Robin J. Shields

Placing microalgae on the biofuels priority list: a review of the technological challenges

Microalgae provide various potential advantages for biofuel production when compared with ‘traditional’ crops. Specifically, large-scale microalgal culture need not compete for arable land, while in theory their productivity is greater. In consequence, there has been resurgence in interest and a proliferation of algae fuel projects. However, while on a theoretical basis, microalgae may produce between 10- and 100-fold more oil per acre, such capacities have not been validated on a commercial scale. We critically review current designs of algal culture facilities, including photobioreactors and open ponds, with regards to photosynthetic productivity and associated biomass and oil production and include an analysis of alternative approaches using models, balancing space needs, productivity and biomass concentrations, together with nutrient requirements. In the light of the current interest in synthetic genomics and genetic modifications, we also evaluate the options for potential metabolic engineering of the lipid biosynthesis pathways of microalgae. We conclude that although significant literature exists on microalgal growth and biochemistry, significantly more work needs to be undertaken to understand and potentially manipulate algal lipid metabolism. Furthermore, with regards to chemical upgrading of algal lipids and biomass, we describe alternative fuel synthesis routes, and discuss and evaluate the application of catalysts traditionally used for plant oils. Simulations that incorporate financial elements, along with fluid dynamics and algae growth models, are likely to be increasingly useful for predicting reactor design efficiency and life cycle analysis to determine the viability of the various options for large-scale culture. The greatest potential for cost reduction and increased yields most probably lies within closed or hybrid closed–open production systems.

Blastomere morphology as a predictive measure of fish egg viability

Abstract For most fish species, blastomere size and shape are uniform during normal cleavage. However, abnormal blastomere morphology has been observed in a variety of studies, either induced directly by chemical or physical Stressors, or observed in eggs collected from plankton or broodstock. It has been proposed that abnormalities are indicative of low egg viability and may provide a useful predictive tool for egg assessment. However, there is little direct evidence relating blastomere morphology to egg survival. This paper reviews the literature on abnormal embryonic development in fish, and presents new findings on the significance of blastomere morphology in the Atlantic halibut, Hippoglossus hippoglossus. Several studies have reported qualitative relationships between egg batch survival and the proportion of normal embryos for a variety of fish species. In order to investigate more closely the relationship between blastomere morphology and fish egg survival, incubation experiments where carried out with eggs of Atlantic halibut. Eggs were individually assessed at the 8-cell stage for 5 blastomere characteristics and incubated to hatch in multiwell microtitre plates. For data pooled from 13 egg batches, the mean score for each blastomere characteristic showed a significant positive linear regression with the number of eggs hatched. Multiple regression analysis, incorporating all five blastomere characteristics, demonstrated a high degree of correlation between the independent variables. While the experimental procedure that was used for halibut eggs is too laborious for routine hatchery application, it is proposed that subjective observations of blastomere morphology could be readily adopted by commercial hatcheries to provide early assessment of egg viability.

Further description of the development of the digestive organs in Atlantic halibut (Hippoglossus hippoglossus) larvae, with notes on differential absorption of copepod and Artemia prey

Abstract Despite the scientific and commercial interest that Atlantic halibut has received over the past decade, little is known of the ontogeny of its digestive tract during larval development. The provision of enriched Artemia as prey has been associated with incomplete metamorphosis while improved development has been achieved using wild plankton e.g., copepods. The aim of the present study was to investigate whether there was any variation during development in the ability of the larvae to digest and incorporate Artemia or copepods. Groups of larvae fed either Artemia enriched with an oil emulsion (Super Selco™) and with a dried protist Schizochytrium (Algamac 2000™) or with a marine calanoid copepod (Eurytemora velox) were sampled weekly from the end of yolksac resorption to the completion of metamorphosis. The sites of absorption were visualised by the presence of vacuoles in the epithelial cells of the digestive tracts mucosa. During the first 30 days of exogenous feeding, before the stomach had formed, there were small inclusions on the apical side of the rectal epithelial cells in both groups. Between 30 and 50 days post first-feeding, at the time when the stomach was differentiating with the appearance of gastric glands, and the four pyloric caecae were starting to elongate, numerous supranuclear vacuoles were observed in the mid- and hind-gut epithelial cells of the copepod-fed group. The rectal epithelial cells showed lipid vacuoles that filled their apical ends suggesting significant absorptive activity. By contrast, few vacuoles were present in the rectum of the Artemia-fed larvae. By the end of metamorphosis (up to 83 days post first-feeding), the copepod-fed larvae had an adult-like digestive tract with functional stomach and highly absorptive intestine. At the same age, the Artemia-fed larvae showed that the rectum was still the major site of absorption; this is a typical larval feature. Furthermore, observations of the guts content confirmed the poor ability of the halibut larvae to digest Artemia in comparison to copepods. Indeed, while no individual copepod could be identified in the digestive chyme past the mid-gut of these larvae, partially digested Artemia could be seen as far as the anus in those fed on Artemia. Although the rate of development and of differentiation of the digestive tract of first-feeding halibut larvae appeared unaffected by the nature of their prey items, our results show a difference in the absorption sites of copepods vs. Artemia. These findings are of considerable significance to the rearing techniques used in the commercial production of halibut juveniles.

Changes in the gut-associated microflora during the development of Atlantic halibut (Hippoglossus hippoglossus L.) larvae in three British hatcheries

The bacterial flora of reared Atlantic halibut eggs, larvae, juveniles and adults from three different UK halibut hatcheries was monitored using a combination of traditional biochemical tests, the BIOLOG GN bacterial identification system, PCR-RFLP of 16S rRNA genes and partial 16S rDNA gene analysis. A gut microflora was detected towards the beginning of the non-feeding yolk-sac stage, with a substantial increase as the larvae started to feed. Bacteria isolated from non-feeding yolk-sac larvae were predominantly non-fermentative Gram-negative rods, in particular Pseudoalteromonas species; by contrast, the presumptive gut microflora of first-feeding larvae was generally dominated by members of the Vibrio genus. There was evidence of a bacterial succession after the larvae started to feed. Initially, first-feeding halibut reared on enriched Artemia were generally colonised by live food-associated bacteria, particularly Vibrio splendidus and V. alginolyticus-type organisms. Genetically similar V. splendidus isolates were recovered from enriched Artemia and the guts of first-feeding halibut larvae in all three hatcheries. However, only a selected proportion of the Artemia-associated bacteria appeared able to establish themselves within the guts of larval halibut. First-feeding halibut larvae were sometimes colonised by opportunistic bacteria. The gut floras of two individual batches of halibut larvae were briefly dominated by a Pseudoalteromonas and a Photobacterium phosphoreum isolate, respectively. Genetically-similar V. salmonicida-type organisms were present in larvae sampled from all three hatcheries, both from copepod and Artemia-fed fish. The guts of the adult halibut analysed had a culturable flora dominated by Photo. phosphoreum.

Fertility and motility of sperm from Atlantic halibut (Hippoglossus hippoglossus) in relation to dose and timing of gonadotrophin-releasing hormone agonist implant

Abstract In broodstocks of Atlantic halibut, Hippoglossus hippoglossus , male and female gamete production often becomes unsynchronised towards the end of the spawning season—milt becomes very viscous and difficult to express while the females are still producing batches of good quality eggs. Gonadotrophin-releasing hormone agonist (GnRHa) has been shown to stimulate spermiation in a number of fish species. Therefore, we conducted two experiments where male halibut were implanted intramuscularly with pellets containing GnRHa. The effect of the pellets was tested at three periods: before, at the height of and at the end of spermiation. In the middle period, GnRHa was tested at two doses (5 and 25 µg/kg bodyweight). Measurements were made of milt hydration, sperm motility and fertilisation rate. Implanted males began spermiation at least 4 weeks before control males. Both doses of GnRHa increased the fluidity of the milt. This effect lasted for at least 20 days in the low dose group and for 40 days in the high dose group. When applied at the end ofthe season, GnRHa reversed the normal trend for the milt to become more viscous. GnRHa treatments did not affect fertilisation rates obtained with the sperm. However, towards the end of the spawning season, sperm motility was enhanced in males treated with the high dose of GnRHa (25 μg/kg) compared to controls. As described previously, plasma concentrations of the gonadal steroids, 5β-pregnane-3β,17,20β-triol 20-sulphate and 17,20α-dihydroxy-4-pregnen-3-one, were significantly enhanced by GnRHa treatment. Concentrations of testosterone on the other hand decreased when spermiating males were treated with GnRHa. Our data suggest that 17,20β-dihydroxy-4-pregnen-3-one or its metabolites are involved in milt hydration, possibly through affecting ion transport. Crown Copyright

Gonadotrophin-releasing hormone agonist raises plasma concentrations of progestogens and enhances milt fluidity in male Atlantic halibut (Hippoglossus hippoglossus)

In two separate spawning seasons, spermiating male Atlantic halibut were implanted with pellets containing gonadotrophin-releasing hormone agonist (GnRHa). Males were bled repeatedly, and milt samples were collected. Blood samples were assayed for free and conjugated steroids: testosterone, 11-ketotestosterone, 17,20α-dihydroxy-4-pregnen-3-one (17,20α-P), 17,20β-dihydroxy-4-pregnen-3-one (17,20β-P), 17,20β,21-trihydroxy-4-pregnen-3-one and steroids with a 17,20β configuration. Towards the end of the first season, pellets were implanted into three wild-caught and three hatchery-reared males. No control fish were available. The major progestogen in plasma was identified as sulphated 5β-pregnane-3β,17,20β-triol (3β,17,20β-P-5β-S). Concentrations of this steroid were stimulated by the GnRHa. Sulphated 17,20β-P was also identified in the plasma, but at 10-fold lower concentrations than 3β,17,20β-P-5β-S. In the middle of the second season, pellets were implanted into five hatchery-reared males; five unimplanted males were used as controls. Levels of androgens fell following GnRHa treatment, levels of progestogens rose briefly, and there was a significant increase in the fluidity of the milt. Of all the measured steroids, free and sulphated 17,20α-P showed the best correlation with milt fluidity.

Enhanced Cellular Immunity in Shrimp ( Litopenaeus vannamei ) after ‘Vaccination’

It has long been viewed that invertebrates rely exclusively upon a wide variety of innate mechanisms for protection from disease and parasite invasion and lack any specific acquired immune mechanisms comparable to those of vertebrates. Recent findings, however, suggest certain invertebrates may be able to mount some form of specific immunity, termed ‘specific immune priming’, although the mechanism of this is not fully understood (see Textbox S1). In our initial experiments, either formalin-inactivated Vibrio harveyi or sterile saline were injected into the main body cavity (haemocoel) of juvenile shrimp (Litopenaeus vannamei). Haemocytes (blood cells) from V. harveyi-injected shrimp were collected 7 days later and incubated with a 1∶1 mix of V. harveyi and an unrelated Gram positive bacterium, Bacillus subtilis. Haemocytes from ‘vaccinated’ shrimp showed elevated levels of phagocytosis of V. harveyi, but not B. subtilis, compared with those from saline-injected (non-immunised) animals. The increased phagocytic activity was characterised by a significant increase in the percentage of phagocytic cells. When shrimp were injected with B. subtilis rather than vibrio, there was no significant increase in the phagocytic activity of haemocytes from these animals in comparison to the non-immunised (saline injected) controls. Whole haemolymph (blood) from either ‘immunised’ or non-immunised’ shrimp was shown to display innate humoral antibacterial activity against V. harveyi that was absent against B. subtilis. However, there was no difference in the potency of antibacterial activity between V. harveyi-injected shrimp and control (saline injected) animals showing that ‘vaccination’ has no effect on this component of the shrimps immune system. These results imply that the cellular immune system of shrimp, particularly phagocytosis, is capable of a degree of specificity and shows the phenomenon of ‘immune priming’ reported by other workers. However, in agreement with other studies, this phenomenon is not universal to all potential pathogens.

Rapid determination of bulk microalgal biochemical composition by Fourier-Transform Infrared spectroscopy

Analysis of bulk biochemical composition is a key in fundamental and applied studies of microalgae and is essential to understanding responses to different cultivation scenarios. Traditional biochemical methods for the quantification of lipids, carbohydrates and proteins are often time-consuming, often involve hazardous reagents, require significant amounts of biomass and are highly dependent on practitioner proficiency. This study presents a rapid and non-destructive method, utilising Fourier-Transform Infrared (FTIR) spectroscopy for the simultaneous determination of lipid, protein and carbohydrate content in microalgal biomass. A simple univariate regression was applied to sets of reference microalgal spectra of known composition and recognised IR peak integrals. A robust single-species model was constructed, with coefficients of determination r(2)>0.95, high predictive accuracy and relative errors below 5%. The applicability of this methodology is demonstrated by monitoring the time-resolved changes in biochemical composition of the marine alga Nannochloropsis sp. grown to nitrogen starvation.

An in vitro and in vivo assessment of the potential of Vibrio spp. as probiotics for the Pacific white shrimp, Litopenaeus vannamei.

Aims:  The objective of the work was to determine whether known strains of nonpathogenic vibrios can act as probiotics for the control of Vibrio infections in the Pacific white shrimp, Litopenaeus vannamei.

Influence of the N:P supply ratio on biomass productivity and time-resolved changes in elemental and bulk biochemical composition of Nannochloropsis sp.

This work reports for the first time the detailed impacts of dual nitrogen (N) and phosphorus (P) stress on growth dynamics and biochemical composition in the Eustigmatophyte Nannochloropsis sp. P-stress concurrent with N-stress had subtle effects on culture bulk biochemical composition, but negatively influenced biomass productivity. However, the N:P supply ratio can be raised to at least 32:1 without compromising productivity (yielding a maximum lipid content of 52% of dry weight and volumetric lipid concentration of 233 mg L(-1)). The maximum biomass and lipid yields per unit of cell-P were 1.2 kg DW (gP)(-1) and 0.54 kg lipid (gP)(-1). The P concentration of many common media is thus in surplus for optimal Nannochloropsis sp. biomass and lipid production, offering potential for significant savings in P usage and improving the sustainability of algal cultivation.