Shown to the right of the Chemical Engineer are four anaerobic fermenters used to evaluate the effects of ozonation on desugared Spent Sulfite Liquor (SSL) with regard to microbial growth and production of Methane and additional yeast protein.
Studies on production of Methane by the action of Methane Bacteria (Methanogens) on ozonated SSL were done in continuous mode. Each fermenter was started by adding a 10% volume of fresh fermenting sewage sludge to a prereduced base medium containing 2.5 g/l sodium formate, 2.5 g/l sodium acetate, 2.0 ml/l (60%) sodium lactate, 2.0 ml/l methanol, and 2.0 ml/l ethanol.
As soon as methane production was observed, a diluted solution of base medium and ozonated SSL was added continuously with a 3.5 day retention time. To acclimate the organisms to SSL the ratio of base medium to SSL was decreased in stepwise fashion to eventually eliminate the base medium completely over a one month period, ultimately arriving at ratio of 75:25% ozonated SSL to distilled water. This feed was continually added to the fermenter from a 4oC refrigerated reservoir. Fermenter pH was controlled at 7.2 by maintaining the feed pH at 8.0 with NaOH.
Volatile and nonvolatile organic acids in the fermenter effluents were analyzed by gas chromatography. Gas production rates were measured and recorded hourly by water displacement in gas-measuring burettes. This gas was also analyzed by gas chromatography for determination of hydrogen, carbon dioxide, methane, and hydrogen sulfide concentrations. See Table 1 for results of organic acids, COD, BOD5, and sulfur analysis (38, 124, 137, 229, 231).
This is a 1000 X microscopic view of Methanobacterium ruminatium a major methane producing bacterium (methanogen) isolated from anaerobic fermenters utilizing ozonated SSL as substrate. This is a Gram stain slide preparation showing the Gram positively stained methanogen rods (blue-black) and culture debris as (pink).
Estimates of methanogenic bacterial populations in the fermenters were made weekly using a modification of the Hungate roll tube technique. The basal medium previously describe plus 2% agar was initially gassed with oxygen-free N2 during preparation, then gassed with a 1:1 mixture of CO2 and H2 as samples were added to roll tubes. These agar roll tubes were then streaked, incubated, and enumerated as described previously in the Microbiology section.
The presence of methanogenic bacteria was initially verified by streak isolating 80 light-colored colonies from fermenting ozonated SSL into separate roll tubes. Approximately 10 colonies were picked from each of 8 roll tubes. Correspondingly, 35 black-dark brown colonies were isolated into roll tubes. After 28 days of incubation at 35 oC, samples of the gas in each tube were analyzed for methane by gas chromatography and Gram stains were also performed. The presence of anaerobic bacteria other than methanogens or sulfur-reducing bacteria was determined by plate count using Brewer's anaerobic agar.
Methane bacteria counts in the fermenters averaged 1.7 x 109 colony-forming units (CFU)/ml. This value was derived by subtracting the count of black-dark brown colonies (also numbering 1.7 x 109) from the total roll tube counts. The validity of this technique was verified when it was found that of the 80 light-colored colonies randomly isolated from roll tubes, 78 were shown to produce methane. None of the 35 black-dark brown colonies were found to produce methane.
Microscopic examination was made of 135 randomly picked light-colored methanogenic colonies. Ninety three percent of these were found to be Gram-positive coccoid to short, lancet-shaped rods, sometimes in chain, and various types of Gram-negative rods, similar to descriptions for representatives of the genus Methanobacterium. This genus is present in many forms of anaerobic digestion. The remaining 7% of the 135 light-colored colonies closely resembled the methanogenic genus Methanococcus. These isolates were small Gram-variable to Gram-negative cocci which grew in clumps, pairs, or as single cells. Methanococcus is also commonly found in most anaerobic digesters.
Microscopic examination of the bacteria growing as black-dark brown colonies revealed all were curved to sigmoid Gram-negative, nonspore forming rods. The production of H2S by each of these isolates was readily detected, indicating the presence of sulfur-reducing bacteria, probably Desulfovibrio spp. (38, 70, 124, 137, 229, 231).
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