Nitrification, denitrification, and dechlorination in bleached kraft pulp mill wastewater

This study deals with combining the biologi cal removal of organic halogens with the removal of nitrogen from bleached kraft pulp mill wastewater in fluidized-bed reactors under nitrifying and denitrifying conditions. Untreated and biotreated bleached kraft pulp mill wastewaters had no detrimental effect on nitrification or denitrification. The nitrifying biofilm reactor, pregrown on synthetic inorganic feed with ammonia, removed without a lag phase adsorbable organic halogens [7.2 mg Cl (g biomass volatile solids)⁻¹day⁻¹] from bleached kraft pulp mill wastewater and selected chlorophenols from synthetic wastewater. Electron microscopical examination of the biofilm showed that bacteria, morphologically similar to the nitrifying species Nitrosomonas or Nitrobacter, and Nitrosospira were dominant. The denitrifying fluidized-bed reactor, pregrown on nitrate and methanol, denitrified without a lag phase bleached kraft pulp mill wastewater. Under denitrifying conditions, 35% of the total organic carbon content of untreated bleached kraft pulp mill waste water was removed. The reducing power delivered by untreated bleached kraft pulp mill wastewater for denitrification was 2 mmol electrons/mmol carbon mineralized. Dechlorination under denitrifying conditions was negligible. Received: 21 November 1996 / Received revision: 27 January 1997 / Accepted: 1 February 1997     

Denitrifying and methanogenic bacteria in the biofilm of a fixed-film reactor operated with methanol/nitrate demonstrated by immunofluorescence and microscopy

 A denitrifying bacterial biofilm population established on a polypropylene substratum of a fixed-film reactor was characterized by microscopy, scanning electron microscopy and immunofluorescence after 120 days of operation. The reactor, operated at pH 7.0, 22°C, and −180 mV with synthetic wastewater containing methanol/nitrate, achieved a denitrification rate of 0.24 mol NO^- ₃ l^-1 day^-1 with a removal efficiency for nitrate of 95%–99% at an organic loading rate of 0.325 mol methanol l^-1 day^-1. The gas produced contained 2%–3% (v/v) methane and 3%–4% (v/v) carbon dioxide in addition to nitrogen. The biofilm contained mainly cells of Methanobrevibacter arboriphilus antigenically related to strain DC, short, flagellated, gram-negatively staining rods of Pseudomonas sp. antigenically related to Pseudomonas stutzeri strain AN11, non-identified pink-pigmented rods and small lemon-shaped cells with mono- and bipolar appendages resembling prosthecate Hyphomicrobium sp. The biofilm analysis provided evidence for a syntrophy between the denitrifying, methylotrophic, bacterial consortium and hydrogenotrophic methanogens, which were identified by antigenic fingerprinting with 17 antibody probes. Received: 11 July 1994/Received revision: 23 September 1994/Accepted: 28 September 1994     

Fermented whey – an inexpensive feed source for a laboratory-scale selenium-bioremediation reactor system inoculated with Thauera selenatis

It is critical that an inexpensive electron- donor/carbon-source be found for selenium bioremedia-tion using the selenate-respiring bacterium, Thauera selenatis. Since acetate is a preferred substrate for growth of this organism, a method was developed for fermenting the lactose in whey to large amounts of acetate. Indigenous whey microorganisms fermented the whey lactose in this manner when grown in continuous culture at a very slow dilution rate (D = 0.05 h⁻¹). The successful use of the fermented whey lactose as the carbon-source/electron-donor feed for a laboratory-scale selenium-bioremediation reactor system, inoculated with T. selenatis, treating selenium-contaminated drainage water was also demonstrated. Selenium oxyanions and nitrate were reduced by 98%. Received: 30 October 1998 / Received revision: 26 January 1999 / Accepted: 5 February 1999     

Improvement of mechanical and biological properties of freeze-dried denitrifying alginate beads by using starch as a filler and carbon source

Freeze-dried, alginate-based beads, used for the immobilization of a denitrifying bacterium (Pseudomonas sp.), were filled with different concentrations (10%, 20%, 30% and 40%, w/w) of granular starch. The beads were incubated under denitrifying conditions in laboratory-scale, flow-through columns and monitored for changes in their physical and denitrifying properties. Freeze-dried beads containing high concentrations of starch were found to have better mechanical and denitrifying properties than beads containing low concentrations of this filler. Nitrate removal by the beads was found to be correlated with their starch content. Nitrite accumulation, as a result of incomplete denitrification, increased with the decrease in starch content of the beads. Nitrite in the outlet of the columns was measured in all types of beads during the initial phase of incubation but was undetectable, with the exception of beads with the lowest starch content, at later stages of incubation. Received: 9 November 1998 / Received revision: 3 February 1999 / Accepted: 5 February 1999     

Complete degradation of tetrachloroethene by combining anaerobic dechlorinating and aerobic methanotrophic enrichment cultures

 Degradation of tetrachloroethene (perchloroethylene, PCE) was investigated by combining the metabolic abilities of anaerobic bacteria, capable of reductive dechlorination of PCE, with those of aerobic methanotrophic bacteria, capable of co-metabolic degradation of the less-chlorinated ethenes formed by reductive dechlorination of PCE. Anaerobic communities reductively dechlorinating PCE, trichloroethene (TCE) and dichloroethenes were enriched from various sources. The maximum rates of dechlorination observed for various chloroethenes in these batch enrichments were: PCE to TCE (341 μmol l^-1 day^-1), TCE to cis-dichloroethene (159 μmol l^-1 day^-1), cis-dichloroethene to chloroethene (99 μmol l^-1 day^-1) and trans-dichloroethene to chloroethene (22 μmol l^-1 day^-1). A mixture of these enrichments was inoculated into an anoxic fixed-bed upflow column. In this column PCE was converted mainly into cis-1, 2-dichloroethene, small amounts of TCE and chloroethene, and chloride. Enrichments of aerobic methanotrophic bacteria were grown in an oxic fixed-bed downflow column. Less-chlorinated ethenes, formed in the anoxic column, were further metabolized in this oxic methanotrophic column. On the basis of analysis of chloride production and the disappearance of chlorinated ethenes it was demonstrated that complete degradation of PCE was possible by combining these two columns. Operation of the two-column system under various process conditions indicated that the sensitivity of the methanotrophic bacteria to chlorinated intermediates represented the bottle-neck in the sequential anoxic/oxic degradation process of PCE. Received: 24 October 1994 / Received revision: 20 January 1995 / Accepted: 23 January 1995     

Nitrate reduction by Citrobacter diversus under aerobic environment

A new aerobic denitrifier, Citrobacter diversus, was isolated from both nitrification and denitrification sludge. To monitor the variation in the concentration of nitrogen oxides, aerobic denitrification by C. diversus was carried out in a batch reactor. When the nitrate concentration was greater than 180 mg N l⁻¹, the nitrate reduction rate became stable. The effect of the C/N ratio on the denitrification activity was also investigated. The results showed that the optimum denitrification activity was obtained when the C/N ratio was 4–5. The range of the C/N ratio was higher than that for traditional anoxic denitrification. The effect of the dissolved oxygen concentration was further studied; and it was found that the range of dissolved oxygen concentrations, both for specific growth rates and for specific denitrification rates, was 2–6 mg⁻¹. From these results, it can be concluded that both the concentration of dissolved oxygen and the C/N ratio are key factors in the aerobic denitrification by C. diversus. Received: 23 November 1999 / Received revision: 4 February 2000 / Accepted: 13 February 2000     

Methanotrophic community structure and activity under warming and grazing of alpine meadow on the Tibetan Plateau

Knowledge about methanotrophs and their activities is important to understand the microbial mediation of the greenhouse gas CH₄ under climate change and human activities in terrestrial ecosystems. The effects of simulated warming and sheep grazing on methanotrophic abundance, community composition, and activity were studied in an alpine meadow soil on the Tibetan Plateau. There was high abundance of methanotrophs (1.2–3.4 × 10⁸ pmoA gene copies per gram of dry weight soil) assessed by real-time PCR, and warming significantly increased the abundance regardless of grazing. A total of 64 methanotrophic operational taxonomic units (OTUs) were obtained from 1,439 clone sequences, of these OTUs; 63 OTUs (98.4%) belonged to type I methanotrophs, and only one OTU was Methylocystis of type II methanotrophs. The methanotroph community composition and diversity were not apparently affected by the treatments. Warming and grazing significantly enhanced the potential CH₄ oxidation activity. There were significantly negative correlations between methanotrophic abundance and soil moisture and between methanotrophic abundance and NH₄–N content. The study suggests that type I methanotrophs, as the dominance, may play a key role in CH₄ oxidation, and the alpine meadow has great potential to consume more CH₄ under future warmer and grazing conditions on the Tibetan Plateau.     

Enumeration of Methanotrophic Bacteria in the Cover Soil of an Aged Municipal Landfill

The enumeration of methanotrophic bacteria in the cover soil of an aged municipal landfill was carried out using (1) fluorescent in situ hybridization (FISH) with horseradish peroxidase-labeled oligonucleotide probes and tyramide signal amplification, also known as catalyzed reporter deposition-FISH (CARD-FISH), and (2) most probable number (MPN) method. The number of methanotrophs was determined in cover soil samples collected during April–November 2003 from a point with low CH₄ emission. The number of types I and II methanotrophs obtained by CARD-FISH varied from 15 ± 2 to 56 ± 7 × 10⁸ cells g⁻¹ absolute dry mass (adm) of soil and methanotrophs of type I dominated over type II. The average number of methanotrophs throughout the cover soil profile was highest during May–September when the cover soil temperature was above 13°C. Methanotrophs accounted for about 50% of the total bacterial population in the deepest cover soil layer owing to higher availability of substrate (CH₄). A lower number of methanotrophs (7 × 10² to 17 × 10⁵ cells g⁻¹ adm of soil) was determined by the MPN method compared to the CARD-FISH counts, thus confirming previous results that the MPN method is limited to the estimation of the culturable species that can be grown under the incubation conditions used. The number of culturable methanotrophs correlated with the methane-oxidizing activity measured in laboratory assays. In comparison to the incubation-based measurements, the number of methanotrophs determined by CARD-FISH better reflected the actual characteristics of the environment, such as release and uptake of CH₄, temperature, and moisture, and availability of substrates.     

Analysis of 16S rRNA and methane monooxygenase gene sequences reveals a novel group of thermotolerant and thermophilic methanotrophs, Methylocaldum gen. nov.

Two methanotrophic bacteria with optimum growth temperatures above 40° C were isolated. Thermotolerant strain LK6 was isolated from agricultural soil, and the moderately thermophilic strain OR2 was isolated from the effluent of an underground hot spring. When compared to the described thermophilic methanotrophs Methylococcus capsulatus and Methylococcus thermophilus, these strains are phenotypically similar to Methylococcus thermophilus. However, their 16S rRNA gene sequences are markedly different from the sequence of Methylococcus thermophilus (∼ 8% divergence) and, together with Methylomonas gracilis, they form a distinct, new genus within the γ-subgroup of the Proteobacteria related to extant Type I methanotrophs. Further phenotypic characterisation showed that the isolates possess particulate methane monooxygenase (pMMO) but do not contain soluble methane monooxygenase. The nucleotide sequence of a gene encoding pMMO (pmoA) was determined for both isolates and for Methylomonas gracilis. PmoA sequence comparisons confirmed the monophyletic nature of this newly recognised group of thermophilic methanotrophs and their relationship to previously described Type I methanotrophs. We propose that strains OR2 and LK6, together with the misclassified thermophilic strains Methylomonas gracilis VKM-14L^T and Methylococcus thermophilus IMV-B3122, comprise a new genus of thermophilic methanotrophs, Methylocaldum gen. nov., containing three new species: Methylocaldum szegediense, Methylocaldum tepidum and Methylocaldum gracile. Received: 2 April 1997 / Accepted: 23 July 1997     

Monitoring nitric oxide from immobilised denitrifying bacteria, Pseudomonas stutzeri, by the use of chemiluminescence

A chemiluminescence detector was used to measure the production of nitric oxide, NO, from the denitrifying bacteria Pseudomonas stutzeri. NO is an intermediate when P. stutzeri converts nitrate into nitrogen gas. The reaction between NO and ozone is selective and sensitive in generating chemiluminescence. Calibrations were made down to 1 nM, with a signal-to-noise ratio of 3. Bacteria were immobilised in alginate beads. Denitrification experiments were made in an anaerobic non-growth medium by adding nitrate to a certain concentration in the reactor. The bacteria were exposed to nitrate in the concentration range 1 pM–5 mM. The lowest concentration to give a measurable NO response was 100 nM. Received: 16 October 1997 / Received revision: 20 January 1998 / Accepted: 24 January 1998     

Denitrifiers associated with methanotrophs and their potential impact on the nitrogen cycle

Here I describe how losses of fixed nitrogen can occur in riparian zones by the activity of denitrifying bacteria associated with methane-oxidizing (methanotrophic) bacteria. Several methanotrophs catalyze nitrogen cycle processes that can occur in riparian buffer zones, including nitrification and nitrogen fixation. Methanotrophs can produce nitric and nitrous oxides during oxidation of ammonium (nitrification), but they cannot carry out denitrification. However, there is good evidence that denitrifying bacteria can be associated with methanotrophs and can use simple carbon compounds released by the methanotrophs as substrates for the denitrification reactions and for growth. Evidence is presented that denitrifiers isolated from methanotrophic gel-stabilized oxygen gradient systems can use methanol, formaldehyde, and formate, all methane oxidation intermediates, to support their denitrification. Such denitrification associated with methanotrophs can release dinitrogen and so contributes to losses of fixed nitrogen, and may also produce the important atmospheric trace gases nitric and nitrous oxides. Data presented also show that some methanotrophs produce nitrogen oxides, including nitrite, nitric oxide, and nitrous oxide, during growth on nitrate. Assimilatory reduction of nitrate appears to be a requirement for the release of these products.     

Enhanced mineralization of pentachlorophenol by κ-carrageenan-encapsulated Pseudomonas sp. UG30

A pentachlorophenol(PCP)-degrading Pseudomonas sp. strain UG30 was encapsulated in κ-carrageenan for use in PCP degradation. Free and encapsulated cells were compared for their ability to dechlorinate and mineralize 100–800 μg/ml sodium pentachlorophenate in broth. Dechlorination was measured with a chloride ion electrode, and mineralization was measured by ¹⁴CO₂ evolution from radiolabelled [U-¹⁴C]PCP. Free and encapsulated Pseudomonas sp. UG30 cells mineralized up to 200 μg/ml and 600 μg/ml PCP, respectively, after 21 days. Encapsulation of UG30 cells provided a protective effect, allowing dechlorination and mineralization of high levels of PCP to occur. Received: 3 May 1996 / Received revision: 4 September 1996 / Accepted: 13 September 1996     

High rates of microbial sulphate reduction in a mesophilic ethanol-fed expanded-granular-sludge-blanket reactor

In a mesophilic (30–35 °C), sulphidogenic, ethanol-fed expanded-granular-sludge-blanket reactor, sulphate, at loading rates of up to 10.0–12.0 g Sl⁻¹␣day⁻¹, was removed with an average efficiency of more than 80%. The pH was between 7.7 and 8.3 and the maximal total dissolved sulphide concentration was up to 20 mM S (650 mg S/l). The alkaline pH was maintained by either a pH-control unit with sodium hydroxide or by stripping part of the sulphide and CO₂ from the recycle with nitrogen gas. The superficial upstream liquid velocity (v _up) was 3.0–4.5 m/h. The ratio of ethanol to sulphur was near stoichiometry. At alkaline pH, the activity of the acetotrophic sulphate-reducing bacteria, growing on acetate, was strongly enhanced, whereas at pH below 7.7 the acetotrophic sulphate-reducing bacteria were inhibited by aqueous H₂S. With regard to the removal efficiency and operational stability, external stripping with N₂ and pH control were equally successful. Received: 2 December 1996 / Received revision: 13 March 1997 / Accepted: 15 March 1997     

Nitrifying granules cultivation in a sequencing batch reactor at a low organics-to-total nitrogen ratio in wastewater

It is possible to cultivate aerobic granular sludge at a low organic loading rate and organics-to-total nitrogen (COD/N) ratio in wastewater in the reactor with typical geometry (height/diameter = 2.1, superficial air velocity = 6 mm/s). The noted nitrification efficiency was very high (99%). At the highest applied ammonia load (0.3 ± 0.002 mg NH₄⁺–N g total suspended solids (TSS)⁻¹ day⁻¹, COD/N = 1), the dominating oxidized form of nitrogen was nitrite. Despite a constant aeration in the reactor, denitrification occurred in the structure of granules. Applied molecular techniques allowed the changes in the ammonia-oxidizing bacteria (AOB) community in granular sludge to be tracked. The major factor influencing AOB number and species composition was ammonia load. At the ammonia load of 0.3 ± 0.002 mg NH₄⁺–N g TSS⁻¹ day⁻¹, a highly diverse AOB community covering bacteria belonging to both the Nitrosospira and Nitrosomonas genera accounted for ca. 40% of the total bacteria in the biomass.     

Effect of dissolved oxygen and carbon–nitrogen loads on denitrification by an aerobic consortium

Four samples of natural ecosystems and one sample from an activated sludge treatment plant were mixed together and progressively adapted to alternating aerobic/anoxic phases in the presence of nitrate in order to enrich the microflora in aerobic denitrifiers. Aerobic denitrifying performances of this mixed ecosystem at various dissolved oxygen concentrations and various carbon–nitrogen loads were evaluated and compared to those obtained with the aerobic denitrifier Microvirgula aerodenitrificans. The consortium and the pure strain exhibited an aerobic denitrifying activity at air saturation conditions (7 mg dissolved oxygen l^–1), i.e. there was co-respiration of the two electron acceptors with significant specific nitrate reduction rates. Dissolved oxygen concentrations had no influence on denitrifying performances above a defined threshold: 0.35 mg l^–1 for the consortium and 4.5 mg l^–1 for M. aerodenitrificans respectively. Under these thresholds, decreasing the dissolved oxygen concentrations enhanced the denitrifying activity of each culture. The higher the carbon and nitrogen loads, the higher the performance of the aerobic denitrifying ecosystem. However, for M. aerodenitrificans, the nitrate reduction percentage was affected more by variations in nitrogen load than in carbon load. Received: 6 December 1999 / Received revision: 8 March 2000 / Accepted: 10 March 2000     

Thermophilic acidification of dairy wastewater

Acidification of simulated dairy wastewater was conducted in an upflow reactor at 55 °C. Results showed that the degree of acidification decreased with the increase in chemical oxygen demand (COD) loading rate, from 60.8% at 4 g l⁻¹ day⁻¹ to 27.1% at 24 g l⁻¹ day⁻¹. Carbohydrate was readily degraded at all loading rates, but degradation of protein and lipid decreased with the increase in loading rate. Most carbohydrate degradation occurred at the reactor bottom, whereas protein was degraded mainly after the carbohydrate became depleted. The predominant acidification products were acetate, propionate, butyrate and ethanol, whereas formate, i-butyrate, valerate, i-valerate, caproate, lactate, methanol, propanol and butanol were present in lesser quantities. The increase in loading rate resulted in the increase of propionate and the decrease of acetate, but had little effect on ethanol and butyrate productions. Only 2.5–8.8% of influent COD was converted to hydrogen and methane. The biomass yield was 0.30–0.43 mg VSS mg⁻¹ COD. Received: 8 December 1999 / Received revision: 14 February 2000 / Accepted: 25 February 2000     

Bioremediation of atrazine-contaminated soil by repeated applications of atrazine-degrading bacteria

Bioaugmentation has previously been unreliable for the in situ clean-up of contaminated soils because of problems with poor survival and the rapid decline in activity of the bacterial inoculum. In an attempt to solve these problems, a 500-l batch fermenter was investigated for its ability to deliver inoculum repeatedly to contaminated soils via irrigation lines. In a field experiment, mesocosms were filled with 350 kg soil containing 100 mg kg⁻¹ atrazine, and inoculated one, four or eight times with an atrazine-degrading bacterial consortium that was produced in the fermenter. After 12 weeks, no significant degradation of atrazine had occurred in soil that was inoculated only once; whereas, mesocosms inoculated four and eight times mineralized 38% and 72% of the atrazine respectively. Similar results were obtained in a laboratory experiment using soil contaminated with 100 mg kg⁻¹ [¹⁴C]atrazine. After 35 days, soil that was inoculated once with 10⁸ cfu ml⁻¹ of the consortium or with the atrazine-degrading bacterium, Pseudomonas sp. strain ADP, mineralized 17% and 35% of the atrazine respectively. In comparison, microcosms inoculated every 3 days with the consortium or with Pseudomonas sp. (ADP) mineralized 64% or 90% of the atrazine over this same period. Results of these experiments suggest that repeated inoculation from an automated fermenter may provide a strategy for bioaugmentation of contaminated soil with xenobiotic-degrading bacteria. Received: 20 November 1998 / Received revision: 8 February 1999 / Accepted: 12 February 1999     

Characterization and optimization of a two-phase partitioning bioreactor for the biodegradation of phenol

An electrochemical reactor employing activated carbon fibers (ACF) was constructed for the disinfection of bacteria in drinking water. The application of an alternating potential of 1.0 V and −0.8 V versus a saturated calomel electrode, for disinfecting and desorbing bacteria, enabled reactor operation for 840 h. Drinking water was passed through the reactor in stop/flow mode: 300 ml/min flow for 12 h and no flow for 12 h, alternately. The bacterial cell density in treated water was always been less than 20 cells/ml. It was also found that the formation of biofilm on the ACF reactor caused an increase in current, enabling the self-detection of microbial fouling. Received: 19 February 1996 / Received last revision: 23 July 1996 / Accepted: 2 September 1996     

Biodehalogenation of low concentrations of 1,3-dichloropropanol by mono- and mixed cultures of bacteria

The degradation of low concentrations of 1,3-dichloro-2-propanol (1,3-DCP) and related halohydrins by whole cells and cell-free extracts of soil bacteria has been investigated. Three bacteria (strains A1, A2, A4), isolated from the same soil sample, were distinguished on the basis of cell morphology, growth kinetics and haloalcohol dehalogenase profiles. Strain A1, probably an Agrobacterium sp., dehalogenated 1,3-DCP with the highest specific activity (0.33 U mg protein⁻¹) and also had the highest affinity for 1,3-DCP (K _m, 0.1 mM). Non-growing cells of this bacterium dehalogenated low concentrations of 1,3-DCP with a first-order rate constant (k ₁) of 1.13 h⁻¹ . The presence of a non-dehalogenating bacterium, strain G1 (tentatively identified as Pseudomonas mesophilius), did not enhance the dehalogenation rate of low 1,3-DCP concentrations. However, the mixed-species consortium of strains A1 and G1 had greater stability than the mono-species culture at DCP concentrations above 1.0 gl⁻¹. Received: 30 April 1996 / Received revision: 30 July 1996 / Accepted: 5 August 1996     

Long-term survival of the plant-growth-promoting bacteria Azospirillum brasilense and Pseudomonas fluorescens in dry alginate inoculant

Two plant-growth-promoting bacteria, Azospirillum brasilense Cd and Pseudomonas fluorescens 313, immobilized in 1983 in two types of alginate-bead inoculant (with and without skim-milk supplement) and later dried and stored at ambient temperature for 14 years, were recovered in 1996. The population in each type of bead had decreased, yet significant numbers survived (10⁵–10⁶ cfu/g beads). Population numbers depended on the bead type and the three independent bacterial counting methods: the conventional plate-count method, indirect enzyme-linked immunosorbent assay and the limited-enrichment technique. Both bacterial species retained several of their original physiological features. When inoculated onto wheat plants, both species colonized and produced plant-growth effects equal to those of the contemporary strain from a culture collection or to their own 1983 records. This study showed that bacteria can survive in alginate inoculant over long periods. Received: 1 May 1998 / Received revision: 24 August 1998 / Accepted: 3 September 1998