Search

Mass mortalities of juvenile Pacific oysters (Crassostrea gigas) in aquaculture operations have been linked to the ostreid herpes virus 1 (OsHV-1) or its variants. This oyster has heritable resilience to the virus which can provide a basis for selective breeding for higher OsHV-1 resistance, however, controlled challenge methods for improving selection response towards increased OsHV-1 resistance are required. The objective of this study was to compare the survival of sibling juvenile oysters exposed to either a laboratory virus challenge or a heat shock challenge (as proxy for the virus challenge) and the survival of the same families on two oyster farms where they were exposed to natural field conditions including the presence of OsHV-1. A strong correlation between the family rankings was observed for the two farm sites. Estimated narrow sense heritabilities (h2) on the underlying liability scale were 0.38 (on-farm survival), 0.45 (laboratory virus challenge survival) and 0.15 (heat shock challenge survival). Only on-farm survival and laboratory virus challenge had a high genetic correlation. The method for measuring heat shock resistance in juvenile oysters produced high variability in survival among families but low estimates of heritability. Overall, the results of this study indicate that the laboratory virus challenge is a simple and relatively effective tool for selective breeding specifically towards OsHV-1 resistance or as part of a controlled multi-trait program. Statement of relevance The OsHV-1 virus is a serious problem for commercial aquaculture of Pacific oysters in numerous countries. This paper tests two ways of evaluating oysters for resistance to OsHV-1 that can be incorporated into selective breeding programs.

A lack of understanding regarding the feeding biology of post-settlement mussels has considerably constrained the hatchery culture of many commercially important mussel species, including the Greenshell™ mussel, Perna canaliculus . Therefore, we evaluated how particles of different size (MySpat artificial diet full particle size range 2–60 μm, sieved MySpat diet with a size range of 2–15 μm; polystyrene beads, 3–15 μm, and microalgae, 4–6.5 μm) affected the retention and capture efficiency in post-settlement juveniles (PSJ). The response of PSJ of various sizes (PSJ-I: 0.3 mm shell length, PSJ-II: 1.0 mm, and PSJ-III: 7.0 mm) was different. Overall, smaller mussels (PSJ-I and PSJ-II) were unable to selectively capture particles by their size on all diets, conversely larger juveniles actively captured smaller particles between 2 and 8 μm. Independently of the PSJ size, the full particle size range MySpat artificial diet had a negative effect on the feeding activity of the mussels, with particles blocking the pallial organs in the non-selective PSJ-I and PSJ-II, and completely inhibiting the feeding activity in the more selective PSJ-III. These results provide useful information to understand the feeding biology and ecology of PSJ of this species that can be used for the improvement of hatchery feeding methods. Artificial and live food particle size can be chosen in order to avoid larger harmful particles (> 15 μm), and the exclusive use of smaller food particles should be considered for PSJ Statement of relevance This article provides information to better understand particle capture process in post-settlement juvenile mussel in order to improve hatchery/nursery feeding practices. The results of this research will allow making informed decisions regarding selection of food particle size in relation to the size of the juvenile mussels and their ability to deal with particles of different sizes. Furthermore will also help to decide the adequate size of transfer for grow-out as well as being useful for the development of suitable artificial diets.

Chaetoceros calcitrans is a small, fast-growing diatom with a high nutritional value for filter feeders. It is often used in hatcheries, particularly in the production of bivalve spat. It is widely produced in batch culture systems ranging from carboys to plastic bags to high volume tanks. Continuous culture of C. calcitrans has generally not been successful due to frequent crashes of the culture. We report the continuous culture of C. calcitrans in a hanging plastic bag system that is suitable for use in commercial hatcheries. Runs of continuous production lasted up to 125 d, with an average bag life of 24.4 d. Crashes occurred at irregular intervals in individual bags and caused the cell concentration to drop by up to three orders of magnitude. Cultures recovered over a few days following a crash. The temporary reduction of production due to crashes in individual bags was compensated by a 30% higher production than required. At dilution rates of 1.0–1.3 volume exchanges d − 1 the system produced 7–13 × 10 6 cells mL − 1 d − 1 . The cell diameter mode as determined by a Coulter Counter was 2.8–3.0 μm. The cells had the typical flat rectangular box shape with well-developed setae. The culturable bacterial population of the algae culture was 10 5 –10 7 colony forming units mL − 1 . Genetic integrity of C. calcitrans and monospecific status of the continuous culture were maintained over 7 sequential bag-to-bag transfers during a 125-day run. The fatty acid profiles of continuous and batch cultures of C. calcitrans were similar. Growth and survival of Greenshell™ mussel ( Perna canaliculus ) larvae were identical when fed with C. calcitrans batch culture or continuous culture delivered directly from the harvest line. When continuous culture was first collected over 24 h, diluted and then delivered to the rearing tanks over 24 h, larval growth was markedly slower (24 d to reach settlement competency compared to 21 d for the other two treatments); however the subsequent settlement success of competent larvae was not statistically different between the three diets (‘Carboy’, ‘Continuous/Harvest line’, ‘Continuous/Feed-out bin’; 33.9–35.5% settled). Concomitant to the differences in larval performance we observed a difference in larval fatty acid profile: lower-performing larvae had a markedly lower proportion of mono-unsaturated fatty acids and a markedly higher proportion of poly-unsaturated fatty acids. Lower performance and different fatty acid profiles of larvae fed the ‘Continuous/Feed-out bin’ diet appeared to be caused by unidentified changes that occurred during storage of the C. calcitrans culture.

Earlier work identified two novel strains of probiotic bacteria, Alteromonas macleodii 0444 and Neptunomonas sp. 0536, for Greenshell™ mussel ( Perna canaliculus ) larvae. Herein, we investigated whether the combination of the two probiotics in a multi-strain mix provided a) enhanced larval production during routine rearing and b) improved larval protection during two separate pathogen-challenge tests ( Vibrio sp. DO1 and Vibrio splendidus ). The response of larvae to multi-strain or single-strain probiotic administration was compared to that of larvae without probiotics. Two concentrations of each probiotic were tested (10 7 and 10 8 CFU ml − 1 ). Addition of 10 8 CFU ml − 1 multi-strain mix in the routine rearing of larvae yielded smaller-sized larvae and a lower feeding rate when compared with the 10 7 CFU ml − 1 and control groups. During the challenge test against Vibrio sp. DO1 and V. splendidus , protection by single-strain probiotic administration was observed at both levels of probiotic administration (10 7 and 10 8 CFU ml − 1 ), with no apparent added protection from multi-strain probiotics. Although 10 8 CFU ml − 1 levels provided protection against pathogen attack, they were also potentially detrimental to normal larval rearing when administered in combination and, as such, where administration of A. macleodii 0444 and Neptunomonas sp. 0536 would be applied together, a multi-strain mix of probiotics at 10 7 CFU ml − 1 , is recommended as the best concentration of each probiotic.

Antibiotic management of aquacultured animals, such as Greenshell™ mussel (GSM) larvae, Perna canaliculus , is undesirable because of health concerns and political pressures; hence, alternatives are needed. Herein, two novel probiotic bacteria were identified and trialled in a GSM larval rearing hatchery. Sequencing of the 16S rRNA gene and phylogenetic analysis identified the strains as Alteromonas macleodii 0444 and Neptunomonas sp. 0536. Both probiotics were evaluated separately at the Glenhaven Aquaculture Centre hatchery facility during routine larval rearing and when the larvae were challenged with both a high (10 7 and 10 6 CFU ml −1 ) and low (10 6 and 10 5 CFU ml −1 ) pathogenic dose of Vibrio sp. DO1 and V. splendidus respectively. In all experiments, probiotic application improved larval survival significantly when administered prior to pathogen exposure. Across all experiments, larvae that were exposed to the high and low dosages of pathogens averaged 14% and 36% survival respectively on the fourth day following pathogen exposure. The administration of probiotics prior to pathogen challenge resulted in larval survival of 50% and 66% respectively. Non-inoculated control larvae and larvae administered the probiotic alone demonstrated 67% and 79% survival respectively. Neptunomonas sp. 0536 appeared to suppress naturally occurring vibrios in the culture environment of healthy GSM larvae. This is the first report of A. macleodii and Neptunomonas sp. as probiotic bacteria in a large scale production facility.

Bacterial pathogens of Greenshell mussel (GSM) larvae can cause batch losses during hatchery production. Twenty-two isolates were screened using a larval bioassay. Two strains were identified as potential pathogens. Phenotypic identification of these strains revealed two non-reactive Gram-negative, oxidase positive rods. Sequencing of the 16S rRNA gene identified Vibrio splendidus and a V. coralliilyticus/neptunius-like isolate as pathogens of GSM larvae, with an ability to cause 83% and 75% larval mortality in vitro, respectively, at a concentration of 10(2) CFU mL(-1). Histopathology indicated that the route of infection was via the digestive system. Using healthy larvae as target hosts, Koch's postulates were confirmed for the two isolates. This is the first report on pathogens of GSM larvae.

This paper describes a simple method for cryopreserving sperm of the Pacific oyster ( Crassostrea gigas Thunberg) in quantities suitable for commercial spat production. Experiments to refine the cryoprotectant mixtures demonstrated the key role of trehalose. Trehalose alone (at 0.45 M final concentration) was an effective cryoprotectant. The addition of 2.5–15% dimethyl sulphoxide (DMSO) in combination with 0.45 M trehalose gave only modest improvement in fertility over trehalose alone ( p =0.056). There was no significant difference in fertility among DMSO concentrations ( p =0.611). Seawater (SW) without cryoprotectant gave very poor results, but yielded some fertilization at very high sperm concentrations (7±1% at 10 7 sperm mL −1 , 21±2% at 3.2×10 7 sperm mL −1 , mean±S.E., n =3). The fertility of unfrozen sperm was 30- to 100-fold higher than that of sperm cryopreserved with DMSO and/or trehalose. For sperm cryopreserved in 4.5-mL cryovials, two simplified freezing methods gave fertilization rates equivalent to sperm cryopreserved by controlled rate freezing ( p =0.386). These methods involved securing the cryovials to aluminium canes and then either placing them into a bath of methanol chilled with dry ice, or holding them on a floating rack 3 cm above liquid nitrogen. A third technique of plunging the cryovials directly into liquid nitrogen gave reduced and variable fertility relative to the methanol/dry ice bath method ( p =0.032). The commercial applicability of the protocols was demonstrated on a batch of 30 million eggs. Fertilization with cryopreserved sperm yielded 81% fertilization, and larval rearing by normal commercial practises yielded 3.7 million settled spat, which was comparable to the 2.5 million spat from a parallel batch fertilized with unfrozen sperm.

A new brevetoxin analog, brevetoxin B5 (BTX-B5) was isolated together with BTX-B1 and PbTx-3 from the New Zealand cockle Austrovenus stutchburyi harvested at an outbreak of neurotoxic shellfish poisoning early in 1993. The structure was elucidated by comparison of its spectral data (NMR and CAD FAB MS/MS) with those of BTX-B1 and PbTx-2, and confirmed by synthesis from PbTx-2 with SeO2 and H2O2. It was detected in the toxic greenshell mussel Perna canaliculus and Pacific oyster Crassostrea gigas collected at the same time.

The McMurdo Ice Shelf is in the northwestern corner of the Ross Ice Shelf, between Ross Island and Brown Peninsula. An area of about 1,500 km2 is known as Dirty Ice, an ablation zone covered by gravel. A large portion of this gravel originates from marine sediment, and much of the shelf ice is frozen seawater (7). During the summer melt, the area is covered by ponds of a wide size range between hummocks with a vertical profile as high as 20 m (12). Ponds form and disappear again over decades, leading to a wave-like cycling of the shelf surface through ponds and hummocks (2). Freezing, thawing, and evaporation often lead to pronounced solute gradients and water column stratification (9a). The bottoms of these ponds are covered with thick mats consisting of cyanobacteria, diatoms, and green algae (11, 12), below which is a layer of anaerobic sediment. Variations in chemical and physical conditions between the ponds lead to community differentiation within and between the mats and to differences in mat morphology, thus creating in a small area a variety of modern unlithified stromatolites unique on Earth (31). The mats harbor a small population of grazers, mainly the rotifer Philodinia gregaria, but their activity does not appear to have a key function in the ecosystem. Apart from the discrete ponds on the ice shelf, there are also ponds in estuaries. These ponds are connected by seawater at high tide and also contain diverse assemblages of algae and cyanobacteria with primary production characteristics similar to those for discrete ponds (8). Previous studies have shown that ponds may vary in conductivity, from highly saline to almost freshwater (12). While considerable knowledge has been gained about the photosynthetic activity and carbon flux attributed to the algal mats of these ponds, together with physical and chemical characteristics (2, 11), no studies on the processes occurring in the underlying pond sediments have been published. In this paper we describe some of the major heterotrophic processes occurring in the sediments of a low-salinity meltwater pond, Orange Pond, near Bratina Island. The rates of terminal anaerobic processes are given together with the contributions of these processes to terminal carbon and electron flow, and we discuss their significance for carbon flux in the mat-sediment ecocouple.

A new brevetoxin B analog, brevetoxin B4 (BTXB4), was isolated as the major toxin in greenshell mussels, Perna canaliculus, collected in New Zealand at the time of the neurotoxic shellfish poisoning incident. The new analog accounted for nearly two-thirds of the mouse lethality of the shellfish and was determined to be a mixture of N-myristoyl-BTXB2 and N-palmitoyl-BTXB2. Copyright © 1999 John Wiley & Sons, Ltd.

A new brevetoxin analog, brevetoxin B2 (BTXB2), was isolated from greenshell mussels, Perna canaliculus, collected at the time of the neurotoxic shellfish poisoning incident in New Zealand. The structure was elucidated based on NMR, CAD FAB MS/MS, and chemical degradation experiments.

Hydrogen and acetate inhibit the microbial degradation of propionate and ethanol in digesting sludge. Propionate oxidation is more sensitive than ethanol oxidation to product activities. This difference can be explained on thermodynamic grounds. It is concluded that the limiting factors for complete anaerobic mineralization of biodegradable organic matter in digesting sludge are to be sought in the boundary conditions for the exergonic oxidation of propionate. The different ecological significances of hydrogen and acetate as inhibitory agents of fatty acid degradation in sludge digestion are discussed. Hydrogen partial pressure and alkalinity are recommended as parameters in monitoring sludge digestion.

A new brevetoxin analog, brevetoxm B3 (BTXB3 1 ). was isolated from the greenshell mussel, Perna canaliculus from New Zealand. In BTXB3 the brevetoxin B skeleton is modified by cleavage of ring D. esterification of the resulting alcohol, and oxidation of the aldehydic terminus.

A new marine toxin, gymnodimine, was isolated from New Zealand oysters, Tiostrea chilensis, and the dinoflagellate Gymnodinium cf. mikimotoi. Its unique structure was elucidated by spectroscopic methods.

A new marine toxin, gymnodimine, was isolated from New Zealand oysters, Tiostrea chilensis, and the dinoflagellate Gymnodinium cf. mikimotoi. Its unique structure was elucidated by spectroscopic methods.

Bacterial diseases remain a large problem in aquaculture hatcheries. The successful design and implementation of protective measures in the hatchery depends on an understanding of the dynamics of the infection process. Developing an in situ experimental protocol for pathogen challenge is therefore of paramount importance. Here, we demonstrated the minimum effective pathogenic dose (MEPD) of Vibrio splendidus (10(5) CFU ml(-1)) and a Vibrio coralliilyticus/neptunius-like isolate, Vibrio sp. DO1 (10(6) CFU ml(-1)), for New Zealand Greenshell mussel (GSM, Perna canaliculus) larvae during hatchery production. In a flow-through water hatchery system, larvae given 1 to 2 h of static water exposure to these pathogen doses showed respective average cumulative mortalities of 58 and 69% on the fourth day following pathogen exposure. After the 1 to 2 h static exposure, larvae were returned to flow-through water. Larvae exposed to a dosage one order of magnitude greater than the MEPD had higher mortalities of 73 and 96% for V. splendidus and Vibrio sp. DO1 respectively. These 4 levels of mortality were significantly greater than those of the non-exposed control larvae which respectively averaged 23 and 35% in experiments involving V. splendidus and Vibrio sp. DO1. Experiments were repeated 4 times to confirm reproducibility. After pathogen exposure, pathogens were detected in the larvae and tank water of treatments with dosages of > or =10(5) CFU ml(-1) (V. splendidus) and 10(6) CFU ml(-1) (Vibrio sp. DO1), but not in treatments with lower pathogen dosages. The challenge protocols are reproducible and provide an opportunity to assess measures for the protection of GSM larvae against infection in the hatchery environment.

A bioassay was developed to screen and select bacterial strains as probiotics for an alternative to antibiotic usage in the management of bacterial pathogens of Greenshell™ mussel (GSM) larvae, Perna canaliculus. Sixty-nine isolates originating from a GSM hatchery environment were tested for probiotic activity in larval pathogen-challenge bioassays conducted in tissue culture dishes (TCDs). Vibrio splendidus and a V. coralliilyticus/neptunius-like isolate, Vibrio sp. DO1, were the tested pathogens. Forty of the tested isolates afforded larval survival significantly greater than pathogen controls (p < 0.05). The bioassay technique achieved a 58% success rate in searching for putative probiotics and highlighted the benefit of including the host animal in the first stage of the screening procedure. The time of inoculation of putative probiotic strains prior to pathogen challenge influenced the outcome of the assay. A pre-exposure period of 20 h revealed a greater number of potential probiotics than a two-hour pre-exposure period. Pilot challenge tests, under normal hatchery conditions, confirmed the usefulness of the TCD screening method in recognising effective probiotics. © 2009 Elsevier B.V. All rights reserved.

Aquaculture production of molluscs is worth US$11 billion per year and represents 65% of World mollusc product. A significant limitation to the industry is loss of stock through bacterial disease. Traditional methods to combat disease with antibiotics have been questioned and alternatives have been sought. The field of probiotics as well as the screening methods used to acquire probiotic strains for the alternative management of disease in aquaculture is discussed. This review provides a comprehensive summary of probiotics in aquaculture with special reference to mollusc culture. © 2007 Elsevier B.V. All rights reserved.

A gram-negative, rod- to oval-shaped, aerotolerant anaerobic bacterium was isolated from an anaerobic enrichment inoculated with sediment taken from below the cyanobacterial mat of a high-salinity pond near Bratina Island on the McMurdo Ice Shelf, Antarctica. The organism was positive for terminal oxidase and catalase and was motile by means of a polar flagellum. Optimal growth of anaerobic cultures occurred at 12 degrees C, at pH 6.5, and at an NaCl concentration of 3% (w/v). Of a variety of polysaccharides tested, only starch and glycogen supported growth. No growth was observed on cellulosic substrates and xylan, and the organism was unable to attack esculin. Monosaccharides and disaccharides, including the cyanobacterial cell-wall constituent N-acetyl glucosamine, were fermented. Per 100 mol of hexose, the following products (in mol) were formed: acetate, 60; formate, 130; ethanol, 56; lactate, 73; CO2, 15; and butyrate, 2. Propionate, ethanol, n-propanol, n-butanol and succinate were not detectable in the culture medium (< 1 mol per 100 mol of monomer). Hydrogen was not detected in the head space (detection limit < 10(-5) atm). Growth yields in aerobic static liquid cultures were slightly higher than those in anaerobic culture, and fermentation favoured acetate at the expense of electron sink products. Growth was inhibited in aerobic shaking cultures, and the organism did not utilize nitrate or sulfate as electron acceptors. The G+C content of the DNA from the bacterium was 42.8 mol%. A phylogenetic analysis indicated that the organism is a member of the gamma-subgroup of Proteobacteria, but that it is distinct from other members of this group based on the sequence of its 16S rRNA gene, mol% G+C, morphology, and physiological and biochemical characteristics. It is designated as a new genus and species; the type strain is star-1 (DSM 10704).

A gram-positive, motile, rod-shaped, strictly anaerobic bacterium was isolated from an enrichment initiated with sediment taken from below the cyanobacterial mat of a low-salinity pond on the McMurdo Ice Shelf, Antarctica. The organism grew optimally at 12 degrees C, at pH 6.5, and at an NaCl concentration of0.5% (w/v). It survived freeze-thawing at low salt concentrations,but not exposure to temperatures over 25 degrees C for more than 20 h or short-term exposure to temperatures50 degrees C. Out of a variety of polysaccharides tested as growth substrates, only xylan supported growth. The organism also grew on a variety of mono- and disaccharides including the cyanobacterial cell wall constituent, N-acetyl glucosamine. Fermentation products on a mol product per 100 mol of hexose monomer fermented basis were: acetate, 72; formate, 72; butyrate, 55; hydrogen, 114; and CO2, 100. Not detectable in the culture medium(2 mol per 100 mol of monomer) were lactate, propionate, ethanol,n-propanol, n-butanol, and succinate. The G+C content of the DNA from the bacterium was 33 mol%, and a phylogenetic analysis indicated that it grouped closely with members of the RNA-DNA homology group 1 of the genus Clostridium. It differed from other species of this genus with regard to growth temperature optimum, substrate range, and fermentation pattern, and is therefore designated as a new species of Clostridium for which the name Clostridium vincentii is proposed. The type strain is lac-1 (DSM 10228).

A method was developed for rapid measurement of soil denitrification under conditions where natural soil structure and aeration status is maintained. Air was continuously recirculated by means of a membrane pump through a soil core and a sample loop of a gas chromatograph equipped with an electron capture detector. Addition of acetylene to the recirculating air permitted measurement of denitrification in the soil core. Because of the rapid distribution of C2H2 and removal of N2O provided by the gas flow, denitrification rates could usually be determined in less than 2 h. By means of external 6-way and 8-way valves, four soil cores could be simultaneously analyzed on one gas Chromatograph equipped with dual detectors. Soil cores could also be stored at 4°C for later analysis without affecting the denitrification rate. The detection limit for denitrification rate measurements was 0.5 ngN g−1 soil day−1 or approximately 2.6 g N ha−1 day−1. Coefficients of variation for repeated measurements on the same soil core were usually less than 15%, but coefficients of variation for repacked or natural cores of the same soil were much higher (70–90%) Disruption of the natural soil structure by sieving increased the denitrification rate in an aggregated clay loam soil, but decreased the rate in a non-aggregated sandy soil. These results illustrate the importance of maintaining natural soil structure during denitrification measurements. The effect of pumping gas through soil was evaluated by comparing denitrification rates in soil cores where C2H2 was allowed to distribute into the soil by passive diffusion with rates obtained by pumping. Lower denitrification rates were observed in the static incubation presumably due to limited diffusion of C2H2 into or N2O out of the denitrifying sites in the soil. This diffusion limitation could be overcome in the static incubations if C2H2 was initially distributed through the soil by pumping. This gas flow method is well suited to the study of soil denitrification rates under nearly natural conditions because the indigenous substrates and anaerobic microsites are preserved, the rapidity in which denitrification rates can be measured, and the high sensitivity and relatively low analytical variability of the method.

SUMMARY. 1 In sediment from Wintergreen Lake, Michigan, denitrification was not detectable by the acetylene inhibition method at in situ nitrate concentrations. When nitrate was added to sediment slurries, denitrification capacities up to 18.8μg N g-1 h-1 were measured. The denitrification capacities decreased with increasing sediment depth and distance from shore. 2 The high denitrification capacities in these sediments which under natural conditions had no supply of nitrate and oxygen suggested that denitrifies with alternative mechanisms for anaerobic energy conversion were present. Nitrous oxide was a significant portion of the N-gas produced immediately after the nitrate addition. Small amounts (4–5% of the total N-gas production) of nitric oxide accumulated in the early phase of nitrate reduction. Presumably after depletion of nitrate and nitrite both N2O and NO were further reduced to N2. 3 About 70%r of the added nitrate was denitrified, and the remainder was assumed to have been reduced to ammonium.

In January 1982, sediment microbial N transformations and inorganic N fluxes across the sediment/water interface were studied at nine sites off the South Island West Coast, New Zealand. The sediments showed a great variety in physical, chemical and biological properties. The sediment organic matter had a molar C N ratio of 5.9–10.9, and the total N P ratio was 1.2–4.0. The denitrification capacity in the top 7.5 cm of sediment was 0.1–77.2 mmol N m −2 day −1 and generally declined with increasing sediment depth. The in situ denitrification rate was 0.02–1.84 mmol N m −2 day −1 and highest activities were generally found in surface sediments and at 6–7.5 cm depth. Denitrification accounted for 82–100% of total nitrate reduction. Net N mineralization was indirectly estimated at 0.6–2.4 mmol N m −2 day −1 , and the experimental determination of this N transformation gave 0.6–3.2 mmol N m −2 day −1 . Denitrification accounted for 3–75% of net N mineralization. The diffusive flux of ammonium and nitrate across the sediment/water interface was 0.1–0.7 and 0.1–0.6 mmol N m −2 day −1 , respectively.

Characteristics of dissimilatory nitrate reduction by Propionibacterium acidi-propionici, P. freudenreichii, P. jensenii, P. shermanii and P. thoenii were studied. All strains reduced nitrate to nitrite and further to N2O. Recovery of added nitrite-N as N2O-N approached 100%, so that no other end product existed in a significant quantity. Specific rates of N2O production were 3 to 6 orders of magnitude lower than specific rates of N2 production by common denitrifiers. Oxygen but not acetylene inhibited N2O production in P. acidi-propionici and P. thoenii. Nitrite reduction rates were generally higher than nitrate reduction rates. The enzymes involved in nitrate and nitrite reduction were either constitutive or derepressed by anacrobiosis. Nitrate stimulated synthesis of nitrate reductase in P. acidi-propionici. Specific growth rates and growth yields were increased by nitrate. At 10 mM, nitrite was toxic to all strains, and at 1 mM its effect ranged from none to total inhibition. No distinction was obvious between incomplete forms of denitrification and dissimilatory nitrate reduction to ammonia. N2O production from nitrite by propionibacteria may represent a detoxication mechanism rather than a part of an energy transformation system.

Methanogenesis by a Syntrophomonas wolfei/ Methanospirillum hungatei coculture was inhibited in presence of ethylene and the hydrogenation catalyst Pd-BaSO4. However, butyrate oxidation by S. wolfei continued and ethylene was reduced to ethane. Per mol of butyrate oxidized, 2.4 mol acetate was produced and 0.8 mol ethylene was reduced. Acetylene, propylene and butene were less effective as H2 acceptors than ethylene, and addition of bromoethanesulfonic acid was necessary to inhibit methanogenesis in the presence of the two longer-chain olefins. Other hydrogenation catalysts were less effective in the order Pd-charcoal < PE-asbestos < Pd-PEI beads < Pt-Al2O3, Pd-CaCO3. Optimal ethylene hydrogenation was achieved with still incubation in presence of 7.2 mg Pd-BaSO4 and 0.7 g sand per ml medium. The higher catabolic rate of S. wolfei in presence of the methanogen indicated that the biological H2 removal mechanism was more efficient than the catalytic olefin reduction.

The acetylene block technique was used to assay denitrification in undisturbed sediment cores of an intertidal mud flat. Nitrogen loss due to this process was estimated at 1 to 41 kg of N/hectare (ha) per year. Anaerobic nitrate-saturated slurry of the same sediment had a denitrification capacity of 1,026 kg of N/ha per year. The acetylene block technique failed at low nitrate concentrations, so that denitrification at average in situ nitrate concentrations could not be determined. Denitrification followed zero-order kinetics at nitrate concentrations high enough to allow successful blockage of N 2 O reduction. Thus, an estimate of in situ rates based on kinetic parameters and in situ nitrate concentrations was impossible. No denitrification was observed in a slurry of the top 1.5 cm under aerated conditions and nitrate saturation. In undisturbed sediment, significant denitrification occurred in few discrete sites within a matrix of undetectable or low activity. Despite numerous errors contributing to the uncertainty of the estimate of in situ rates, the result obtained by this method was considered more valuable than the determination of denitrification capacities. Methods which include severe changes of physical and chemical parameters may frequently lead to overestimates of denitrification rates.

Among five hydrogenation catalysts, palladium on charcoal was the most reactive one when suspended in anaerobic culture medium, and Lindlar catalyst (Pd on CaCO 3 ) was the most reactive one when suspended in the gas phase of culture tubes. Palladium on charcoal in the culture medium (40 to 200 mg 10 ml −1 ) completely inhibited growth of Neocallimastix frontalis and partly inhibited Ruminococcus albus . Lindlar catalyst (40 to 200 mg per tube) suspended in a glass pouch above the culture medium did not affect the rate of cellulose degradation or the ratio of fermentation products by these organisms. Acetylene added to tubes containing Lindlar catalyst in pouches, and either of the two organisms in monoculture or coculture with Methanospirillum hungatei , was reduced to ethylene and then ethane, followed by hydrogen production. Similar results were obtained with 1-pentene. Neither acetylene nor 1-pentene affected cellulose degradation but both inhibited methanogenesis. In the presence of Lindlar catalyst and propylene or 1-butene, fermenter-methanogen cocultures continued to produce methane at the same rate as controls and no olefin reduction occurred. Upon addition of bromoethanesulfonic acid, methanogenesis stopped and olefin reduction took place followed by hydrogen evolution. In a gas mixture consisting of propylene, 1-butene, and 1-pentene, the olefins were reduced at rates which decreased with increasing molecular size. These results demonstrate the technical feasibility of combining in one reactor the volatile fatty acid production by anaerobic digestion with chemical catalyst-mediated reductions, using the valuable by-product hydrogen.

Acetylene inhibition and 13N methods were used to assay digested sludge for its potential to denitrify and to reduce nitrate to ammonium. At nitrate concentrations below 10 μM, the reduction of N2O to N2 was not inhibited by acetylene concentrations as high as 80 kPa, though at higher nitrate concentrations acetylene was an effective inhibitor. NO, N2O, and N2 were produces immediately after addition of nitrate or nitrite, indicating that denitrifying enzymes were present. NO was maintained at a concentration of 2–5 nM, while nitrate or nitrite were being reduced, but this gas was depleted once the ionic N oxide substrates were exhausted. Acetylene had little effect on appearance and disappearance of NO. It was also noted that NO was readily consumed by chemical reactions in the anaerobic sludge. Added N2O was reduced without a lag, but pasteurized samples did not consume N2O although they produced it. Fresh digested sludge reduced 60–70% of the added 13NO3− to 13NH4+ with the rest of the NO3−-N presumably lost to denitrification. This agrees well with the nitrate partitioning observed by the acetylene inhibition method in which 30–40% of the NO3−-N was recovered as N2O. Denitrification capacity persisted in both digested sludge and methanogenic enrichment culture which had been grown in a chemostat for 2.5 years with acetate and ammonium as the solid carbon and nitrogen source. This suggests that denitrifiers with capacities for alternative anaerobic energy metabolism may be more common than now known.