Thermophilic anaerobic digestion for sewage sludge stabilization

The microbially mediated biochemical reactions that occur during anaerobic digestion processes for methane production from soluble carbon energy substrates are well known, but in spite of this, the interactions within the multi-species cultures responsible for the overall process require more detailed elucidation. When the process feed comprises mixed, solid, carbon energy substrates, as in the case of waste sewage sludge stabilization, many aspects of both the process biochemistry and microbiology are unresolved. This mini-review seeks to identify some of these unresolved questions, particularly with respect to operation at thermophilic temperatures.     

Simultaneous methanogenesis and phototrophic bacterial growth in relatively dry sewage sludge under light

Anaerobically digested sewage sludge with a variety of moisture content, namely 81%, 86%, 90% and 98%, were anaerobically cultured at 35 degrees C under light. Phototrophic bacteria grew in the 86% moisture sludge (bacteriochlorophyll a, 0.46 g/L), 90% sludge (bacteriochlorophyll a, 0.36 g/L) and 98% sludge (bacteriochlorophyll a, 0.04 g/L) with methane production. Phototrophic bacteria could not grow in the 81% moisture sludge (bacteriochlorophyll a 0.004 g/L). Phototrophic bacteria could assimilate about 46% of the extracellular ammonium in the 90% moisture sludge. Phototrophic bacteria utilized organic compounds competing with methanogens; therefore, methane yield from the 90% moisture sludge under the light conditions was lower than that under the dark conditions. Phototrophic bacteria could grow in anaerobically digested sludge with relatively low moisture content, and assimilated extracellular ammonium in the sludge. The quality of digested sludge with phototrophic bacterial biomass for fertilizer could be improved compared with that without phototrophic bacterial biomass.     

Interaction of acetogens and methanogens in anaerobic freshwater sediments

Anaerobic decomposition processes in the profundal sediments of Blelham Tarn (English Lake District) are often limited during late summer by the input of organic carbon. The concentration of acetate in the interstitial water fell from about 100 microM (immediately after sedimentation of the spring diatom bloom) to a relatively constant value of about 20 microM in late summer, during which acetate utilization appeared to be balanced by production. Addition of chloroform and molybdate caused an accumulation of cold acetate in large sediment cores and of [14C]acetate in small cores to which [14C]bicarbonate had been added. In both cases chloroform caused the greater accumulation, implying that acetoclastic methanogens were the more active consumers. The conversion of 14CO2 to [14C]acetate was inversely related, with depth, to its conversion to 14CH4. Methanogenesis from CO2 decreased during late summer, whereas acetogenesis and acetoclastic methanogenesis increased over the same time period. The production of acetate from CO2 was generally equivalent to less than 10% of the acetate carbon utilized but could be as high as 25% of that value. Hydrogen consumption by acetogens could be as high as 50% of that utilized in methanogenesis. The role of acetogenic bacteria in anaerobic processes may therefore be of greater significance in lakes such as Blelham Tarn than in more eutrophic systems.     

Interaction of acetogens and methanogens in anaerobic freshwater sediments.

Anaerobic decomposition processes in the profundal sediments of Blelham Tarn (English Lake District) are often limited during late summer by the input of organic carbon. The concentration of acetate in the interstitial water fell from about 100 microM (immediately after sedimentation of the spring diatom bloom) to a relatively constant value of about 20 microM in late summer, during which acetate utilization appeared to be balanced by production. Addition of chloroform and molybdate caused an accumulation of cold acetate in large sediment cores and of [14C]acetate in small cores to which [14C]bicarbonate had been added. In both cases chloroform caused the greater accumulation, implying that acetoclastic methanogens were the more active consumers. The conversion of 14CO2 to [14C]acetate was inversely related, with depth, to its conversion to 14CH4. Methanogenesis from CO2 decreased during late summer, whereas acetogenesis and acetoclastic methanogenesis increased over the same time period. The production of acetate from CO2 was generally equivalent to less than 10% of the acetate carbon utilized but could be as high as 25% of that value. Hydrogen consumption by acetogens could be as high as 50% of that utilized in methanogenesis. The role of acetogenic bacteria in anaerobic processes may therefore be of greater significance in lakes such as Blelham Tarn than in more eutrophic systems.     

Sequential anaerobic degradation of 2,4-dichlorophenol in freshwater sediments

2,4-Dichlorophenol (2,4-DCP) was anaerobically degraded in freshwater lake sediments. From observed intermediates in incubated sediment samples and from enrichment cultures, the following sequence of transformations was postulated. 2,4-DCP is dechlorinated to 4-chlorophenol (4-CP), 4-CP is dechlorinated to phenol, phenol is carboxylated to benzoate, and benzoate is degraded via acetate to methane and CO2; at least five different organisms are involved sequentially. The rate-limiting step was the transformation of 4-CP to phenol. Sediment-free enrichment cultures were obtained which catalyzed only the dechlorination of 2,4-DCP, the carboxylation of phenol, and the degradation of benzoate, respectively. Whereas the dechlorination of 2,4-DCP was not inhibited by H2, the dechlorination of 4-CP, and the transformation of phenol and benzoate were. Low concentrations of 4-CP inhibited phenol and benzoate degradation. Transformation rates and maximum concentrations allowing degradation were determined in both freshly collected sediments and in adapted samples: at 31 degrees C, which was the optimal temperature for the dechlorination, the average adaptation time for 2,4-DCP, 4-CP, phenol, and benzoate transformations were 7, 37, 11 and 2 days, respectively. The maximal observed transformation rates for these compounds in acclimated sediments were 300, 78, 2, 130, and 2,080 micromol/liter(-1)/day(-1), respectively. The highest concentrations which still allowed the transformation of the compound in acclimated sediments were 3.1 m/M 2,4-DCP, 3.1 mM 4-CP, 13 mM phenol, and greater than 52 mM benzoate. The corresponding values were lower for sediments which had not been adapted for the transformation steps.     

Survival of Trichinella spiralis larvae in sewage sludge anaerobic digesters

The survival of some bacteria, viruses, protozoans, and helminths through the sewage digestion process has been a question of considerable concern among researchers throughout the world. Among the most resistant organisms are some of the pathogenic roundworms and tapeworms. Encysted larvae of Trichinella spiralis are sometimes present in animal tissues discarded as waste from slaughterhouses, restaurants or other sources. In experimental anaerobic sewage digesters, encysted larvae of T. spiralis, in rat muscle, were able to survive a maximum of 96 hr in a "batch" digester. In a digester "fed" daily with small numbers of encysted larvae, "draw-off" remained infective for white rats throughout a 16-day experimental period. Potentially infective material could be present when there is continuous "draw-off" from the anaerobic digesters.     

Inactivation of Salmonella during anaerobic digestion of sewage sludge

The inactivation of Salmonella duesseldorf in sewage sludge during anaerobic digestion was investigated at 35 and 48°C with mean retention periods of between 10 and 20 days. Digesters were fed daily with raw sludge containing added Salm. duesseldorf after removal of digested sludge. During steady operation, the levels of Salm. duesseldorf in the digested and the feed sludge were determined and their specific rates of decay were estimated. The latter were: (i) greater at 48°C than at 35°C for the same retention time; (ii) similar for retention periods greater than 15 d, but lower for 10 d; (iii) greater when the level of salmonellas in the feed was lower. Gas production, a measure of steady state, was gradually lost when the mean retention period was reduced to 6.7 d. In experiments in which a single dose of Salm. duesseldorf was added to digesting sludge, the inactivation appeared to follow first-order kinetics at 35°C and the decimal decay rate, 1.6/d, was similar to that in the daily feeding experiments (1.4/d) with larger and similar inocula of Salm. duesseldorf. At 48°C, however, the rate of inactivation declined with decreasing time from inoculation suggesting that the culture contained cells differing in thermal resistance. The degrees and rates of inactivation of salmonellas in those experiments were greater than in full-scale digesters, because the latter seldom operated under conditions ideal for inactivation or because indigenous salmonellas are more resistant.     

Hydrogenase activity in aged, nonviable Desulfovibrio vulgaris cultures and its significance in anaerobic biocorrosion

Batch cultures of Desulfovibrio vulgaris stored at 32 degrees C for 10 months have been found to retain 50% of the hydrogenase activity of a 1-day culture. The hydrogenase found in old cultures needs reducing conditions for its activation. Viable cell counts are negative after 6 months, showing that the hydrogenase activity does not depend on the presence of viable cells. These observations are of importance in the understanding of anaerobic biocorrosion of metals caused by depolarization phenomena.     

Sulfide-induced dissimilatory nitrate reduction to ammonia in anaerobic freshwater sediments

Abstract: Different reduced sulfur compounds (H₂S, FeS, S₂O₃²⁻) were tested as electron donors for dissimilatory nitrate reduction in nitrate-amended sediment slurries. Only in the free sulfide-enriched slurries was nitrate appreciably reduced to ammonia (     ), with concomitant oxidation of sulfide to S⁰ (     ). The initial concentration of free sulfide appears as a factor determining the type of nitrate reduction. At extremely low concentrations of free S²⁻ (metal sulfides) nitrate was reduced via denitrification whereas at higher S²⁻ concentrations, dissimilatory nitrate reduction to ammonia (DNRA) and incomplete denitrification to gaseous nitrogen oxides took place. Sulfide inhibition of NO- and N₂O- reductases is proposed as being responsible for the driving part of the electron flow from S²⁻ to NH₄⁺.     

Sequential anaerobic degradation of 2,4-dichlorophenol in freshwater sediments.

2,4-Dichlorophenol (2,4-DCP) was anaerobically degraded in freshwater lake sediments. From observed intermediates in incubated sediment samples and from enrichment cultures, the following sequence of transformations was postulated. 2,4-DCP is dechlorinated to 4-chlorophenol (4-CP), 4-CP is dechlorinated to phenol, phenol is carboxylated to benzoate, and benzoate is degraded via acetate to methane and CO2; at least five different organisms are involved sequentially. The rate-limiting step was the transformation of 4-CP to phenol. Sediment-free enrichment cultures were obtained which catalyzed only the dechlorination of 2,4-DCP, the carboxylation of phenol, and the degradation of benzoate, respectively. Whereas the dechlorination of 2,4-DCP was not inhibited by H2, the dechlorination of 4-CP, and the transformation of phenol and benzoate were. Low concentrations of 4-CP inhibited phenol and benzoate degradation. Transformation rates and maximum concentrations allowing degradation were determined in both freshly collected sediments and in adapted samples: at 31 degrees C, which was the optimal temperature for the dechlorination, the average adaptation time for 2,4-DCP, 4-CP, phenol, and benzoate transformations were 7, 37, 11 and 2 days, respectively. The maximal observed transformation rates for these compounds in acclimated sediments were 300, 78, 2, 130, and 2,080 micromol/liter(-1)/day(-1), respectively. The highest concentrations which still allowed the transformation of the compound in acclimated sediments were 3.1 m/M 2,4-DCP, 3.1 mM 4-CP, 13 mM phenol, and greater than 52 mM benzoate. The corresponding values were lower for sediments which had not been adapted for the transformation steps.     

Effects of nickel and lead and a support material on the methanogenesis from sewage sludge

The effects of the addition of two heavy metals (nickel and lead) and a support material (purified sepiolite) on the methanogenesis have been evaluated in two types of domestic sewage sludges. A higher toxic effect of the metals was observed on the production of methane from loading sludge than from the anaerobic digester sludge, nickel being more toxic than lead in all cases studied. Antagonistic effects between both heavy metals were obtained when the loading sludge was supplemented with several concentrations of these metals. The addition of sepiolite to the loading sludge reduced toxic effects of both metals, which contrasts with the results obtained using sludges from anaerobic digester.     

Effects of solids retention time on methanogenesis in anaerobic digestion of thickened mixed sludge

When a bench-scale digester fed thickened mixed sludge was operated over an SRT range of 4-20 days, removal efficiencies for total chemical oxygen demand and volatile suspended solids declined with decreasing SRT (especially <10 days), but methanogenesis was stable for SRT as low as 5 days. Quantitative PCR analyses showed that methanogens declined steadily for SRT<10 days, with the acetate-cleaving Methanosaetaceae becoming more dominant. Clone-library analyses indicated significant shifts in bacterial population from 20 to 4 day SRT: declining Chloroflexi (28 to 4.5%) and Syntrophomonas (9 to 0%), but increasing Bacteroidetes (12.5 to 20%) and two acetogenic genera belonging to the phyla Firmicutes and Spirochaetales (6.3 to 12%). Thus, the decrease in the apparent hydrolysis constant (khyd-app) with higher SRT and the process limiting size of Methanosaetaceae with the lower SRT are proactive signs for defining rate limitation in anaerobic digestion.     

Effect of sulfate on carbon and electron flow during microbial methanogenesis in freshwater sediments.

The effect of sulfate on methane production in Lake Mendota sediments was investigated to clarify the mechanism of sulfate inhibition of methanogenesis. Methanogenesis was shown to be inhibited by the addition of as little as 0.2 mM sulfate. Sulfate inhibition was reversed by the addition of either H2 or acetate. Methane evolved when inhibition was reversed by H2 additions was derived from 14CO2. Conversely, when acetate was added to overcome sulfate inhibition, the evolved methane was derived from [2-14C]acetate. A competition for available H2 and acetate was proposed as the mechanism by which sulfate inhibited methanogenesis. Acetate was shown to be metabolized even in the absence of methanogenic activity. In the presence of sulfate, the methyl position of acetate was converted to CO2. The addition of sulfate to sediments did not result in the accumulation of significant amounts of sulfide in the pore water. Sulfate additions did not inhibit methanogenesis unless greater than 100 mug of free sulfide per ml was present in the pore water. These results indicate that carbon and electron flow are altered when sulfate is added to sediments. Sulfate-reducing organisms appear to assume the role of methanogenic bacteria in sulfate-containing sediments by utilizing methanogenic precursors.     

Isolation of anaerobic oxalate-degrading bacteria from freshwater lake sediments

Enrichment cultures that anaerobically degraded oxalate were obtained from lake sediment inocula. From these, 5 pure cultures of anaerobic oxalate-degrading bacteria were isolated and partially characterized. The isolates were Gram-negative, non-sporeforming, non-motile, obligate anaerobes. Oxalate was required for growth and was stoichiometrically converted to formate; ¹⁴CO₂ was also recovered when ¹⁴C-oxalate was added. Maximal growth occurred when the oxalate concentration was 50 mM. Acetate stimulated growth in the presence of oxalate, however, ¹⁴C-experiments indicated that acetate was only utilized for cell carbon.The isolates were either spiral-shaped or rod-shaped organisms. The first morphotype grew much more slowly than the second and exhibited 13-fold lower cell yields. These isolates represent a new strain of oxalate-degrading bacteria. The second morphotype was similar to the anaerobic oxalate-degrading bacteria previously found in rumen. This report extends the known habitats in which anaerobic oxalate-degrading organisms have been found to include aquatic sediments.     

Proteolytic activity of a rumen anaerobic fungus

Abstract A strain of the anaerobic phycomycetous fungus Neocallimastix frontalis isolated from the rumen of a sheep had a high proteolytic activity which became predominantly extracellular during growth. Proteolytic activity appeared to be due to a metalloprotease, as it was inhibited by 1,10-phenanthroline, EDTA and other chelators but not by phenylmethylsulphonyl fluoride (PMSF). Inhibition by EDTA was fully reversed by the addition of Zn²⁺, Ca²⁺ or Co²⁺, whereas addition of metal ions in the presence of 1,10-phenanthroline restored only a little activity. p -Chloromercuribenzoate (PCMB) was also inhibitory in dialysed supernatant fluid. N-α-p-Tosyl- l -lysine chloromethylketone (TLCK) inhibited proteolysis, suggesting that the protease(s) has a trypsin-like specificity, but benzoylarginine p -nitroanilide was not hydrolysed. Protease activity has a broad pH profile with a maximum at pH 7.5. Gel fractionation indicated that most of the activity was in a high- M _r form.     

Developments in pre-treatment methods to improve anaerobic digestion of sewage sludge

During wastewater treatment, most organic matter is transferred to a solid phase commonly known as sludge or biosolids. The high cost of sludge management and the growing interest in alternative energy sources have prompted proposals for different strategies to optimize biogas production during anaerobic sludge treatment. Because of the high solid content and complex structure of sludge-derived organic matter, methane production during digestion is limited at the hydrolysis step. Therefore, large digester volume and long retention times of over 20 days are necessary to achieve adequate stabilization. Pre-treatments can be used to hydrolyze sludge and consequently improve biogas production, solids removal and sludge quality after digestion. This paper reviews the main pre-treatment processes, with emphasis on the most recent developments. An overview of the different technologies is presented, discussing their effects on sludge properties and anaerobic digestion. Future challenges and concerns related to pre-treatment assessment and implementation are also addressed.     

污泥厌氧消化中同型产乙酸菌富集研究

在市政污泥厌氧消化过程中,采用三阶段选择性富集同型产乙酸菌,通过监测培养过程中pH值、挥发性脂肪酸、关键酶的活性以及不同碳源利用率等,研究同型产乙酸菌在系统中的累积情况.实验数据表明:经过40 d的富集培养,pH值稳定在8.0,乙酸含量逐渐趋于稳定,占COD的比例为46.4％,同型产乙酸菌对底物的利用率达到了87％,乙酸激酶和乙酰磷酸转移酶呈现明显上升的趋势.结果显示,通过本方法可在市政污泥中有效地富集同型产乙酸菌.     

Hydrogenase activity in aged, nonviable Desulfovibrio vulgaris cultures and its significance in anaerobic biocorrosion.

Batch cultures of Desulfovibrio vulgaris stored at 32 degrees C for 10 months have been found to retain 50% of the hydrogenase activity of a 1-day culture. The hydrogenase found in old cultures needs reducing conditions for its activation. Viable cell counts are negative after 6 months, showing that the hydrogenase activity does not depend on the presence of viable cells. These observations are of importance in the understanding of anaerobic biocorrosion of metals caused by depolarization phenomena.