J. van Rijn

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

Abstract 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.

Nitrate removal in aquariums by immobilized pseudomonas.

Biological denitrification of nitrate to nitrogen gas was examined in a freshwater and a marine aquarium. Nitrate removal in the aquarium water was accomplished with denitrifiers immobilized in a freeze‐dried, alginate‐starch matrix. Starch served as a bacterial carbon source and cellular matrix‐strengthening filler. Freeze‐dried beads were placed in canisters through which nitrate‐rich aquarium water was recirculated. The freshwater aquarium (100 L) contained goldfish (Carassius auratus) at a total biomass of 390 g, whereas cichlids (Oreochromis mossambicus) were kept at a similar stocking density in the marine aquarium. Denitrification resulted in low ambient nitrate concentrations in both aquariums. The specific nitrate removal rate of the freshwater beads was significantly higher (50 μg of NO3‐N/bead/day) than that of seawater beads (5 μg of NO3‐N/bead/day). Differences in ambient nitrate concentrations between both aquariums and diffusion limitation of nitrate to the active denitrification sites within the beads might explain these observed differences.

Scanning electron microscopy of biofilm formation in denitrifying, fluidised bed reactors

Changes in bacterial colonisation on initially clean sand grains in an anaerobic fluidised bed reactor (FBR) were studied by scanning electron microscopy (SEM). The FBR was part of a laboratory-scale treatment system for nitrate removal from fish culture water. Denitrifying bacteria in the FBR were supplied with anaerobically digested fish feed as carbon and energy source and with nitrate as electron acceptor. Bacterial biofilm formation started rapidly with single rods settling in the fissures and cavities on the sand grains surface. Rods (Pseudomonas-like and Zoogloea-like) divided and accumulated and after about 10 days were integrated in a biofilm which almost covered the entire grains. Apart from single rods, colonies of bacteria resembling Zoogloea developed from the second day onwards. After approximately 10 days, the zoogloeal colonies were overgrown by bacteria resembling Pseudomonas. A steady increase in these cells was observed, eventually leading to a situation in which the majority of grains were totally covered with a compact biofilm comprised of these bacteria. The biofilm increased in thickness with time and around day 20, sloughing was observed at parts of the biofilm surface. These sloughed areas were colonised once more with bacteria according to a similar succession pattern observed on the initially clean sand grains. The colonisation pattern on sand in the laboratory-scale FBR was compared to the colonisation on sand grains in an FBR, operating in a pilot-plant of a closed intensive fish culture system. The bacterial biofilm structure was similar in both reactors. In the pilot-plant FBR all different stages of colonisation, observed in the laboratory-scale FBR, occurred simultaneously. Also in the pilot-plant FBR, bacteria resembling Zoogloea and Pseudomonas were most abundant.

Enumeration and factors influencing the relative abundance of a denitrifier, Pseudomonas sp. JR12, entrapped in alginate beads.

The relative abundance of the denitrifier, Pseudomonas sp. JR12, was examined in an alginate-based entrapment complex under non-sterile, denitrifying conditions. Immuno-labeling of the Pseudomonas inoculant followed by flow cytometry (FCM) was used for determination of the relative abundance of this bacterium under the various incubation conditions. Additional information on the relative abundance of the inoculant was obtained by a quantitative enzyme-linked immunosorbent assay (ELISA) and results obtained by FCM and ELISA were compared. Ambient nitrate levels controlled the successful, long-term proliferation of the inoculant. At low ambient nitrate levels, Pseudomonas sp. remained the dominant microorganism during incubation. Higher ambient nitrate concentrations, attained by either decreasing the inoculum size of Pseudomonas sp. or raising inlet nitrate concentrations of the medium supplied to the incubation vessels, resulted in a gradual shift toward other, nitrite-accumulating denitrifiers. Thus far, most studies on the use of entrapped microorganisms for bioremediation purposes have been conducted under controlled laboratory conditions. Based on this study, conducted under non-sterile laboratory conditions, it is concluded that in-situ bioremediation using entrapped target microorganisms is bound to fail without a proper understanding of the factors that cause the target microorganism to out-compete undesired microbial invaders. Furthermore, based on the close agreement between the two detection methods used, it is concluded that flow cytometry provides a rapid and accurate tool for the detection of the relative abundance of immuno-labeled target organisms in heterogeneous microbial populations.

Changes in Mechanical, Structural, and Denitrifying Properties of Entrapped Pseudomonas stutzeri Bacteria Preparations

The denitrifier, Pseudomonas stutzeri, entrapped in chitosan beads, was incubated under denitrifying conditions in a column receiving a continuous supply of full‐growth medium. Biochemical, structural, and mechanical properties of the beads were studied during the first 11 days of incubation; nitrate removal was followed for 45 days. Under these conditions they contracted and became lighter with time. Contraction was not uniform in all planes. Mechanical compressive properties of beads strengthened over the first 9 days and weakened thereafter. Concommittantly, structural changes of the beads were observed; denitrification and nitrite accumulation were lower in entrapped than in free cells.

Detection and removal of dissolved hydrogen sulphide in flow‐through systems via the sulphidation of hydrous iron (III) oxides

Abstract A novel automated warning and removal system for hydrogen sulphide in aqueous flow‐through systems has been developed based on the sulphidation of ferrihydrite sorbed to zeolite substrate. The system consists of a small flow‐through reaction cartridge with photo‐sensors positioned at the base. During the reaction, sulphide is initially oxidised to elemental sulphur by the ferrihydrite, and Fe2+ is subsequently released to solution. This Fe2+ then reacts with additional dissolved sulphide to form solid phase iron monosulphide. The colour change from orange ferrihydrite to black iron monosulphide is continuously monitored by the photo‐sensors, which provide a rapid and reproducible response (via a voltage change) to pulses of sulphidic water. The response of the photo‐sensors is linear with respect to inflowing sulphide concentration, while the most rapid response to dissolved sulphide occurs at a flow rate of approximately 200 ml min‐1 (equivalent to a hydraulic loading rate of 21 cm min‐1). The presence of phosphate in solution substantially decreases reaction rates due to adsorption to reactive surface sites. However, the response time of the photo‐sensors remains sufficient to provide a rapid indication of sulphidic conditions even in systems with high concentrations of dissolved phosphate. The cartridge has the advantage of partially or completely removing sulphide (depending on flow rate and substrate mass) from an initial pulse of water. At the optimal flow rate for the successful use of the cartridge as a sulphide warning system (200 ml min‐1), required substrate masses for the complete removal of dissolved sulphide (over the experimental range of 0–1000 μM) are relatively small (0.5–2 kg).

Accumulation of humic-like and proteinaceous dissolved organic matter in zero-discharge aquaculture systems as revealed by fluorescence EEM spectroscopy

Recirculating aquaculture systems (RAS), offering many economic and fish husbandry benefits, are characterized by an accumulation of dissolved organic matter (DOM) and, specifically, humic substances (HS). As reported in a number of studies, HS may affect biological activity in both invertebrates and vertebrates. Given the accumulation of HS in RAS, it is therefore of great interest to characterize DOM and, specifically, its HS fraction in the RAS. The present study was aimed at characterizing long-term changes in fluorescent DOM composition in the culture water of RAS systems, which were operated in a novel, zero water exchange mode. Two such zero-discharge recirculating systems (ZDS) were examined: a freshwater system, stocked with hybrid tilapia (Oreochromis aureus x Oreochromis niloticus) and a marine system, stocked with gilthead seabream (Sparus aurata). Excitation-emission matrices (EEMs) of fluorescence, coupled with parallel factor analysis (PARAFAC), were used to characterize and quantify the different DOM components in the ZDS. In the culture water, one tryptophan-like and four HS-like components were identified. The fluorescence intensities of three of the HS-like components as well as the tryptophan-like component increased at comparable rates during ZDS operation while a much slower accumulation of these compounds was observed in a parallel operated, flow-through, freshwater aquarium. The ZDS examined in this study comprised a sludge digestion stage where a considerable accumulation of all fluorescent components was detected. A HS-like components and a tryptophan-like component in blood of tilapia from the freshwater ZDS were similar to components found in the culture water. Blood levels of both components were higher in fish cultured in the DOM-rich ZDS than in fish raised in the control, flow-through freshwater aquarium. Fluorescence of the HS-like component found in the fish blood increased also with time of ZDS operation. The finding that fish blood contains a HS-like fluorescent component may have important implications for the understanding of the physiological effects of HS in fish and the possible benefits of these substances in aquaculture.

Starch as a filler, matrix enhancer and a carbon source in freeze-dried denitrifying alginate beads

Publisher Summary This chapter describes the experiments conducted to highlight the use of starch as filler, matrix enhancer, and a carbon source in freeze-dried denitrifying alginate breads. In these experiments, freeze-dried, alginate-based beads used for the immobilization of a denitrifying bacterium (Pseudomonas sp.) were filled with different concentrations 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 the starch content of the beads. Nitrite accumulation, because 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 exception of beads with the lowest starch content, at later stages of incubation. The results showed that that it is possible to improve the mechanical properties of beads made of natural polymers by using similar methods to those applied in the field of synthetic polymers. Not only the mechanical properties but also the biological properties of the freeze-dried alginate beads were improved by using starch as both a filler and carbon source. Beads containing the highest concentration of starch (40%) revealed a superior performance with respect to both longevity and denitrification, as compared to beads with less starch.

The protective effect of humic‐rich substances on atypical Aeromonas salmonicida subsp. salmonicida infection in common carp (Cyprinus carpio L.)

When challenged with atypical Aeromonas salmonicida subsp. salmonicida, exposure of the common carp (Cyprinus carpio L.) to different humic-rich compounds resulted in a significant reduction in infection rates. Specifically, in fish exposed to (i) humic-rich water and sludge from a recirculating system, (ii) a synthetic humic acid, and (iii) a Leonardite-derived humic-rich extract, infection rates were reduced to 14.9%, 17.0% and 18.8%, respectively, as compared to a 46.8% infection rate in the control treatment. An additional set of experiments was performed to examine the effect of humic-rich components on the growth of the bacterial pathogen. Liquid culture medium supplemented with either humic-rich water from the recirculating system, the synthetic humic acid or the Leonardite humic-rich extract resulted in a growth reduction of 41.1%, 45.2% and 61.6%, respectively, as compared to the growth of the Aeromonas strain in medium devoid of humic substances. Finally, in a third set of experiments it was found that while the innate immune system of the carps was not affected by their exposure to humic-rich substances, their acquired immune system was affected. Fish, immunized against bovine serum albumin, displayed elevated antibody titres as compared to immunized carps which were not exposed to the various sources of humic substances.