Phosphine Formation from Sewage Sludge Cultures

Anaerobe (1999) 5, 525±531 Article No. anae.1999.0199

ECOLOGY/ENVIRONMENTAL MICROBIOLOGY

Phosphine Formation from Sewage Sludge Cultures Barbara V. Rutishauser and Reinhard Bachofen* University of Zurich, Institute of Plant Biology, Department of Microbiology, Zollikerstr. 107, CH-8008 Zurich, Switzerland

Gas-chromatographic analysis of the volatile compounds in the headspace of cultures inoculated with sewage sludge indicated phosphine production over a period of several days, a reaction which is clearly microbiologically mediated. The amounts of phosphine recovered from 40-mL specimens sewage sludge enrichment were in a pg-range of 5 to 100 in the presence of (Received 11 March 1999, glucose as carbon source and phosphate as phosphorus source. Phosphine accepted in revised form accumulation was found to be stimulated by increasing the phosphate 21 May 1999) concentration of the medium. Hypophosphite and lecithin as phosphorus sources instead of phosphate as well as mannitol and succinic acid instead Key Words: phosphine formation, of glucose as carbon sources affected the phosphine formation. # 1999 Academic Press sewage sludge, gas-chromatography

Introduction The existence of volatile components in the biogeochemical cycle of phosphorus is suggested if one compares the cycles of nitrogen and arsenic, elements of the fifth main group of the periodic system, with the nearby phosphorus. For all of them biologically reduced or methylated forms exist [1]. The existence of biologically formed phosphine, the phosphorus analogon to gaseous ammonia or arsine has been discussed starting about hundred years ago. Putrefaction processes as well as different soil types had been said to produce phosphine [2,3], but other early investigations denied these results [4]. However, with modern analytic methods, i.e., gas-chromatography, phosphine was recently found to be ubiquitous in anaerobic environments. It was detected in gases

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evolving from sewage sludge treatment plants and wetland soils as well as from common soils [5±8], even in the headspace of a bacterial culture [5]. Matrix bounded phosphine was found by Gassmann and Glindemann in several anaerobic ecosystems such as fluvial and marine sediments, in the contents of rumen and in feces of animals and human beings [9,10]. Phosphine evolving from landfills and animal slurry treatment plants was detected as well as its presence in the atmosphere [11±13]. The mechanism for the formation of the detected phosphine is still under debate [14]. While phosphine evolution has been reported from microbial habitats, so far no correlation between microbial growth and phosphine production has been established. Neither the culture in which phosphine was detected in the gas space has been specified nor has the origin of the phosphine detected been looked for [5]. Recent results about microbial hypophosphite and phosphite oxidation support the hypothesis of a full phosphorus redox cycle and suggest that the phosphine evolution detected in anaerobic habitats is # 1999 Academic Press

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due to microbial processes [15]. If bacteria are capable of oxidizing hypophosphite (‡III) to phosphate (‡V), the probability that organisms exist that carry out the reduction of phosphate to phosphine (7III) is quite high. In this report we investigated the accumulation of phosphine in the headspace of a bacterial enrichment over a time period of several days and show the microbiological origin of the phosphine detected. The influence of the phosphate concentration of the medium on phosphine formation was studied as well as the effect of the composition of the growth medium.

Materials and Methods Inoculum and culture conditions The sewage sludge for inoculation came from the Maggi company sewage sludge treatment plant in Kemptthal (Switzerland). Samples were taken from the sludge storage pond of the company. The sampling site was chosen because the phosphorus concentration in this particular sewage sludge treatment plant was above average and, if existing, bacteria turning non-volatile phosphorus compounds into phosphine could be expected there. The sewage sludge was cultured in a medium containing 10 g/L D-(‡)-glucose, 10 g/L KH2PO4, 0.8 g/L NH4Cl, 0.4 g/L Fe(II)Cl2, 2 g/L MgCl2.6H2O, 1 g/L Ca(H2PO4)2.2H2O (medium gluc PO4). The medium does not contain any other substantial electron acceptor besides phosphate. Other electron acceptors present in the sewage sludge, however, cannot be excluded. The low phosphate medium contained 1 g/L KH2PO4 (medium gluc low PO4). Further 8 g/L NaH2PO2 (medium gluc PO2) and 93.08 g/L lecithin (60%) (medium gluc lecithin) were used instead of KH2PO4 as alternative phosphorus sources, respectively. Mannitol [10.12 g/L mannitol (medium mannitol PO4)] and succinic acid [9.84 g/L succinic acid (medium succ PO4)] were used as alternative carbon sources. The amounts of carbon and phosphorus sources added were calculated such that there were equal amounts of carbon and phosphorus as with glucose and KH2PO4. Oxygen was removed from the media by bubbling nitrogen through the liquid after autoclavation. The pH was adjusted to 7 through addition of deoxygenated sodium hydroxide solution. The cultures were grown as batch cultures at 308C in the dark under nitrogen in screw cap serum bottles sealed with phosphine inert viton stoppers. For the experiments with high and low phosphate concentration of the medium, 500-mL serum bottles containing 80 mL medium were inoculated with 4 mL sewage sludge. To compare the effects

of the different sources of phosphorus and carbon, 125-mL serum bottles containing 40 mL medium inoculated with 2 mL sewage sludge were used. All the other experiments were carried out in 250-mL serum bottles with 40 mL medium and 2 mL sewage sludge inoculum. The inoculum was withdrawn from the sealed sampling bottle with plastic syringes. The medium for the enrichment of methanogenic bacteria was prepared according to Balch [16]. Chromatographic determination of phosphine and methane Phosphine determination and identification was carried out with two different gas-chromatographic systems. For most measurements an HP 5890 II gaschromatograph (Hewlett Packard) with an NPD (nitrogen±phosphorus detector), a detector particularly sensitive to phosphorus compounds, was used. The instrument was equipped with a cryogenic trapping system worked out by D. Glindemann and G. Gassmann [12,13]. To eliminate possibly present acidic gases such as hydrogen sulfide, gas samples were injected into the trapping system through a tube filled with solid sodium hydroxide. Chromatographic separation was performed isothermally at 1008C on a GSQ-column (30 m60.53 mm I.D.) (J‡W Scientific). The detector temperature was 2508C. Helium was used as carrier gas with a gas flow rate of 4.8 mL/min. Alternatively a DANI 86.10 HT gas chromatograph (DANI Strumentazione analitica S.p.A Monza-Italy) with FPD (flame photometric detector) was used combined with the purge-and-trap technique described for the analysis of volatile organic sulfur compounds by Henatsch and JuÈttner [17]. A glass tube filled with Chromosorb 106 (Supelco) cooled with solid carbon dioxide was used as trap for phosphine. To remove acidic gases, a glass tube filled with solid sodium hydroxide was placed in front of the trapping tube. Chromatographic separation was carried out isothermally at 358C on a GSQ-column (30 m60.53 mm I.D) (J‡W Scientific). The detector temperature was 2208C, the injector temperature 2508C. Helium was used as carrier gas with a gas flow of 14.85 mL min. The identification of phosphine was obtained from comparing the retention times with a phosphine standard as well as by co-chromatography and spiking the samples with a phosphine standard. Methane was measured using the DANI 86.10-HT gaschromatograph (DANI Strumentazione analitica S.p.A Monza-Italy) with FID (flame ionization detector) on the same GSQ-column (30 m60.53 mm I.D) (J‡W Scientific) isothermally at 358C. The injector temperature was 2508C, the detector temperature 2208C.

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Sampling procedure For analysis with the GC-NPD 40 mL samples of headspace gas above the sewage sludge cultures were taken with a gas tight 60-mL plastic syringe. Because the different cultures showed different pressure conditions, 40 mL of the headspace were taken at original pressure. In that way, not the absolute amounts of phosphine produced by the cultures are compared, but rather the amounts in 40 mL of headspace at the pressure prevailing in the culture bottles. So the different pressures have been considered without knowing their exact value and the samples can be compared directly. To purge the whole sample from the inlet system, 50 mL of air were applied after the sample. For the sample analysis with the GC-FPD, the whole headspace above the cultures was purged upon the trapping tube. The formation of methane in the gas space above the cultures was determined by injecting 500 mL of the headspace gas. Protein determination The protein concentration was determined according to Bradford [18], with bovine serum albumin (BSA) as a protein standard (BioRad).

Results and discussion Phosphine accumulation in the headspace of sewage sludge enrichment cultures in medium gluc PO4 In the headspace of the sewage sludge enrichments, phosphine formation due to bacterial activity was observed. Phosphine was identified and separated from the other gases present in the headspace above the sewage sludge culture by means of the gas-chromatography (Figures 1 and 2). Significant amounts of phosphine were noted after 20 to 30 h of incubation reaching a maximum after about 50 to 60 h (Figure 3). Inoculation with sterilized sewage sludge or sewage sludge added to medium supplemented with formaldehyde or mercuric chloride resulted in a complete absence of the phosphine peak after 3 and 6 days of incubation, respectively. This indicates that metabolic activity of the bacteria is a prerequisite for the formation of phosphine in the headspace of the cultures. The kinetics of phosphine accumulation followed a typical microbial growth curve. However, the bulk of the organisms of the mixed population exhibited exponential growth between 10 and 20 h while phosphine production was retarded (Figure 3).

Figure 1. Representative chromatogram of headspace gases of a sewage sludge culture grown on medium gluc PO4 (a) and a phosphine standard treated the same way as the sample (b) analysed with the GC-FPD. The second peak on chromatogram (a) was not identified.

In contrast the accumulation of the dominant gas present in anaerobic environments, methane, follows about the course of the growth assessed by protein determination (Figure 3). It seems that phosphine production is either due to an organism growing slower than most of the bacteria in the culture or phosphine is formed in a non growth coupled pathway. Some reports on phosphine point to the fact that in anoxic habitats phosphine always coexists with methane [10]. This leads to the speculation that methanogenic bacteria are responsible for the phosphine formation. However, when methanogenic bacteria in sewage sludge cultures were inhibited with bromoethanesulfonic acid (C2H4BrNaO3S), methane production was impaired while phosphine formation

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B.V. Rutishauser and R. Bachofen seems to be due only to the fact that both processes take place under similar environmental conditions. Investigation of factors influencing phosphine formation from sewage sludge cultures To further examine the requirements and optimal conditions for phosphine formation, experiments with a lower phosphate concentration of the medium and such with different carbon sources and phosphorus sources were carried out. (i) Effect of the phosphate concentration on phosphine formation. Sewage sludge cultures grown in the medium with high phosphate show higher phosphine accumulation than cultures grown in low phosphate (Figure 4). Cultures with low phosphate formed less biomass, measured as total protein formed, than cultures with high phosphate (data not shown), indicating that the cultures develop differently. With low phosphate, more pressure was built up in the culture bottles than with high phosphate content which indicated that the total amount of gases formed was greater in the former. Furthermore, the two cultures were also different on visual examination. These observations indicate that the different media stimulate growth of different organisms or the production of different metabolites by the same organisms. A high phosphate concentration increases not only phosphine formation but also bacteria able to take advantage of the excess phosphate for growth.

Figure 2. Representative chromatogram of 40 mL of headspace gas of a sewage sludge culture grown on medium gluc PO4 at attenuation 1 (a) and a phosphine standard applied in 50 mL of air at attenuation 2 (b) analysed with the GC-NPD. Peak 5 is phosphine, peaks 1 and 2 are not identified, peaks 3 and 4 correspond to ethene and ethane, respectively.

remained. Likewise enrichment of methanogenic bacteria from the sewage sludge and subsequent transfer into gluc PO4 medium did not lead to phosphine formation. Both observations indicate that the methanogens are not involved in phosphine formation. The coexistence of phosphine and methane

(ii) Effect of alternative sources of phosphorus and carbon on phosphine accumulation. Hypophosphite was chosen as alternative phosphorus source because it is more reduced than phosphate and possibly an intermediate in the reduction process from phosphate to phosphine. Lecithin was employed as representative of organic phosphorus sources as phosphine may also originate from decomposition of organic phosphorus compounds. Phosphine formation in cultures grown on hypophosphite and lecithin as sole phosphorus sources besides Ca(H2PO4)2.2H2O after 3, 6 and 10 days of incubation is listed in Table 1. With phosphate as phosphorus source the phosphine production reached its peak after about 3 days. At this time, cultures with hypophosphite as phosphorus source showed less phosphine production than the cultures with phosphate. In contrast, an increase of the phosphine content was observed after further incubation. Considering the large sampling volume combined with a small overpressure compared to the cultures grown on phosphate, a continuous phosphine production must be assumed. On phosphate, no further increase

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Figure 3. Time course of phosphine formation in the headspace of a sewage sludge enrichment culture grown on gluc PO4 medium as analysed by GC-NPD (a) and its growth curve as obtained by protein determination (^) and methane formation in the headspace (~) (b).

of the phosphine was observed after 3 days of incubation. The maximum amounts of phosphine measured above cultures grown on phosphate are approximately twice as large as above cultures grown on hypophosphite. Over 10 days of incubation the total amount of phosphine formed in cultures grown on hypophosphite was only slightly smaller than in cultures grown on phosphate. This indicates that the phosphine production and possibly growth of cultures grown on hypophosphite are slower than in cultures grown on phosphate. From these findings it cannot be concluded if hypophosphite is an intermediate in the reduction of phosphate to phosphine. If hypophosphite is involved in phosphine formation as intermediate product, an accelerated phosphine formation would have been expected with hypopho-

sphite. Since too many unknown factors influencing the process affect the mixed culture, no conclusions can be made on the basis of the data obtained. Cultures grown on lecithin as phosphorus source lack detectable phosphine in the headspace after 3 days of incubation. Judging from the great pressure prevailing in the culture bottles, as remarked during the sampling procedure, and the peaks obtained by gas-chromatographic analysis of the compounds present in the headspace, gases other than phosphine have been formed vigorously. After further incubation some phosphine has been formed. Lecithin led only to the formation of a fraction of the amounts of phosphine formed by cultures grown on phosphate or hypophosphite. Thus lecithin does probably not support phosphine production directly. Phosphine

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B.V. Rutishauser and R. Bachofen Table 2. Phosphine content of 40 mL of the headspace of sewage sludge enrichment cultures grown on mannitol and succinic acid as carbon sources and analysed by GC-NPD after 3 and 6 days of incubation Phosphine [pg] Days of incubation 3 6

Figure 4. Time course of phosphine formation in the headspace of a sewage sludge enrichment culture grown on gluc medium PO4 (*) and gluc low PO4 (*), respectively, as analysed by GC-NPD.

may result from the decomposition of organic bound phosphate followed by reduction as suggested in the older literature [2,19]. As alternative carbon sources mannitol and the nonfermentable succinic acid have been selected. Mannitol was described in 1927 by Rudakov as a carbon source especially stimulating phosphine formation [3]. In our hands mannitol as carbon source yielded similar amounts of phosphine as glucose (Table 2). Succinic acid was chosen as representative of nonfermentable carbon sources. If phosphine formation takes place over a respiratory mechanism with phosphate as electron acceptor similar to sulfate reduction or nitrate reduction, which both run off with non-fermentable carbon sources as electron donor, phosphine development during cultivation on succinic acid may be an indication of phosphate respiration. Succinic acid instead of glucose as carbon source led to phosphine formation but less than with glucose. It reached similar values after six days of incubation as cultures with hypophosphite as phosphorus source (Table 2), which do not increase after further incubation (data not shown). In contrast to the cultures grown on fermentable carbon sources, the culture medium remained neutral during the incubation as it was not affected by fermentation products. A non-fermentable carbon source could point to phosTable 1. Phosphine content of 40 mL of the headspace of sewage sludge enrichment cultures grown on phosphate, hypophosphite and lecithin as phosphorus source and analysed by GC-NPD after 3, 6 and 10 days of incubation Phosphine [pg] Days of incubation 3 6 10

Gluc PO4

Gluc PO2

Gluc lecithin

6.482+1.924 2.386+1.338 1.765+0.096

1.833+1.059 1.958+0.831 3.341+1.729

± 1.035+0.595 1.505+1.022

Mannitol PO4

Succ PO4

5.117+2.497 2.048+0.877

1.833+0.245 1.969+0.245

phate respiration as the mechanism for bacterial phosphine formation similar to the respiratory reduction of selenate or arsenate [20]. If phosphate respiration were really the mechanism for phosphine formation, the smaller production of phosphine with succinic acid as carbon source could be explained by the less negative redox potential of the site at which electrons are fed into the respiratory chain. With succinic acid as carbon source less electrons from the same amount of carbon are available for the reduction of phosphate, as 1 mol of glucose supplies 24 electrons for the formation of 3 mol phosphine whereas 1 mol succinic acid only supplies 14 electrons for the formation of 1.75 mol phosphine. With succinic acid and low phosphate in the medium phosphine formation was below the detection limit. This confirms the result found with glucose, i.e., that high phosphate in the medium strongly stimulates the process. Acknowledgements Dr G. Gassmann is thanked for generously making his gaschromatograph available to us and his useful advice concerning phosphine analytics. We also thank Dr D. Glindemann for helpful hints concerning phosphine detection, and the Swiss National Science Foundation (3100-039597. 93/1) for financial support.

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