Mineral and non-carbon nutrient utilization and recovery during sequential phototrophic-heterotrophic growth of lipid-rich algae.

A critical factor in implementing microalgal biofuels for mass production is the nutrient requirements. The current study investigated the fate of macro- and micronutrients and their availability in a sequential phototrophic-heterotrophic production process for the lipid rich microalga Auxenochlorella protothecoides. More than 99 % (by weight) of overall process nutrients were supplied during the initial photoautotrophic stage reflecting its significantly larger volume. Under photoautotrophic growth conditions only 9-35 % of supplied Mn, S, Fe, N, Mg, and Cu and less than 5 % of P, Mo, Co, B, Zn, and Ca were consumed by the algae. The rest of these nutrients remain in the spent growth media during the culture concentration-down from an 800 L phototrophic pond to a 5 L heterotrophic fermenter. In contrast, Zn, Mo, Mn, Mg, Ca, and N were exhausted (90-99 % removal) during the first 25 h of the heterotrophic growth stage. The depletion of these key nutrients may have ultimately limited the final biomass density and/or lipid productivity achieved. Approximately 10-20 % of the total supplied S, Mn, Fe, N, and Cu and 5 % of Ca and Zn were assimilated into algal biomass. Several elements including N, P, Mn, B, Cu, Ca, Mg, S, and Fe were released back into the liquid phase by anaerobic digestion (AD) of the residual biomass after lipid extraction. The nutrients recovered from the AD effluent and remaining in the spent medium should be recycled or their initial concentration to the phototrophic stage decreased to enhance process economics and sustainability for future commercialization of algal-derived biofuels.

Characterization of a Taura syndrome virus isolate originating from the 2004 Texas epizootic in cultured shrimp.

A comprehensive investigation of the Taura syndrome virus (TSV) isolate that caused epizootics in shrimp farms in Texas in 2004 (Texas isolate) revealed that this virus was more virulent in laboratory bioassays than the TSV reference isolate, Hawaii 1994, causing severe symptom development and rapid mortality. Histopathology of moribund animals demonstrated epithelial necrosis within the stomach, appendages, general body cuticle and gills, and the surviving animals demonstrated moderate to numerous lymphoid organ spheroids. Purified virions showed icosahedral morphology, with a diameter of 31 nm. Comparative genome analysis showed that the Texas isolate is more closely related to TSV isolates from Thailand and China than to the Hawaii isolate. The predicted tertiary structures of the inhibition of apoptosis protein (IAP) and protease domains of the Texas isolate are very similar to those of the Hawaii isolate. However, the RNA-dependent RNA polymerase (RdRp) of the Texas isolate has significant structural differences from the Hawaii isolate due to point mutation(s) in the RdRp gene. Changes in the RdRp tertiary structure might contribute to the replication fidelity, virulence and ecological adaptability of the Texas isolate.

Functional characterization of putative promoter elements from infectious hypodermal and hematopoietic necrosis virus (IHHNV) in shrimp and in insect and fish cell lines.

Infectious hypodermal and hematopoietic necrosis virus (IHHNV) of shrimp contains a linear single-stranded DNA genome of approximately 4.1kb with three putative open reading frames (ORFs) on the same DNA strand designated, the Left, Middle, and Right ORFs. The Left ORF codes for non-structural protein and the Right ORF codes for capsid protein, whereas the role of the Middle ORF is still unknown. Two putative promoters, designated P2 and P61, were detected upstream of the Left ORF and Right ORF, respectively. We evaluated the activities of these two promoters with or without a transcriptional enhancer element via the use of firefly luciferase reporter constructs in insect and fish cells, and in shrimp tail muscle. In insect and fish cells, the P2 promoter was stronger than the P61 promoter. The presence of the SV40 enhancer element negatively affected P2 but not P61 promoter activity in insect cells. However, in fish cells, the SV40 enhancer element dramatically increased the activities of both promoters. In shrimp, there was no significant difference in luciferase expression driven by these two promoters. In shrimp tail muscle, the presence of SV40 enhancer element in the construct had no significant effect on the P2 promoter and a negative effect on the P61 promoter. The IHHNV P2 and P61 promoters were found to be constitutive promoters that can drive gene expression in both invertebrate and vertebrate hosts.

Anaerobic digestion of lipid-extracted Auxenochlorella protothecoides biomass for methane generation and nutrient recovery.

This study evaluated methane production and nutrient recovery from industrially produced, lipid extracted algal biomass (LEA) of Auxenochlorella protothecoides using semi-continuous anaerobic digestion (AD) at different organic loading rates (OLRs) and hydraulic retention times (HRTs). It was shown, that AD can improve biofuel production efficiency and sustainability, especially for scaled processes, through up to 30% increase in energy generation (up to 0.25 L of methane per g of LEA volatile solids) and partial nutrient recovery and recycling. The nutrient recycling with the AD effluent may reduce the cost of the supplied fertilizers by up to 45%. However, methane production was limited to nearly 50% of theoretical maxima potentially due to biomass recalcitrance and inhibition effects from the residual solvent in the LEA. Therefore, further AD optimization is required to maximize methane yield and nutrient recovery as well as investigation and elimination of inhibition from solvent residues.

Expression of a foreign epitope on infectious pancreatic necrosis virus VP2 capsid protein subviral particle (SVP) and immunogenicity in rainbow trout.

Infectious pancreatic necrosis virus (IPNV) is a major viral pathogen of salmonid fish and causes serious economic losses to salmonid aquaculture. Previously, we demonstrated that the IPNV capsid protein, VP2, expressed in yeast self-assembles into subviral particles (SVPs) and injection of these IPNV rVP2 SVPs into rainbow trout elicits an immune response. Immunized fish had reduced viral loads compared to unimmunized fish when challenged with IPNV. To evaluate the suitability of IPNV rVP2 SVPs for future development of multivalent vaccines, a linear epitope of a human oncogene, c-myc, was cloned into the IPNV rVP2 SVP backbone as a model epitope and expressed in yeast. Western blot analyses with anti-c-myc and anti-IPNV antibodies provided positive identification of both the c-myc and VP2 epitopes on the c-myc VP2 SVPs. Transmission electron microscopy of purified chimeric c-myc VP2 SVPs revealed the formation of approximately 20nm particles. Rainbow trout immunized with c-myc VP2 SVPs elicited both anti-c-myc and anti-IPNV immune responses. When immunized fish were challenged with IPNV, the viral load in the c-myc VP2 SVP immunized fish was significantly lower than the sham-vaccinated controls. The results indicate that IPNV rVP2 SVPs can tolerate the insertion of foreign epitopes without affecting either the antigenic potential of the epitopes of the backbone protein or the inserted foreign epitope. This opens the possibility of using the IPNV rVP2 SVP platform to express epitopes of other viruses, which could pave the way for development of multivalent subunit vaccines or novel marker vaccines.

Antigenicity of infectious pancreatic necrosis virus VP2 subviral particles expressed in yeast.

Infectious pancreatic necrosis (IPN) virus, the etiologic agent of infectious pancreatic necrosis in salmonid fish, causes significant losses to the aquaculture industry. The gene for the viral capsid protein (VP2) was cloned into a yeast expression vector and expressed in Saccharomyces cerevisae. Expression of the capsid gene in yeast resulted in formation of approximately 20nm subviral particles composed solely of VP2 protein. Anti-IPNV antibodies were detected in rainbow trout vaccinated either by injection of purified VP2-subviral particles (rVP2-SVP) or by feeding recombinant yeast expressing rVP2-SVP. Challenge of rVP2-SVP immunized trout with a heterologous IPNV strain and subsequent viral load determination demonstrated that both injection and orally vaccinated fish had lower IPNV loads than naive or sham-vaccinated fish. This study demonstrates the ability of rVP2-SVPs to induce a specific immune response and the ability of immunized fish to reduce the viral load after an experimentally induced IPNV infection.