Conversion of Paraffin Oil to Alcohols by Clostridium ...

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Mar. 1995, p. 1153–1155 0099-2240/95/$04.0010 Copyright q 1995, American Society for Microbiology

Vol. 61, No. 3

Conversion of Paraffin Oil to Alcohols by Clostridium thermosaccharolyticum SANDRA M. LANDUYT,1* EDWARD J. HSU,2 BI-TAR WANG,2 2 AND SAN-SAN TSAY Penn Valley Community College, Kansas City, Missouri 64111,1 and National Taiwan University, Taipei, Taiwan, Republic of China2 Received 12 September 1994/Accepted 13 December 1994

Solventogenic cells of Clostridium thermosaccharolyticum converted the paraffin oil overlay, used to maintain anaerobic conditions, into butanol, ethanol, and isopropanol only under oxyduric conditions. Figure 1 shows a correlation among stepwise synchronous growth in cell number (curve A), the corresponding concentration of solvents (curve B), and the utilization of paraffin oil (curve C) by C. thermosaccharolyticum under culture condition 4 (Table 1). At the end of synchronous growth, greater than 80% of the cells were elongated a minimum of four to five times, but the concentration of the combined solvents produced was relatively low (0.4%) and there was no change in the layer of paraffin oil. After 25 h, the synchrony was lost; however, the cells continued to elongate five to ten times the vegetative length. This was when the paraffin layer began to decrease, and by 30 h the layer of oil decreased more than 4 ml. A thin emulsion interphase formed between the oil and the culture fluid. When the temperature was shifted at 45 h, the elongated cells continued differentiation, with 10 to 20% of the sporangia showing signs of swollen heads. The total solvent concentration, predominately butanol and ethanol in a 1:1 ratio, increased nearly 10-fold to 3.7% at 115 h. The relative amounts of butanol, ethanol, and isopropanol varied throughout the solventogenic phase from a ratio of 1:1:1 at 105 h to 2:1:1 at 175 h. The cell density at the peak of solventogenesis (10.6%) was 4.40 3 109 cells per ml, of which 80 to 90% were elongated four to five times and 50 to 60% of the elongated cells were refractile sporangia. Fewer than 1% were short, vegetative cells. After incubation was stopped and the culture was centrifuged to break the emulsion layer apart, it was evident that 10 ml of the paraffin oil had been consumed. Additional experimentation was carried out, using a New Brunswick (C-30) Bio-flo fermentor, to scale up and improve solubilization. The fermentation vessel (inner diameter, 10 cm) contained 1,500 ml of xylan-Ca-gluconate medium and 400 ml of paraffin oil. The continuous mixing and scraping were set at 200 rpm, and the culture was purged hourly for 15 min with the N2 at a flow rate of 125 ml/min, using a peristaltic pump (New Brunswick model PA-56). Because of the continuous mechanical mixing and scraping, which resulted in faster cell division and differentiation of the organism, it was necessary to stop cell division at 24 h. Similar results regarding the extent of differentiation of the cells were observed, but the solubilization of the paraffin oil occurred much earlier. At 16 h, the oil layer was turbid and decreased from 400 to 380 ml with no emulsion layer. This 20-ml decrease was preceded by approximately 12 generations of stepwise growth. At 44 h, only 40 ml of the turbid oil was evident above the interphase, and by 95 h the emulsion layer was 110 mm thick, with no clear oil apparent. When the agitation and incubation were stopped at 188 h and centrifugation

We previously observed that synchronized cells of Clostridium thermosaccharolyticum produced high solvent concentrations in a medium that contained xylan, calcium-gluconate, and paraffin oil (7). In this study, different culture conditions were tested to optimize the conversion of paraffin oil to butanol and ethanol. It was found that the solventogenic cells of this Clostridium sp. solubilized and utilized paraffin oil only in the presence of more than one fermentable carbohydrate, hourly N2 purging with scraping of the fermentation vessel, and minute amounts of O2. These conditions resembled the initial oxidation step of saturated aliphatic hydrocarbons, a strictly aerobic process that occurs predominantly by mixed populations in the presence of other organic compounds (2, 11). Biodegradation of saturated aliphatic hydrocarbons represents one of the primary mechanisms by which petroleum and other hydrocarbon pollutants may be eliminated from the environment. The synchronization of C. thermosaccharolyticum in dilution bottles containing xylan medium has been described (9, 10). Synchronized growth and differentiation of the organism were carried out under the conditions presented in Table 1. All experiments were carried out in triplicate. Anaerobic conditions were maintained by a paraffin oil overlay and hourly purging with 99.9% industrial grade N2, except where indicated in Table 1. The 0.1% impurities in the N2 gas contained 50 ppm of O2 (Oxygen Service, Kansas City, Mo.). Shaking of the culture vessel was used to ensure complete mixing of the substrate with the cell mass and to prevent aggregation of individual cells. It was previously found that during synchronous elongation and growth the cells clumped together and the culture fluid appeared flocculated. When these cells were viewed following capsule staining, there was a region surrounding the individual cells, approximately two times the thickness of the cells, that did not stain, indicative of capsule material (8). Cell division was stopped at 45 h by shifting the temperature to 358C; the fermentation was continued for an additional 130 h. Culture samples were obtained hourly by using a pipette inserted into the bottom of the culture. Samples were centrifuged at 2,200 3 g in a Sorvall small-angle centrifuge with an SP/X rotor (Sorvall, Inc., Norwalk, Conn.) for 30 min to separate the cell mass from the culture fluid. Solvent concentrations were determined by gas-liquid chromatography analysis (9, 10) and were reported as the sum of the individual products.

* Corresponding author. 1153

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NOTES TABLE 1. Culture conditions for synchronized growth and bioconversion

Culture conditions

Carbon source

Paraffin oil overlay

1

Xylan-Ca-gluconate

Added following temp shift

None

None

2

Xylan-Ca-gluconate

Added from beginning

Hourly prior to temp shift (30 ml/min)

Hourly prior to temp shift

3

Paraffin oil

Added from beginning

Hourly throughout incubation (60–80 ml/min)

Hourly throughout incubation

4

Xylan-Ca-gluconate

Added from beginning

Hourly throughout incubation (60–80 ml/min)

Hourly throughout incubation

was performed, a decrease of 105 ml of paraffin oil was observed. The solvent concentration, however, was only 5.6%. A set of dilution bottles containing 10% corn starch as the sole carbon source without paraffin oil was inoculated with the fermentor-prepared cells (harvested at 24 h) to determine whether these cells were capable of converting a much higher concentration of carbohydrate to alcohols. Manipulations and incubation of these cultures were carried out inside an anaerobic glove box (Labconco, Kansas City, Mo.). The dilution bottles were placed on a magnetic stir plate (Bell-Stir MultiStir; Bellco Glass, Inc.; Vineland, N.J.) throughout the incubation period to permit slow mixing (50 rpm) of the substrate and the elongated cells. Following 141 h of incubation, 5.6% solvents were produced, and a second batch of corn starch (7%) was added. When the cultures were harvested at 245 h, the solvent concentration reached 9.6%. Synchronized cultures prepared under culture conditions 1 to 3 (Table 1) were unsatisfactory with regard to conversion of paraffin oil to alcohols. The highest solvent concentration was 1.0 to 1.2% under culture conditions 1 and 2. Under condition 2, solubilization with a decrease in paraffin oil (4 ml) was only observed before the temperature shift. No growth, differentiation, solubilization, or utilization of the paraffin oil was ever observed under culture condition 3 (no fermentable carbohydrate present). Although clostridia degrade sugars by the Embden-Meyerhof-Parnas pathway, sugar acids such as gluconate are utilized via a modified Entner-Doudoroff pathway; the central inter-

FIG. 1. Conversion of paraffin oil by synchronously prepared solventogenic cells. Curve A, total cell count; curve B, total solvents produced (percentages, volume/volume); curve C, milliliters of paraffin oil consumed.

Nitrogen purging

Scraping of growth vessel

mediate is pyruvate, which is converted to acetyl coenzyme A CO2, and reduced ferredoxin by pyruvate ferredoxin oxidoreductase (1). Over the past several years, we have developed methods to ensure differentiation of C. thermosaccharolyticum to 100% refractile spores, including utilization of xylanCa-gluconate medium, in which the sporangia produced solvents (4–10). Vegetative cells of C. thermosaccharolyticum are known to utilize more than 18 different carbohydrates (3, 5), and only sporulating cells appear capable of converting carbohydrates to solvents. In this investigation, it was established that only the solventogenic, nondividing cells were capable of converting paraffin oil to solvents. It would appear that xylan and Ca-gluconate, not paraffin oil, were utilized for growth and stage I of sporulation during the first 25 h (Fig. 1, curves A and B). The sporangia continued to differentiate and subsequently solubilized and converted the paraffin oil to solvents (Fig. 1, curves B and C). Banthorpe (2) described the main genera of microorganisms known to metabolize saturated, aliphatic hydrocarbons, but none of those described were anaerobic organisms. Despite the fact that clostridia are obligate anaerobes, they are not so aero-intolerant that they cannot survive an occasional encounter with O2 (12), and perhaps some species can utilize O2 as a reactant for the initial oxidation of paraffin oil. In the present study, it would seem that the 50 ppm of O2, in the N2, was sufficient to serve as a reactant for the initial oxidation of the paraffin oil overlay by the sporangia. It has also been shown that the degradation of hydrocarbons, particularly alkanes, by microorganisms is accompanied by excretion of emulsifying agents (11). The emulsification observed in the study reported here may be due to biosurfactants that were produced by the synchronized cells during the first 25 h of incubation. In summary, the following observations were made. (i) The synchronized cells that were intermittently purged with industrial grade N2 emulsified the paraffin oil overlay and were highly solventogenic (10.6%). (ii) The synchronized cells prepared without paraffin oil and N2 purging did not solubilize or utilize the subsequently added paraffin oil and produced only low concentrations of solvents (1.0 to 1.2%). (iii) Starch added at high concentrations was more efficiently converted to solvents when added in two stages, a process that imitates the san-dan-shikomi (three-step addition) of sake manufacturing. (iv) The manner in which the solvents were produced following exponential growth resembled the production of secondary metabolites, even though the concentrations were reflective of primary metabolites. This investigation was funded in part by the Chinese Petroleum Corporation and the Chung-Hwa Pulp Corporation.

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NOTES REFERENCES

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