 547-5962.
140
whereas the subsequent application of alum significantly improved water clarity
(Albright, 2000).
This study examined the contents of commercial microbial products to determine
their consistency with earlier research and present-day advertising, and then to investigate
their effectiveness. Commercial preparations were cultured, microorganisms isolated and
identifications were attempted using standard methods (Rodina, 1972; Bukovsan, 1986;
Austin, 1988; Leboffe, 1996; Prescott, 1996). Additionally, algae were cultured in flasks
and the formulations were tested, according to manufacturer directions and in excess, to
determine algaecidal character. Finally, product content was examined for sand, total
phosphorous, and nitrates-nitrites.
MATERIALS AND METHODS
A one liter bottle of Bacta-Pur Klear®, a one pound (.455 kg) box of Organica
Pond Clarifier® and a three pound (1.36 kg) box of Pond Saver® were obtained from the
previously identified commercial sources. Taking note of Pond Saver's advertised claim
of four billion colony-forming units (CFUs) per gram, dilutions of Pond Saver were
initiated per Appendix B (Table 1) to verify the CFUs and to identify colony types.
Appendix B, Table 2 was derived from Organica, Incorporated claims of 400 billion
bacteria per pound. Assuming Bacta-Pur Klear would provide similar CFUs per dosage,
dilutions of that product were initiated per Appendix B, Tables 3, 4, and 5. Liquid
Difco®brain heart infusion (BHI) nutrient agar was mixed with 0.1 ml of dilutions 2, 3,
and 4 for each product using the pour plate methodology (Bukovsan, 1986) and incubated
at 20° (+1- 2°) C. Additionally, a O.1ml sample of pure Bacta-Pur Klear was mixed with
the BHI nutrient agar and incubated identically. Observations were made at 24 and 48
hours and noted.
After initial observations, a revision of Appendix B, Table 5, was designed
(Appendix B, Table 6) to provide satisfactory colony count ranges for Bacta-Pur Klear.
To avoid widely varying CFUs in plates cultured from dilutions ofthe dry
products (i.e., Pond Saver and Pond Clarifier), products were hydrated prior to use.
Aquarium bubble stones (AquaFizzle® Art. #A-762) and clear plastic aquarium tubing
(Blue Ribbon Pet Products Inc.®) plugged with fiber plugs (Brentwood Salon Care® Prod.
No. 504200) were autoclaved at 121°C and 15 psi for 20 minutes. Air was then pumped
through the tubing, plugs, and bubble stones into the Dilution 0 bottles for 24 hours
before it was used to form susequent dilutions. Buffered water was prepared per APHA,
AWWA, WPCF (1992) in order to ensure pH requirements of manufacturers. Each
dilution was stirred for a minimum of one minute with a sterilized Teflon® covered
magnetic stirrer and then shaken for 30 seconds prior to further dilution and inoculation.
To avoid "cooking" any heat sensitive bacteria, spread plates (as opposed to pour
plates) were used. This was accomplished using an ethyl alcohol washed and flamed "L"
141
shaped bent stirring rod (Drygalski spatula) which was used to push the O.lml sample
over the entire surface of the petri plate (Bukovsan, 1998; Austin, 1988). The ethyl
alcohol was cooled in an ice bath and two separate spatulas were alternately used to
prevent overheating with continued use and flaming.
Dilutions of all products were streaked on Difco Plate Count Agar (PCA), Difco
Brain Heart Infusion Agar, and NWRI agar prepared per APHA, AWWA, WPCF (1992).
All plates were incubated at 200 (+/- 2 ° C).
Additionally, an attempt, patterned after Burnham (1973), was made to grow the
bacteria from Bacta-Pur Klear and Pond Saver on media prepared from fresh algae
(tentatively identified as Ankistrodesmus faicatus (Palmer, 1962)). Water from a BFS
aquarium that had maintained a monospecific bloom of green algae for several months
was filtered using a Whatman® 4.7cm glass microfibre filter (cat no. 1820047) and a
vacuum pump. When the filter became clogged, the algae and filter were placed in a
screw-top 250-ml Erlenmeyer flask and filled to the 125-ml mark with a solution of agar
made from 25 g of agar and 500 ml of water heated to boiling. These flasks were then
autoc1aved at 121 DC and 15 psi for 20 minutes. The media was then used in spread
plates.
Anabaena sp. (tentatively identified asA. constricta [cf Palmer, 1962]) and
Chiorella pyrenoidosa (identified by Ward's Natural Science Establishment
Incorporated®) were obtained from Ward's Natural Science Establishment Inc. Attempts
were made to culture these algae using a variety of media, light regimens, and mixing
techniques. After initial testing, the Chiorella pyrenoidosa was cultured using a modified
Bristol's solution (Ward's Natural Science Establishment, Inc., undated, after Bold 1949).
This media was Bristol's solution (80%) with minor alterations to micronutrients and
20% soil-water medium (Ward's Natural Science Establishment, Inc., undated).
Additionally, C. pyrenoidosa was grown on Ward's supplied Proteose Agar slants that
were made of this media supplemented with proteose peptone and agar (Ward's Natural
Science Establishment, Inc., undated). The Anabaena sp. was cultured using a proteose
peptone Bristol's solution medium (Seeber, 1982). Both algae were cultured with a light
regimen which maintained a lighted cool-white fluorescent light 18 inches (45.5 cm) from
the bottom of the flasks for 16 hours out of every 24 (5 :00 AM to 9:00 PM; by use of a
Paragon TI+MITE® timer) to simulate summer epilimnion conditions. No shaking was
used. Anabaena sp. flasks were swirled once every four or five days. C. pyrenoidosa
flasks were not mixed or swirled. Lighting procedures were established based on Zeikus
(1998) and on recommendations from Ward's Natural Science Establishment, Inc.
technical representatives.
Bacteria cultured were preliminarily compared to the documented characteristics
of the bacteria likely to be encountered (Appendix C).
142
Living cultures of A. constricta and C. pyrenoidosa were exposed to various
dilutions of the bacterial products. These products were tested in extreme concentrations
(>2000x recommended first dose). Later they were tested in their recommended first
dosage (as calculated in Appendix D). Absorbency at 664nm, as determined with the use
of the BFS Milton Roy® Spectrometer, was used as an indicator of algae culture
concentration as that wavelength has the maximum absorbency for Chlorophylla, per
APHA, AWWA, WPCF (1992).
The three products were analyzed by Albright (1999) for total phosphorous and
nitrate+nitrites per APHA, AWWA, WPCF (1992) using the ascorbic acid method
following persulfate digestion and the cadium reduction method, respectively.
RESULTS AND DISCUSSION
Most ofthe Pond Saver and Pond Clarifier did not dissolve when mixed with
water. This was the case whether the material was soaked for minutes or days, bubbled or
not. Gritty particulate matter, which looked like common sand, resulted from soaking
Pond Saver and Pond Clarifier. This particulate matter made creating dilutions difficult
and made counting resulting colonies tedious. Bacta-Pur Klear, on the other hand, had no
particulate component.
Product CFU raw data are contained in Appendix E. The best CFU count
obtained for each product is as follows:
•
•
•
Pond Saver:
Pond Clarifier:
Bacta-Pur Klear:
1O,000,000/g (as opposed to 4 billion claimed).
300,000/g (as opposed to 882,000 claimed).
9,000/ml.
Twelve bacteria and one fungus (most likely a contaminant; see "Discussion and
Conclusions") were cultured from the products. They are described in Appendix F.
More colonies grew using the Drygalski spread plate technique than did using the
pour plate technique.
The cooked algae agar media produced only a small proportion ofthe colonies
produced with the other media. PCA media produced the largest colony counts with
Pond Saver while BHI Nutrient Agar produced the largest number of colonies with the
other products.
Products had immediate visually noticeable clarifying impacts on C. pyrendoidosa
when added in extreme dosages. The impacts on Anabaena sp. were not so pronounced.
Tables 1-4 compare spectrophotometric readings (absorbancy at 664 nm) of the algae
cultures immediately before and 72 hours after the addition of the algicidal products at
extreme (Tables 1 and 2) and recommended (Tables 3 and 4) dosages. That wavelength
143
was used because chlorophyll a has a maximum absorbancy in that range. The intent was
that absorbancy readings would provide relative changes in chlorophyll a concentrations
(a proxy for algae densities) following product addition. However, it became evident
that, due to the nature of the product, suspended particulate material associated with the
products caused turbidity. This undoubtedly affected absorbancy readings and confuses
the contribution by algae and the products themselves.
An additional problem was encountered when using Anabaena sp. It tended to
grow in a filamentous fonn. Because it could not be homogenized throughout the
sample, spectrophotpmetric readings did not accurately reflect its densities.
Total phosphorous and nitrates-nitrites found in the products are identified in
Table 5.
Table 1. Anabaena culture absorbency at 664 nm with extreme product dosage.
Distilled Water Blank:
Control Broth:
Pond Saver:
Pond Clarifier:
Bacta-Pur Klear:
Before
Product
Inoculation
After
72 Hours
0.000
0.135
0.125
0.235
0.223
0.000
0.102
2.418
0.424
0.361
% of Original
Clarity
Absorbency
After 72 Hours
75%
1934%
180%
162%
NA
NA
Table 2. C. pyrenoidosa culture absorbency at 664 nm with extreme product dosage.
Distilled Water Blank:
Control Broth:
Pond Saver:
Pond Clarifier:
Bacta-Pur Klear:
Before
Product
Inoculation
After
72 Hours
0.000
0.013
0.012
0.013
0.012
0.000
0.024
0.268
0.024
0.209
% of Original
Clarity
Absorbency
After 72 Hours
185%
2233%
185%
1742%
NA
NA
144
Table 3. Anabaena culture absorbency at 664 run with recommended first product dosage.
Before
Product
Inoculation
Distilled Water Blank:
Control Broth:
Pond Saver:
Pond Clarifier:
Bacta-Pur Klear:
0.000
0.225
0.109
0.072
0.250
% of Original
Absorbency
After 72 Hours
After
48 Hours
0.000
0.235
0.092
0.058
0.160
104%
84%
81%
64%
Clarity
NA
NA
+
+
+
Table 4. C. pyrenoidosa culture absorbency at 664 nm with recommended first product dosage
Before
Product
Inoculation
Control Slant with no algae:
Control Slant:
Pond Saver:
Pond Clarifier:
Bacta-Pur Klear:
0.000
0.815
0.955
0.518
0.429
After
48 Hours
% of Original
Absorbency
After 72 Hours
0.000
0.825
0.770
0.642
0.666
101%
81%
124%
155%
Clarity
NA
NA
+
Table 5. Total phosphorous and nitrate-nitrites for products
Pond Saver
Pond Clarifier
Baeta-Pur Klear
Total Phosphorous
Nitrate+Nitrites
3830mg/kg
210mg/kg
310mg/liter
.06g/kg
.17g/kg
1.13g/liter
DISCUSSION AND CONCLUSIONS
Identification of Species
As discussed in Ford (1993), remarkably little is known about the bacteria of fresh
water. While many of the bacteria found in sediments and in water are known, it is not
clear that the ones most easily cultured and most often observed are the most active in the
aquatic environment (Ford, 1993). In three separate iterations of culturing the three
products in this study, the noted bacteria were consistently observed. Other smaller
colonies may well have contained other species not noted as different. It is possible that
some species of bacteria found in the formulations did not culture on any of the three
145
media used, although those media were chosen because they are reputed to ensure the
best possible growth.
DNA advances have changed the taxonomy of bacteria, complicating
identification of those that are algicidal. As noted earlier in this paper, many of the
bacteria involved have been reclassified. Although the translation between current
taxonomy and the older names has been made in most cases, some of the old names have
no known (to the author) correspondence (e.g., Cellvibrio spp.). Additionally, current
volumes of "Bergey's" (Kreig and Holt, 1984; Sneath, 1986; Staley, J. T., 1989;
Williams, 1989) associate characteristics with candidate species opposite to those
described in the original literature (e.g., Gram staining results for Arthobacter sp. and
oxidase reaction results for Flavobacterium spp.).
Pour and Spread Plate Techniques.
The probable cause of spread plate growth outperforming pour plate growth was
the heat sensitivity of the aquatic microorganisms involved although strict aerobic
requirements may have been responsible for some of the differences observed.
The Drygalski spatula technique worked best in this investigation, but is
problematic. The spatula tended to overheat with continued use and flaming. The weight
of the spatula tended to abrade the surface of the medium if the media was soft, making
colony counting tedious. Using the spatula left the plate much more exposed to air
currents - and contaminants (e.g., the Penicillium sp. cultured) than when using a
bacteriological loop. This was due to its size and the requirement to spread the droplet
across the entire media surface. If moisture was present inside the petri plate, the addition
of the O.lml innoculum would cause drops to fall off the media onto the petri plate cover
when the plate was inverted. This removed product from contact with the media and
resulted in lowered CFU counts.
CFUs Claimed. An immediate judgment might be made disputing the veracity of
advertising claims of four billion CFUs per gram for Pond Saver and 400 billion CFUs
per pound for Pond Clarifier; however, difficulties growing aquatic bacteria on nutrient
rich data are well documented (Ford, 1993 after Buck, 1979). Additionally, some of the
bacteria cultured grew aggressively on the media while others barely showed their
presence in 48 hours. When attempts were made to count plates at 72 hours, lower CFU
counts resulted due to the aggressive bacteria growing over/around the slower growing
bacteria. This research was done to validate advertised claims. CFU counts were
performed repeatedly to overcome issues such as those experienced using the spread plate
technique discussed above. It is unfortunate that none of the purveyors of products
claiming to provide bacterial suppression of aquatic algae would share with the BFS their
methodologies for culturing and testing their products (Lord, unpubl; Albright, 1998). It
can only be concluded that CFU count claims cannot be verified.
146
Media Perfonnance
Bacterial Media. NWRI was, by far, the most difficult media to prepare of
the three used. Preparation ofthe others was trivial by comparison. Counting colonies
with NWRI was easier than with any other media. Its clarity made counting
straightforward. PCA was the darkest tinted, but tended to be softer causing problems
with the Drygalski spatula technique. NWRI media did not produce more colonies than
the other media contrary to APHA, AWWA, WPCF (1992). In spite of its second best
perfonnance in culturing Pond Saver bacteria, BHI nutrient agar had to be used on all
isolated bacterial types to obtain cell and colony morphologies that were consistent with
that found in most of the literature.
Algae Media. Anabaena sp. tended to grow slowly and was clumpy in all
media, but most consistently in proteose peptone supplemented Bristol's solution. It
failed to grow on the Ward's supplied Proteose Agar slants. C. pyrenoidosa would grow
in a variety of media, but most consistently on the Proteose Agar slants followed by the
soil-water supplemented modified Bristol's solution.
Product Impact on Tested Algae. In recommended dosages, Pond Saver appears
to curtail the growth of C. pyrenoidosa and Anabaena sp. Both Pond Clarifier and Bacta­
Pur Klear appear to curtail Anabaena sp. while aiding the growth of C. pyrenoidosa.
Excessive doses of all products appear to cloud the water tested while inhibiting algae.
Time limitations and a paucity of cultured algae prevented testing in replicate.
Most Likely Product Origins. If made totally soluble, the total phosphorous
content of a kg of Pond Saver is enough to produce a "nuisance algal bloom"
(Mackenthun 1965) in a 255,333 liter pond using the criteria outlined in Mackenthun
(1965) and Sawyer (1969). For Pond Clarifier, the size pond so polluted would be 14,000
liters and for Bacta-Pur Klear the size would be 20,667liters. Similarly, the nitrate-nitrite
component will support algae blooms. Safennan and Morris (1963) noted that algicidal
bacteria were much more common in "bottom and bank: muds" than in effluent "pond
water". Given the sand, phosphorus and nitrate-nitrite content of Pond Saver and Pond
Clarifier, it is likely that they are extractions from sand filters used to clarify sewage
treatment effluent (Other researchers cited herein [e.g., Reim, et al., 1974; Daft et al.,
1973] also collected their bacteria in ponds associated with sewage treatment.) That
being said, Ford (1993) notes that billions of bacteria can be found in every cubic
centimeter of sediment. This makes the products' claims appear reasonable - and the
products' contents unremarkable. Bacta-Pur Klear origins are much less apparent, but it
could be produced by settling and filtering the waters outputted from such sand filters.
Duration of effect. As noted in Martin (1976), Delucca and McCracken (1977),
Cole (1982), and Burnham (1984), and as is apparent in other research (Berger, 1979),
certain bacteria have been found to favor one algae over another. This could lead to the
apparent suppression of algae in a body of water when, in fact, the improved transparency
is actually visible evidence of algae succession facilitated by the introduction of the
147
bacteria. The long-tenn impact of the introduction of certain bacteria (e.g., Pseudomonas
sp., Flavobacterium sp., and Xanthomonas sp.) may actually be to lessen transparency.
Additionally, researchers have noted that certain algae lysing bacteria are "good
parasites" (Bukovsan, 1998) in that they either fail to kill all their hosts (Code and Starr,
1978) or that they have no impact on heterocysts which would ensure future blooms
(Shilo, 1970). Other researchers have noted that the bacilli exude their algae killing
products while creating spores in their postlogrithmic phase (Reim et al., 1974) which
implies a sine wave predator-prey model. Daft et al. (1973) note that algae and bacteria
numbers fluctuated in "a similar manner" while Burnham et al. (1984) note predator-prey
cycles of about nine days.
Recommendations regarding use. While these products may suppress a particular
algae bloom, they do so in a manner that leaves a pond susceptible to blooms involving
other algae species while facilitating subsequent blooms with increased nutrients. The
long-tenn health of most ponds would be facilitated with a dose of alum (Anon., 1990) as
opposed to the products evaluated here.
Recommended future research.
• Bacteria in these compounds should be identified to species if possible to
confinn or refute suspected origins and to identify potential pathogens.
• Product impact on algae should be tested in replicate.
• Soluble phosphorous content of these products, in recommended usage
concentrations, should be evaluated.
• If vendor production and CFU counting procedures become available, further
attempts should be made to validate or correct CFU counts claimed.
• Given the suspected source of these products, they should be tested for the
presence of heavy metals.
CONCLUSION
As a result of the foregoing, it can be concluded that these products probably can
offer ephemeral relief from certain types of pond algae blooms. However, nutrients will
increase which will exacerbate future blooms. Additional work is recommended to fully
identify product contents.
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155
Appendix A:
Bacteria taxa identified as likely components of commercial microbial products.
Achromobacter sp. (Mamkaeva, 1966; Kreig and Holt, 1984) formerly used name
some Alcaligenes and some Acinetobacter;
Actinomycetes (Safferman and Morris, 1962 and 1963).
Alcaligenes sp. (Martin, 1976);
4. Arthrobacter sp. (Berger, et a!', 1979);
5. Bdellovibrio bacteriovorus (Burnham, 1973; Burnham et al., 1976);
6. Cellvibrio spp. (Granhall and Berg, 1972);
7. Flavobacterium spp. (Mamkaeva, 1966; Delucca and McCraken, 1977; Mayfield
and Inniss, 1978);
8. Flavobacterium breve (Kreig and Holt, 1984) formerly known as Bacillus brevis
(Reim et aI., 1974);
9. Myxobacterales spp., possibly Myxococcus spp. or Polyangium spp. (Shilo, 1970;
Daft, et aI., 1973);
10. MyxobacteriumCP-l (Wu,etal., 1968; Daft,etal., 1973; Daft,etal., 1975 and
Daft, et al., 1984);
11. Myxococcus fulvus (Burnham, 1984);
12. Myxococcus xanthus (Burnham et a!., 1981 and Burnham, et aI., 1984);
13. Pseudomonas sp. (Blasco, 1965; Mamkaeva, 1966; Burnham, 1973; Delucca and
McCraken, 1977; Mayfield and Inniss, 1978);
14. Serratia sp. (Mamkaeva, 1966);
15. Serratia marcescens (Darveau and Lynch, 1977);
16. Vampirovibrio chlorellavorous (Kreig and Holt, 1984) fomerly known as
Bdellovibrio chlorellavorus (Code and Starr, 1978);
17. Xanthomonas sp. (Blasco, 1965; Delucca and McCraken, 1977).
1.
for
2.
3.
156
Appendix B: Dilution Calculations
Table 1. Pond Saver CFU-Dilution conversions for 100 ml bottles.
4 billion CFUs per gram
Advertised Advertised
CFUslBottle
CFUS/O. 1ml
Inserted
Blank #
4,000,000,000 4,000,000
1 gram
Dilution 0
40,000,000
40,000
Dilution 1 1ml from 0
400,000
400
Dilution 2 1ml from 1
40,000
40
Dilution 3 0.1mlfrom 1
4,000
4
Dilution 4 1ml from 2
Table 2. Pond Clarifier CFU-Dilution Conversions for 100 ml bottles.
400 billion
400 billion
400 billion
882000
CFUs per pound
CFUs per
0.453515 kg
CFUs per 453.5147 g
CFUs per
1g
Blank #
Dilution 0
Dilution 1
Dilution 2
Dilution 3
Dilution 4
Inserted
10 grams
1m! from 0
0.1ml from 0
1ml from 1
0.1m! from 1
Advertised
Advertised
CFUs/Bottle CFUs/0.1 ml
8,820,001
8,820
88,200
88
8,820
9
882
1
88
0
Table 3. Bacta-Pur Klear Application Conversions.
Rate:
100 ml per 1000 liters
=
0.1 mill iter
Table 4. Pond Saver Application Conversions
liters = 1.057*gallons*4 kg = 2.2046 pounds
3 pounds
=
325,000 gallons
6.6138
=
Kg
1,374,100
Rate:
3 pounds per 325,000 gallons
liters
=
4.81319E-06 kglliter
0.004813187 glJiter
19,252,747 CFUs/liter
157
Table 5. Initial Estimates for Bacta-Pur Klear CFU-Dilution Conversions.
No claim to CFUs per volume
Assuming CFUs per treatment close to Pond Saver:
Blank #
Dilution 0
Dilution 1
Dilution 2
Dilution 3
Estimated
Estimated
CFUs/Bott/e CFUs/O.1ml
Inserted
19,252,747
19,253
0.1 ml Bacta-Pur
192,527
193
1ml from 0
19
19,253
0.1ml from 0
2
1,925
1ml from 1
Table 6. Revised Dilutions for Bacta-Pur Klear.
Blank #
Dilution 0
Dilution 1
Dilution 2
Dilution 3
Inserted
1.0ml Bacta-Pur
0.1 ml Bacta-Pur
1ml from 0
0.1ml from 0
158
Appendix C:
Characteristics of bacteria taxa identified as likely components of commercial microbial
products.
o Actinomycetes
• Form: hyphae with branching (Holt, 1977).
• Various spore types but not endospores (Holt, 1977).
• Gram positive rods.
• Irregular staining often presents beaded appearance (Sneath, 1986).
• Non sporing (Sneath, 1986).
• Straight or wavy slender branching filaments (Sneath, 1986).
• Faculative anaerobic (Sneath, 1986).
• Mature colonies (7-14 days) are either
• Rough and dry, or
• Smooth and soft to mucoid.
• White to gray-white or creamy white (Sneath, 1986).
• Glucose fermenter (Sneath, 1986).
• Indole negative (Sneath, 1986).
o Alcaligenes sp.
• Gram negative rods (1 x 3-4 urn) singly or paired.
• "Four 'degenerately peritrichous' flagella with subpolar or lateral insertion."
• Aerobic.
• Colonies: "white, round, smooth, and convex."
• Negative for acid production from: arabinose, fructose, galactose, glucose, inulin,
lactose, maltose, mannitol, salicin, sorbitol, sucrose, and trehalose.
• Positive for Nitrate ~ Nitrite and motility.
• Reduces litmus milk.
• Negative for catalase, indole, methyl red, Voges-Proskauer, HzS, urease production,
starch hydrolysis, gelatin liquification, and citrate (Martin, 1976).
• Holt (1977) and Krieg and Holt (1984) identify one alcaligenes, Pseudomonas
alcaligenes:
• Gram negative rod.
• 0.5 x 2.0 - 3.0 urn.
• Flagellate.
• Oxidase positive.
o Arthrobacter sp.
• Hydroxylamine production positive.
• Nitrite production positive (Berger, et al., 1979).
• Cell morphology changes markedly during life cycle (Holt, 1977; Sneath, 1986).
• Gram positive, but may
• Appear Gram negative with Gram positive granules (Holt, 1977).
• Become Gram positive coccus in growth cycle (Sneath, 1986).
• No fermentation (Holt, 1977).
• Catalase positive (Holt, 1977; Sneath, 1986).
• Strict aerobe (Holt, 1977).
• No endospore (Sneath, 1986).
159
•
Stationary phase: typically large coccus: 0.6 - 1.Oum (Sneath, 1986).
o Bdellovibrio bacteriovorus.
Burnham et al. (1976) does not provide identifying characteristics.
Gram: negative (Holt, 1977; Krieg and Holt, 1984).
Morphology: rods with sheathed flagellum (Holt, 1977; Krieg and Holt, 1984).
Fermentation: negative (Holt, 1977).
Aerobic (Holt, 1977).
Most often parasitic (Holt, 1977).
Catalase positive (Holt, 1977).
Colonies on yeast extract and peptone:
• Small to pinpoint (Holt, 1977).
• Pale yellow to orange; (Krieg and Holt, 1984).
o Cellvibrio spp.
• Granhall and Berg (1972) do not provide identifying characteristics.
• Not noted in Volumes 1-4 of "Bergey's" (Kreig and Holt, 1984; Sneath, 1986; Staley, J.
T., 1989; Williams, 1989).
• Noted in Hatt (1982) which notes two species: C. gilvus and C. polyotrophicus but
provides no identifying characteristics.
• Noted in Gibbons et al. (1981) which notes four species: c.jlavescens, C. gilvus, C.
polyotrophicus, and C. violacea but provides no identifying characteristics.
o Cytophaga sp. AKA myxobacter e.g., Myxococcus fulvus and Myxococcus xanthus
(Burnham, 1984).
• Noted to be closely related to Flavobacterium, but not described in Kreig and Holt
(1984).
o Flavobacterium spp.
• Gram negative (Delucca and McCraken, 1977).
• Short rods (Delucca and McCraken, 1977).
• Typically 0.5 x 1.0 x 3.0 urn long (Kreig and Holt, 1984).
• No endospores (Kreig and Holt, 1984).
• Aerobic (Kreig and Holt, 1984).
• Lactose fermentation: negative (Delucca and McCraken, 1977).
• Colonies:
• Smooth, flat with entire margins (Delucca and McCraken, 1977).
• Typically yellow to orange; usually translucent; circular; smooth and shiny with
entire edges (Kreig and Holt, 1984).
• Glucose: negative (Delucca and McCraken, 1977).
• Sucrose: negative (Delucca and McCraken, 1977).
• Lactose: negative (Delucca and McCraken, 1977).
• Manitol: negative (Delucca and McCraken, 1977).
• Sacchrose: negative (Delucca and McCraken, 1977).
• Maltose: negative (Delucca and McCraken, 1977).
• Citrate: negative (Delucca and McCraken, 1977).
• Indole: negative (Delucca and McCraken, 1977).
• Motile: negative (Delucca and McCraken, 1977; Kreig and Holt, 1984).
• H2S Production: positive (Delucca and McCraken, 1977).
• Oxidase:
• Negative (Delucca and McCraken, 1977).
•
•
•
•
•
•
•
•
160
• Positive (Kreig and Holt, 1984).
• Catalase positive (Kreig and Holt, 1984).
• Phophotase positive (Kreig and Holt, 1984).
• Mayfield and Inniss, 1978 does not provide identifying characteristics.
o Flavobacterium breve.
• Gram negative (Krieg and Holt, 1984).
• Rods ~0.5 x 1.0 - 2.0 urn (Krieg and Holt, 1984).
• Colonies on nutrient agar:
• Entire edge.
• Convex.
• Circular.
• Smooth.
• Shining.
• Pinpoint to 2.5mm.
• Nonhemolytic (Krieg and Holt, 1984).
• Spores central and oval (Reim et aI., 1974).
• Mannitol acid positive (Reim et al., 1974).
• Deanimation of phenylalanine negative (Reim et al., 1974).
• Decomposition of tyrosine positive (Reim et aI., 1974).
• Glucose acid positive (Reim et al., 1974).
• Growth in 5% NaCI negative (Reim et al., 1974).
• Width: 0.6-0.9 urn; length: 1.5-4.0 urn.
• Gram: variable.
• Nonmotile (Krieg and Holt, 1984).
• Starch hydrolysis: negative.
• Volges-Pros.: positive.
• Indole: negative.
• Decomposition of casein: negative (Holt, 1977).
o Myxobacterium CP-l
• Gram:
• Negative rods (Daft, et al., 1984).
• Weak staining (Sneath, 1986).
• Strong acid fast (Sneath, 1986).
• "Aflagellate" (Daft, et al., 1984).
• Colonies:
• Yellow pigmented (Daft, et al., 1984).
• Not visible for two days (Sneath, 1986).
• "Strictly Aerobic" (Daft, et aI., 1984; Sneath, 1986).
• Non motile (Sneath, 1986).
• Catalase positive (Sneath, 1986).
• Ammonia as sole nitrogen source: negative (Daft, et al., 1984).
• Nitrate as sole nitrogen source: negative (Daft, et al., 1984).
• Hydrolyze starch: positive (Sneath, 1986).
• Liquefy gelatin: positive (Sneath, 1986).
• Glucose as sole carbon: negative (Sneath, 1986).
• Starch as sole carbon: negative (Sneath, 1986).
161
o Myxococcus spp.
• Rods with rounded ends (Holt, 1977).
• Myxospores: refractile and spherical or ellipsiodal (Holt, 1977).
• Motile (Holt, 1977).
• Strict aerobes (Holt, 1977).
o Myxococcus fulvus
• Burnham (1984) does not provide identifying characteristics.
• Colony flesh to reddish orange (Holt, 1977).
• Oxidase negative (Holt, 1977).
o Myxococcus xanthus
• Yellowish to yellowish orange pigment (Burnham et at., 1981; Holt, 1977).
• Myxospore diameter of 1.8 um (Burnham et al., 1981).
• Forms small fruiting bodies without a stalk (Burnham et al., 1981).
• Oxidase negative (Holt, 1977).
• Burnham, et al., 1984 does not provide identifying characteristics.
o Pseudomonas spp.
• Gram negative (Delucca and McCraken, 1977; Kreig and Holt, 1984).
• Rods.
• Short (Delucca and McCraken, 1977).
• Straight or slightly curved (Kreig and Holt, 1984).
• 0.5 - 1.0 x 1.5 - 5.0 um (Kreig and Holt, 1984).
• White circular colonies (Delucca and McCraken, 1977).
• Aerobic or nitrate users (Kreig and Holt, 1984).
• Normally fail to grow in acid conditions (Kreig and Holt, 1984).
• Widely distributed in nature (Kreig and Holt, 1984).
• Lactose fermentation: negative (Delucca and McCraken, 1977).
• Glucose: positive (Delucca and McCraken, 1977).
• Sucrose: negative (Delucca and McCraken, 1977).
• Lactose: negative (Delucca and McCraken, 1977).
• Manitol: negative (Delucca and McCraken, 1977).
• Sacchrose: negative (Delucca and McCraken, 1977).
• Maltose: negative (Delucca and McCraken, 1977).
• Citrate: positive (Delucca and McCraken, 1977).
• Indole: negative (Delucca and McCraken, 1977).
• Motile:
• Positive (Delucca and McCraken, 1977).
• With polar flagella (Kreig and Holt, 1984).
• H 2 S Production: positive (Delucca and McCraken, 1977).
• Oxidase:
• Positive (Delucca and McCraken, 1977).
• Usually positive (Kreig and Holt, 1984).
• Catalase: positive (Kreig and Holt, 1984).
• Mayfield and Inniss, 1978 does not provide identifying characteristics.
o Serratia marcescens
• Darveau and Lynch, 1977 does not provide identifying characteristics.
• Gram: negative.
162
•
Rods:
• Straight.
• 0.5 - 0.8 x 0.9 - 2.0 urn.
• Facultative anaerobic.
• Motile.
• Colonies:
• Pink, white, or red.
• Opaque, somewhat irridescent.
• 1.5 - 2.0 mm diameter colonies overnight on nutrient agar.
• Catalase positive (Kreig and Holt, 1984).
1:1 Vampirovibrio chlorellavorous
• Gram negative (Code and Starr, 1978; Krieg and Holt, 1984).
• Rods and cocci (Krieg and Holt, 1984).
• Pleomorphic: vibrios 0.3 urn wide & cocci 0.6 urn wide (Code and Starr, 1978).
• Flagella: single, unsheathed (Code and Starr, 1978).
• Unable to grow in any media other than live algae cells (Code and Stan, 1978).
1:1 Xanthomonas sp.
• Gram negative (Kreig and Holt, 1984).
• Rods:
• Short (Delucca and McCraken, 1977).
• Straight (Kreig and Holt, 1984).
• 0.4 - 0.7 x 0.7 - 18 urn (Kreig and Holt, 1984).
• Usually singles (Kreig and Holt, 1984).
• Colonies: yellow and smooth (Kreig and Holt, 1984).
• Aerobic (Kreig and Holt, 1984).
• Catalase positive (Kreig and Holt, 1984).
• Lactose fermentation: negative (Delucca and McCraken, 1977).
• Colonies:
• Yellow to orange pigmentation (Delucca and McCraken, 1977).
• "Punctiform which were raised with entire margins" (Delucca and McCraken,
1977,).
• Glucose: positive (Delucca and McCraken, 1977).
• Sucrose: negative (Delucca and McCraken, 1977).
• Lactose: negative (Delucca and McCraken, 1977).
• Manitol: negative (Delucca and McCraken, 1977).
• Sacchrose: negative (Delucca and McCraken, 1977).
• Maltose: negative (Delucca and McCraken, 1977).
• Citrate: negative (Delucca and McCraken, 1977).
• Indole: negative (Delucca and McCraken, 1977).
• Motile:
• Positive (Delucca and McCraken, 1977; Kreig and Holt, 1984).
• By single flagella (Kreig and Holt, 1984).
• H 2S Production: positive (Delucca and McCraken, 1977).
• Oxidase:
• Negative or weak (Kreig and Holt, 1984).
• Negative (Delucca and McCraken, 1977).
163
Appendix D: Calculations for equivalent recommended first dosages.
Table 1. Pond Saver Conversions for Algae Inoculation.
Recommended First Application: 3# per acre/foot
1 pound
=2.205**-1
kg
3 pounds
1 acre/foot
=(3/2.205) kg =
1.360544218 kg
=325,000 gallons
1 gallon = 3.785 x 10**-3 cubic meters
325,000 gallons
3# per acre/foot
=3.785 * 325 =
=1.36kg/1230.13cu meters =
1230.125 cubic meters
0.001106021 kg/cu meters =
1.10602E-06 kg/liters =
0.001106021 g/Iiters = mg/ml
1.10602E-06 g/ml
0.000110602 g/100ml
Moving 1 ml with Each Dilution:
Dilution Bottle 0:
Dilution Bottle 1:
Dilution Bottle 2:
0.01106 g/100ml
0.000111 g/1 ml
0.000111 g/100ml
Table 2. Pond Clarifier Conversions for Algae Inoculation.
Recommended First Application: 8# per acre/foot
1 pound
=2.205**-1
8 pounds
1 acre/foot
kg
=(8/2.205) kg =
3.628117914 kg
=325,000 gallons
1 gallon = 3.785 x 10**-3 cubic meters
325,000 gallons = 3.785 * 325 =
1230.125 cubic meters
8# per acre/foot = 3.63kg/1230.13cu meters =
0.00294939 kg/cu meters
2.94939E-06 kg/liters =
=
164
0.00294939 g/liters =mg/ml
2.94939E-06 g/ml
0.000294939 g/100ml
Moving 1 ml with Each Dilution:
Dilution Bottle 0:
Dilution Bottle 1:
Dilution Bottle 2:
0.029494 g/100ml
0.000295 g/1 ml
0.000295 g/100ml
Table 3. Bacta-Pur Klear Conversions for Algae Inoculation.
Recommended First Application: 100 ml product 1 1000 liters water
100 ml 1 1000 liters =
0.0001 ml/ml
0.01 ml/100ml
Moving 1 ml with Each Dilution:
Dilution Bottle 0:
Dilution Bottle 1:
Dilution Bottle 2:
1 ml/100ml
0.01 ml/1ml
0.01 ml/100ml
165
Appendix E: CFU Counts.
Table 1. Pond Saver 24 Hour CFU Count.
Dilution Replicate
0
a
b
0
c
0
1
a
1
b
c
1
2
a
2
b
2
c
3
a
3
b
c
3
4
a
4
b
4
c
BHI Count
Lawn
Lawn
Lawn
Lawn
Lawn
Lawn
38
41
40
29
36
27
1
3
2
PCA Count NWRI Count
Lawn
Lawn
Lawn
Lawn
Lawn
Lawn
Lawn
Lawn
Lawn
Lawn
Lawn
Lawn
47
TNTC (-230)
28
TNTC
43
TNTC
30
60
47
68
39
62
2
8
2
11
1
3*
* broken agar
Table 2. Pond Saver 48 Hour CFU Count.
Dilution Replicate
a
0
b
0
c
0
1
a
1
b
c
1
2
a
2
b
2
c
3
a
3
b
3
c
4
a
4
b
4
c
BHI Count
Lawn
Lawn
Lawn
Lawn
Lawn
Lawn
43
49
46
32
38
26
2
3
2
*
broken agar
PCA Count NWRI Count
Lawn
Lawn
Lawn
Lawn
Lawn
Lawn
Lawn
Lawn
Lawn
Lawn
Lawn
Lawn
59
TNTC
34
TNTC
76
TNTC
55
93
100
70
81
94 *
5
14
15
6
12 *
14
166
Table 3. Pond Clarifier 24 Hour CFU Count.
Dilution
0
0
0
1
1
1
2
2
2
3
3
3
4
4
4
Replicate
a
b
c
a
b
c
a
b
c
a
b
c
a
b
c
BHI Count
TNTC
TNTC
TNTC
2
3
4
0
1
9
0
0
0
0
0
0
PCA Count NWRI Count
TNTC
81
104
TNTC
103
TNTC
1
0
1
0
4
0
0
1?
1
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
Table 4. Pond Clarifier 48 Hour CFU Count.
Dilution
0
0
0
1
1
1
2
2
2
3
3
3
4
4
4
Replicate
a
b
c
a
b
c
a
b
c
a
b
c
a
b
c
BHI Count
PCA Count NWRI Count
TNTC
TNTC
TNTC
TNTC
TNTC
TNTC
TNTC
TNTC
TNTC
51
52
46
1
5
30
1
1?
1
0
0
0
3
9
5
11
0
2; 3?
1?
0
0
0
0
0
0
* broken agar
7
*
1; 2?
2
1
1
1
0
0
0
0
167
Table 5. Bacta-Pur Klear 24 Hour CFU Count.
Dilution
0
0
0
Replicate
1
1
2
2
2
b
3
3
3
a
b
c
a
c
a
b
c
a
b
c
BHI Count
27
30
19
0
0
2
0
0
0
0
0
0
PCA Count NWRI Count
7
6
3
8
18
4
2
0
0
1
0
0
0
0
0
0
1?
0
0
0
0
0
0
0
Table 6. Bacta-Pur Klear 48 Hour CFU Count.
Dilution
0
0
0
1
1
1
2
2
2
3
3
3
Replicate
a
b
c
a
b
c
a
b
c
a
b
c
BHI Count
60
54
45 *
9
3
4
0
0
0
0
0
0
PCA Count NWRI Count
10
9
8
10
24
9*
3
0
1
1
1
0
0
0
0
0
0
1
0
0
0
0
0
0
* broken agar
168
Appendix F: Microorganisms cultured from products.
o
BPK-A:
• Source: Bacta-Pur Klear.
• Circular colony with raised yellowish-cream center with an entire cream "halo" edge.
• Gram negative rods -1.5 urn x .75 urn.
• Potentially: Achromobacter sp., Arthrobacter sp., Bdellovibrio bacteriovorous, Cellvibrio spp.,
Flavobacterium spp., Flavobacterium breve, Myxobacterium CP-l,Mvxobacterales spp.,
Myxococcusfulvus, Myxococcus xanthus, Serratia spp., Serratia marcescens, or Xanthomonas
spp.
o BPK-B 1:
•
•
•
•
Source: Bacta-Pur Klear.
Circular white flat colony with serrate-lobate edge.
Gram negative rods --4.5 urn x .75 urn.
Potentially: Achromobacter sp., Arthrobacter sp., Cellvibrio spp., Myxobacterales spp.,
Pseudomonas spp., or Serratia spp.
o BPK-B 2 :
•
•
•
•
Source: Bacta-Pur Klear.
Circular white flat colony with serrate-lobate edge.
Gram positive rods with endospores towards one end.
Identified by as Bacillus sp. (Bukovsan, 1999).
o BPK-C:
•
•
•
•
o
Source: Bacta-Pur Klear.
Irregular white flat colony with lobate edge.
Gram positive rods -7.0 - 8.0 urn x .50 urn with no obvious endospores.
Potentially: Achromobacter sp., Actinomycetes,Arthrobacter sp., Bdellovibrio bacteriovorous,
Cellvibrio spp., or Myxobacterales spp.
PC-A:
• Source: Pond Clarifier.
• Black irregular raised colony with lobate edge.
• Gram negative rods -1.5um x .75 urn.
• Potentially: Achromobacter sp., Arthrobacter sp., Bdellovibrio bacteriovorous, Cellvibrio spp., or
Myxobacterales spp.
o PC-B:
•
•
•
•
o
Source: Pond Clarifier.
Cream circular colony with articulate umbonate ridges (often looks like a dIY, partially collapsed
blister) and a lobate edge.
Gram negative sheathed rods.
Potentially: Achromobacter sp., Arthrobacter sp., Bdellovibrio bacteriovorous, Cellvibrio spp.,
Myxobacterium CP-l, Myxobacterales spp., Myxococcus fulvus, Myxococcus xanthus,
Pseudomonas spp., Serratia spp., Serratia marcescens, or Xanthomonas spp.
PC-C:
• Source: Pond Clarifier.
• Yellowish-cream colony with a filamentous edge.
• Gram negative rods -2.25 - 3.75 urn x .75 urn.
169
•
Potentially: Achromobacter sp., Arthrobacter sp., Bdellovibrio bacteriovorous, Cellvibrio spp.,
Myxobacterium CP-l, Myxobacterales spp., Myxococcusfulvus, A-fyxococcus xanthus, Serratia
spp., or Xanthomonas spp.
o
PC-D:
• Source: Pond Clarifier.
• As BPK-A: Circular colony with raised yellowish-cream center with an entire cream "halo" edge
• Gram negative rods ~7.5 - 6.0 urn x .75 urn.
• Potentially: Achromobacter sp., Arthrobacter sp., Bdellovibrio bacteriovorous, Cellvibrio spp.,
Flavobacterium spp., Myxobacterium CP-l,Myxobacterales spp., Myxococcusfidvus,
Myxococcus xanthus, Serratia spp., or Xanthomonas spp.
o
PC-E:
• Source; Pond Clarifier.
• Small circular white granular colony with alobate edge.
• Gram negative rods -1.0 urn x .2 urn.
• Potentially; Achromobacter sp., Arthrobacter sp., Bdellovibrio bacteriovorous, Cellvibrio spp.,
Myxobacterales spp., Serratia spp., or Serratia marcescens.
o PS-A;
•
•
•
Source; Pond Saver.
Circular colony with a pink center and bottom with fuzzy appearing gray surrounding the center
and a yellowish fuzzy edge.
Identified as Penecillium sp. (Marr, 1999).
o PS-B:
•
•
•
•
Source: Pond Saver.
As BPK-B: Circular white flat colony with serrate-lobate edge.
Gram negative rods ~3.0 urn x .75 urn.
Potentially: Achromobacter sp., Arthrobacter sp., Bdellovibrio bacteriovorous, Cellvibrio spp.,
Myxobacterales spp., Pseudomonas spp., or Serratia spp.
o PS-C:
•
•
•
•
CJ
Source: Pond Saver.
Smaller but as PC-B: Cream circular colony with articulate umbonate ridges (often looks like a
dry, partially collapsed blister) and a lobate edge.
Gram negative rods ~3.0 urn x .5 urn.
Potentially: Achromobacter sp., Arthrobacter sp., Bdellovibrio bacteriovorous, Cellvibrio spp.,
Myxobacterium CP-l, Myxobacterales spp., Myxococcus fulvus, Myxococcus xanthus,
Pseudomonas spp., Serratia spp., or Xanthomonas spp.
PS -D:
• Source: Pond Saver.
e
Irregular white flat colony with lobate edge.
• Gram positive rods in chains withendospores ~3.0 urn x 1.0 urn.
• Potentially: Actinomycetes,Arthrobacter sp., Bdellovibrio bacteriovorous, Cellvibrio spp., or
Myxobacterales spp.