Survivability of freeze- and spray-dried probiotics and their effects on the growth and health performance of broilers.

Background and Aim: Many strains of probiotics have been exploited and used as animal dietary supplements for broiler production. The efficacy and survival of probiotics during production may reflect better activities of the probiotics in the host. This study investigated the effects of freeze- and spray-drying on the survivability and properties of probiotics and their ability to improve the growth and health performance of broilers. Materials and Methods: Probiotic powders of four strains of lactic acid bacteria, Enterococcus faecium CA4, Enterococcus durans CH33, Ligilactobacillus salivarius CH24, Pediococcus acidilactici SH8, and Bacillus subtilis KKU213, were prepared using rice bran/chitosan/carboxy methyl cellulose as the carrier. The survival of each probiotic strain was investigated under stress conditions, including freeze-drying, spray-drying, and simulated gastrointestinal conditions. The body weight gain (BWG) and intestinal histomorphology were determined to assess broiler growth performance. Results: All dried probiotics yielded a high survival rate during freeze-drying (95.8-98.6%) and spray-drying (94.4-98.2%). In addition, an analysis of the main effect revealed that the effectiveness of freeze-drying was higher than that of spray-drying in minimizing the loss of cell viability. The antimicrobial activity of all immobilized dried probiotic strains against Salmonella was maintained. The immobilized probiotics tolerated a low pH value of 2.0 and 0.5% (w/v) bile salt. Probiotic administration of a mixture of the five dried probiotics to 1-day-old hatched male broilers at early and late ages resulted in potential colonization in the broiler intestine, and enhancements in the BWG, lipid metabolism, and gut health (villus height and cryptal depth) were observed in the probiotic-treated groups. Conclusion: The administration of three doses of the spray-dried probiotic mixture at days 15, 17, and 19 after hatching was sufficient to achieve long-term growth and health benefits in broilers. This finding might provide a cost-effective alternative to the administration of commonly used antibiotics in broiler production.

Improvements in the color, phytochemical, and antioxidant properties of frozen ripe mango pieces using calcium chloride dipping and chitosan coating.

This study aimed to investigate the influences of a dipping/coating composed of calcium chloride (CaCl2 ) or chitosan on the quality of ripe mango pieces during frozen storage for 6 months. The fruits were dipped in solutions with concentrations of 0.5% and 1% for different times (15 or 30 min for CaCl2 and 1 or 15 min for chitosan). We found that treatment with 1% CaCl2 for 30 min significantly retarded the color changes with the highest L* (p < 0.05) and the lowest of b* and ∆E (p ≥ 0.05). Interestingly, treatment with 0.5% CaCl2 for 30 min significantly preserved the contents of total phenolics and total flavonoids and the antioxidant activities at values higher than the control levels, as determined by DPPH and ABTS assays (p < 0.05). Moreover, treatment with 0.5%-1% chitosan for 1 min effectively delayed the loss of moisture and weight. The results indicate that dipping in CaCl2 is an alternative simple food processing technique for improving the quality of ripe mango pieces during frozen storage that effectively delays the color changes and preserves the antioxidant content and activity. HIGHLIGHTS: The coating of frozen ripe mango pieces with CaCl2 and chitosan was first investigated. CaCl2 effectively retarded the color change during storage and after thawing. Chitosan effectively delayed the loss of moisture and weight of mango pulp. Coating with 0.5% CaCl2 for 30 min maintained the phytochemicals and antioxidant activities. Coating treatment can preserve mango qualities and could be commercialized with cost savings. PRACTICAL APPLICATION: The present article proposes a strategy that effectively delays the physicochemical changes and preserves the nutritional properties of mango fruit and could be commercialized with cost savings. A frozen mango can either be consumed (ready-to-eat frozen mango) or used as a food raw material.

Low Cd-accumulating rice grain production through inoculation of germinating rice seeds with a plant growth-promoting endophytic bacterium.

This study investigated the effects of the plant growth-promoting endophytic bacterium Cupriavidus taiwanensis KKU2500-3 on the growth of KDML105 rice plants and cadmium (Cd) accumulation in grains. The rice plants were cultivated in soils with 20 and 50 ppm Cd under greenhouse conditions for two consecutive years. At both levels, Cd reduced rice growth and development. Under Cd stress, KKU2500-3 colonized the root surface and interior of rice plants at the early growth stage, and this colonization remained until the late stage. The colonized bacteria increased the pigment contents but reduced the root-to-aboveground translocation of Cd. In soil with 20 ppm Cd, the phytochelatin content of the bacteria-inoculated rice was lower (32.3-89.3%) than that of uninoculated rice. In soil with 50 ppm Cd, the bacteria-inoculated rice exhibited higher glutathione reductase (5-63%) and proline (5-115%) levels, a higher reduced glutathione (GSH)/0.5 oxidized glutathione (GSSG) ratio (4-212%) and decreased lipid peroxidation (1-19%) compared with uninoculated rice. The root-to-grain translocation factor of inoculated rice in soil with 50 ppm Cd was significantly lower than that of inoculated rice in soil with 20 ppm Cd, and this finding was consistent with the 38.6% and 75.1% reductions in Cd accumulation observed in grains from soils with 20 and 50 ppm Cd, respectively. The Cd content of KDML105 grains grown in soil with 50 ppm Cd was 0.36 ppm, which is below the Codex standard for polished rice (0.4 ppm). The levels of available P, Zn, and SO42- also affect Cd availability in soil, and colonized KKU2500-3 showed varying responses to different Cd levels. Thus, bacterial inoculation, the Cd level and soil properties play important roles in Cd accumulation in KDML105 rice grains. The role of C. taiwanensis KKU2500-3 on the production of low-Cd-accumulating rice in paddy fields contaminated with a range of Cd levels should be further investigated.

A cadmium-tolerant endophytic bacterium reduces oxidative stress and Cd uptake in KDML105 rice seedlings by inducing glutathione reductase-related activity and increasing the proline content.

The effect of the endophytic Cupriavidus taiwanensis KKU2500-3 on the Cd toxicity of KDML105 rice seedlings was investigated in a 10 µM CdCl2 hydroponic system. As demonstrated after bacterial inoculation of germinating rice seeds, KKU2500-3 colonized all rice plant parts. In RB (Rice + KKU2500-3) and RBC (Rice + KKU2500-3+Cd), KKU2500-3 effectively colonized and was detected at a markedly higher number in the root surface and interior than in shoots and leaves. The activities of antioxidant enzymes ascorbate peroxidase (APOX), glutathione reductase (GR), and superoxide dismutase (SOD) and the proline content in inoculated rice were higher in roots and aboveground tissues. RBC exhibited a higher reduced-to-oxidized glutathione ratio in roots and leaves (3-55%) but a lower malondialdehyde content (8-78%). Phytochelatins (PCs) were detected in all rice tissues, but their levels in RBC were 13-70% lower than those in RC (Rice + Cd), demonstrating that the induction of PCs in rice was unrelated to KKU2500-3. The Cd levels in roots and shoots were lower in RBC than RC, and the root-to-shoot Cd translocation factor was 0.6-62.2% lower. At 30 DAT, the Cd levels in RBC roots and shoots were 30.2% and 73.7% lower, respectively, than those in RC. Colonized KKU2500-3 activated GR and increased the proline content to overcome rice Cd toxicity. These effects may trap Cd in plant cells and reduce its translocation. Hence, KKU2500-3 synergistically interacts with rice to detoxify Cd at early growth stages, and KDML105 rice grains with low Cd accumulation could be produced if this interaction is maintained until late growth stages.

Responses to arsenic stress of rice varieties coinoculated with the heavy metal-resistant and rice growth-promoting bacteria Pseudomonas stutzeri and Cupriavidus taiwanensis.

Arsenic (As)-contaminated rice paddy fields are spreading globally, and thus, rice grains with low As accumulation at a safe level for consumption is profoundly needed. Rice is highly susceptible to As accumulation, and the responses to As vary among rice varieties. Here, combinations of the AsIII-oxidizing bacteria Pseudomonas stutzeri strains 4.25, 4.27, or 4.44 and Cupriavidus taiwanensis KKU2500-3 were investigated with respect to their responses to As toxicity and rice growth promotion during the early growth stage. All bacterial strains enhanced antioxidant enzyme activities, including SOD, CAT, APX, GPX, and GR, under As stress in vitro. Uninoculated and coinoculated rice seedlings of three rice varieties (KDML105, RD6, RD10) were cultivated in hydroponic solution without and with a combination of toxic AsIII and less toxic AsV for 30 days. Compared with uninoculated seedlings, the inoculated seedlings showed higher growth parameters and lower As contents in roots, shoots and throughout the plants. The bioconcentration factor (BCF) and translocation factor were reduced in inoculated seedlings. The effective response of rice to As toxicity influenced by bacteria was highest in KDML105, followed by RD6 and RD10. The root sulfide content was correlated with As accumulation in roots, shoots, and total seedlings and the BCFs. P. stutzeri 4.44 and C. taiwanensis KKU2500-3 were the most promising combinations for application in KDML105 cultivation under As-contaminated conditions. Understanding the basic response of rice coinoculated with effective bacteria at the early stage will provide guidelines for rice cultivation under As conditions at other scales.

Bacillus subtilis and lactic acid bacteria improve the growth performance and blood parameters and reduce Salmonella infection in broilers.

AIM: The aim of the study was to determine the potentials and effects of Bacillus subtilis and lactic acid bacteria (LAB) as probiotics on broiler growth, health, and Salmonella infection. MATERIALS AND METHODS: To evaluate the inoculum size applicable for broilers, 1-day-old broilers were orally fed fresh cultures of single strains and a B. subtilis KKU213/Pediococcus pentosaceus NP6 mixture at 108 and 1012 colony-forming unit (CFUs)/mL/chick. The body weight gain (BWG), Salmonella contamination level and total Bacillus and LAB abundances in the crop and intestine were measured. Subsequently, 1-day-old broilers were orally fed of KKU213, CH403, and Pediococcus acidilactici SH8 at 1010 CFUs/mL, followed by inulin. After 35 days, the BWG, Bacillus and LAB abundances in the cecum, blood parameters, and KKU213 colonization were assessed. RESULTS: The broilers fed single strains or KKU213+NP6 exhibited a higher BWG and a higher crop LAB abundance than the controls (p<0.05). Probiotic feeding decreased the intestinal Salmonella abundance and correspondingly increased the LAB abundance. The broilers fed the mixed culture (KKU213+CH403+SH8) followed by prebiotics showed lower mortality, higher blood high-density lipoprotein levels, and lower blood uric acid levels than the controls (p<0.0004). Probiotic feeding significantly increased the Bacillus and LAB counts (p<0.05). A CE330 isolate obtained from the cecum after 35 days of KKU213 feeding was closely related to B. subtilis KKU213. CONCLUSION: B. subtilis KKU213 is a potent probiotic strain that can survive, colonize and reduce Salmonella infection in broilers and improve their growth and health. This strain, combined with different LAB can act synergistically in the gut and promote broiler growth.

Mitigation of arsenic toxicity and accumulation in hydroponically grown rice seedlings by co-inoculation with arsenite-oxidizing and cadmium-tolerant bacteria.

Arsenic (As) contamination of rice grain is a serious problem worldwide. The objective of this study was to mitigate As toxicity and accumulation in hydroponically grown KDML105 rice seedlings using bacteria isolated from heavy metal-contaminated soils. Seven strains (KKU2500-1, -2, -3, -9, -12, -16 and -22) of 24 cadmium (Cd)-tolerant bacteria produced high levels of inorganic sulfide and thiol-rich compounds in As-supplemented media. The strains were allowed to colonize rice seedlings growing in arsenite [As(III)]- or arsenate [As(V)]-supplemented Hoagland's nutrient solutions. Colonization by strains KKU2500-3 and -12 led to increases in plant growth parameters and similarly reduced As translocation into shoots [translocation factor (TF) = 0.05] in the As(V)-supplemented solution. Strains KKU2500-1 and - 12 also greatly reduced As translocation into shoots (TF = 0.16-0.20) in As(III)-supplemented solution. KKU2500-3 and - 12 co-colonized onto seedlings with the As(III)-oxidizing isolates 4.25, 4.27, 4.40 and 4.44, and the strain combinations KKU2500-12/4.25, KKU2500-3/4.25, KKU2500-3/4.27 and KKU2500-3/4.44 resulted in higher growth parameters for plants grown in As [As(III)+As(V)]-supplemented solution than other combinations. Moreover, the combinations KKU2500-3/4.25 and KKU2500-3/4.44 greatly reduced As translocation (TF = 0.15 and 0.12, respectively), and this decreased As accumulation in shoots was significantly correlated with increased sulfide stimulation in roots and nutrient solution. These results indicate that these co-inoculated bacteria can mitigate As toxicity, translocation and accumulation in KDML105 seedlings and thus demonstrate synergistic activity in rice plants, and this effect can be further developed in field trials.

Reduction of cadmium uptake in rice endophytically colonized with the cadmium-tolerant bacterium Cupriavidus taiwanensis KKU2500-3.

The effects of the cadmium (Cd)-tolerant bacterium Cupriavidus taiwanensis KKU2500-3 on the growth, yield, and Cd concentration in rice grains were investigated in the rice variety Phitsanulok 2 (PL2), which was cultivated in a hydroponic greenhouse. The numbers of Cd-tolerant bacteria isolated from the roots and shoots of plants under the RB (rice with bacteria) and RBC (rice with bacteria and Cd) treatments ranged from 2.60 to 9.03 and from 3.99 to 9.60 log cfu·g-1 of PL2, respectively. This KKU2500-3 strain was successfully colonized in rice, indicating that it was not only nontoxic to the plants but also became distributed and reproduced throughout the plants. Scanning electron microscopy analysis revealed attachment of the bacterium to the root surface, whereas the internally colonized bacteria were located in the vascular tissue, cell wall, and intercellular space. Although the Cd contents found in PL2 were very high (189.10 and 79.49 mg·kg-1 in the RC (rice with Cd) and RBC roots, respectively), the Cd accumulated inside the rice seeds at densities of only 3.10 and 1.31 mg·kg-1, respectively; thus, the bacteria reduced the Cd content to 57.74% of the control content. Therefore, the colonizing bacteria likely acted as an inhibitor of Cd translocation in PL2.

Molecular and enzymatic characterization of alkaline lipase from Bacillus amyloliquefaciens E1PA isolated from lipid-rich food waste.

Bacillus amyloliquefaciens E1PA is a lipase-producing strain that was originally isolated from lipid-rich food waste, and the production of its lipase was found to be induced by vegetable oils. The E1PA lipase was successfully expressed and secreted in a heterologous Escherichia coli host and was ultimately purified. The conserved pentapeptide motif Ala-His-Ser-Met-Gly was observed at positions 108-112. The purified recombinant lipase was stable over a pH range of 4.0-11.0 at 40 °C and exhibited maximal activity at pH 10. The recombinant E1PA lipase hydrolyzed a wide range of acyl esters (C4-C18). However, the highest activity (3.5 units mg(-1)) was observed when the p-nitrophenyl ester of myristate (C14) was used as a substrate. Compared to the lipases produced by Bacillus spp., the E1PA lipase displayed a structural molecular mass excluding the leader sequence (19.22 kDa) and a pI (9.82) that were similar to those reported for B. amyloliquefaciens lipases and lipase subfamily I.4 but that were quite distinct from those of lipase subfamily I.5 (approximately 43 kDa, pI 6). These results suggested that Bacillus lipases are closely related. Although the recombinant E1PA lipase digested only certain oils, the wild-type E1PA lipase degraded a variety of oils, including blended and re-used cooking oils. The recombinant and wild-type forms of the E1PA lipase were able to digest heterogeneous lipid-rich food waste at similar levels; this result suggests that this lipase can function even when it solely consists of its structural enzyme component. The enzyme exhibited lipid hydrolysis ability as either an intracellular domain of the recombinant protein or an extracellular domain secreted by the E1PA strain. However, the recombinant lipase showed higher activity than the wild-type E1PA lipase, indicating that the recombinant protein from E. coli possessed effective lipase activity. Thus, the inducible alkaline E1PA lipase exhibited the ability to act on a broad spectrum of substrates, and the effective form produced in the heterogeneous host can be further developed for several applications, such as biodiesel production and lipase production.

Antibacterial activity and genotypic-phenotypic characteristics of bacteriocin-producing Bacillus subtilis KKU213: potential as a probiotic strain.

The antimicrobial activity and probiotic properties of Bacillus subtilis strain KKU213, isolated from local soil, were investigated. The cell-free supernatant (CFS) of a KKU213 culture containing crude bacteriocins exhibited inhibitory effects on Gram-positive bacteria, including Bacillus cereus, Listeria monocytogenes, Micrococcus luteus, and Staphylococcus aureus. The antibacterial activity of the CFS precipitated with 40% ammonium sulfate (AS) remained even after treatment at 60 and 100 °C, at pH 4 and 10 and with proteolytic enzymes, detergents and heavy metals. When analyzed by SDS-PAGE and overlaid with the indicator strains B. cereus and S. aureus, the 40% AS precipitate exhibited inhibitory activity on proteins smaller than 10 kDa. However, proteins larger than 25 kDa and smaller than 10 kDa were still observed on a native protein gel. Purified subtilosin A was prepared by Amberlite XAD-16 bead extraction and HPLC and analyzed by Nano-LC-QTOF-MS. Its molecular mass was found to be 3.4 kDa, and it retained its antibacterial activity. These results are consistent with the detection of the anti-listerial subtilosin A gene of the sbo/alb cluster in the KKU213 strain, which is 100% identical to that of B. subtilis subsp. subtilis 168. In addition to stable and cyclic subtilosin A, a mixture of many extracellular antibacterial peptides was also detected in the KKU213 culture. The KKU213 strain produced extracellular amylase, cellulase, lipase and protease, is highly acid-resistant (pH 2) when cultured in inulin and promotes health and reduces infection of intestinally colonized broiler chickens. Therefore, we propose that bacteriocin-producing B. subtilis KKU213 could be used as a potential probiotic strain or protective culture.

Changes in the proteome of the cadmium-tolerant bacteria Cupriavidus taiwanensis KKU2500-3 in response to cadmium toxicity.

Cupriavidus taiwanensis KKU2500-3 is a cadmium (Cd)-tolerant bacterial strain that was previously isolated from rice fields contaminated with high levels of Cd. In 500 µmol/L CdCl2, the KKU2500-3 strain grew slower and with a more prolonged lag-phase than when grown in the absence of Cd. A proteomic approach was used to characterize the protein expression in the Cd-tolerant bacteria C. taiwanensis KKU2500-3 during growth under Cd stress. When compared with the untreated cells, a total of 982 differentially expressed protein spots were observed in the CdCl2-treated cells, and 59 and 10 spots exhibited >2- and >4-fold changes, respectively. The level of up- and downregulation varied from 2.01- to 11.26-fold and from 2.01- to 5.34-fold, respectively. Of the 33 differentially expressed protein spots analyzed by MALDI TOF MS/MS, 19 spots were successfully identified, many of which were involved in stress responses. The most highly upregulated protein (+7.95-fold) identified was the chaperone GroEL, which indicated that this factor likely contributed to the bacterial survival and growth in response to Cd toxicity. Detection of the downregulated protein flagellin (-3.52-fold) was consistent with the less effective ATP-mediated and flagella-driven motility. The flagella-losing cells were also observed in the Cd-treated bacteria when analyzed by scanning electron microscopy. Thus, the Cd-stressed cells may downregulate pathways involving ATP utilization in favor of other mechanisms in response to Cd toxicity. When the KKU2500-3 strain was grown in the presence of Cd, H2S was not detected, suggesting a possible role of the sulfur in precipitation with Cd. Apart from a general response, no specific process could be determined using the present proteomic approach. However, the potential role of protein folding-mediated GroEL, flagella-mediated motility and CdS biotransformation in Cd toxicity response observed in this study as well as the extent of Cd-tolerant mechanisms using other methods could facilitate the future application of this strain in addressing Cd environmental contamination.

Cadmium-tolerant bacteria reduce the uptake of cadmium in rice: potential for microbial bioremediation.

We selected 24 bacterial isolates that could tolerate up to 2500 µM CdCl2 from the soil of rice fields downstream from a zinc-mineralized area contaminated with a high level of cadmium (Cd). In the presence of 500 µM CdCl2, all isolates grew slower and with a prolonged lag-phase compared to in the absence of Cd. Cd-binding capacity was high and ranged from 6.38 to 9.38 log[Cd(atom)]/cell. The stability of Cd complexes in bacteria was affected by 1mM EDTA. In 500 µM CdCl2, all isolates produced 0.7 to 4.8-fold more inorganic sulfide and 0.6 to 2.2-fold more thio-rich compounds containing SH groups. Out of 24 Cd-tolerant bacterial isolates, KKU2500-3, -8, -9 and -20 were able to promote the growth of Thai jasmine rice (Kao Hom Mali 105) seedlings in the presence of 200 µM CdCl2, and KKU2500-3 produced the highest numbers of fibrous root. Interestingly, these 4 isolates increased Cd tolerance and decreased the accumulation of Cd in rice by 61, 9, 6, and 17% when grown in the presence of 200 µM CdCl2. Of the 4 isolates, KKU2500-3 produced more inorganic sulfide when grown in CdCl2 at 500-2000 µM. XANES analyses indicated that this isolate precipitated a detectable amount of cadmium sulfide (CdS) when grown in 500 µM CdCl2. Thus, the isolate KKU2500-3 could possibly transform toxic, soluble CdCl2 into non-toxic, insoluble CdS. These 4Cd-tolerant bacterial isolates were identified via 16S rDNA sequencing and classified as Cupriavidus taiwanensis KKU2500-3 and Pseudomonas aeruginosa KKU2500-8, -9, and -20.

Removal of mercury from sediment by ultrasound combined with biomass (transgenic Chlamydomonas reinhardtii).

As one of the most pervasive environmental problems, Hg pollution in sediment is particularly difficult to remediate because it cannot be decomposed. The application of ultrasound combined with biomass (transgenic Chlamydomonas reinhardtii (C. reinhardtii), a green alga) for the removal of Hg from model and contaminated sediments (Al(2)O(3), α-HgS, and PACS-2 marine sediment) was investigated in this study. Ultrasound was found to enhance Hg release from Al(2)O(3), α-HgS, and PACS-2 marine sediment into the aqueous phase compared to mechanical shaking. A transgenic C. reinhardtii (2AMT-2) expressing a plasmamembrane-anchored metallothionein polymer effectively recovered Hg(II) released into the aqueous phase by sonication over a broad pH range from 2.0 to 9.0. The results showed that this combined technique of ultrasound and alga biomass (2AMT-2) engineered for enhanced metal recovery was effective to remove Hg from solids and sediments, especially from Al(2)O(3) and α-HgS with no natural organic matter. The results of this study are discussed with respect to the development of in situ remediation techniques for Hg-contaminated sediments.

Phycoremediation of heavy metals using transgenic microalgae.

Microalgae account for most of the biologically sequestered trace metals in aquatic environments. Their ability to adsorb and metabolize trace metals is associated with their large surface:volume ratios, the presence of high-affinity, metal-binding groups on their cell surfaces, and efficient metal uptake and storage systems. Microalgae may bind up to 10% of their biomass as metals. In addition to essential trace metals required for metabolism, microalgae can efficiently sequester toxic heavy metals. Toxic heavy metals often compete with essential trace metals for binding to and uptake into cells. Recently, transgenic approaches have been developed to further enhance the heavy metal specificity and binding capacity of microalgae with the objective of using these microalgae for the treatment of heavy metal contaminated wastewaters and sediments. These transgenic strategies have included the over expression of enzymes whose metabolic products ameliorate the effects of heavy metal-induced stress, and the expression of high-affinity, heavy metal binding proteins on the surface and in the cytoplasm of transgenic cells. The most effective strategies have substantially reduced the toxicity of heavy metals allowing transgenic cells to grow at wild-type rates in the presence of lethal concentrations of heavy metals. In addition, the metal binding capacity of transgenic algae has been increased five-fold relative to wild-type cells. Recently, fluorescent heavy metal biosensors have been developed for expression in transgenic Chlamydomonas. These fluorescent biosensor strains can be used for the detection and quantification of bioavailable heavy metals in aquatic environments. The use of transgenic microalgae to monitor and remediate heavy metals in aquatic environments is not without risk, however. Strategies to prevent the release of live microalgae having enhanced metal binding properties are described.

Microalgal vaccines.

A variety of recombinant vaccines and vaccine delivery systems are currently under development as alternatives to vaccines produced in animals that are primarily administered by injections. These nonanimal alternatives do not transmit animal pathogens, are often rapid to develop, and can be produced on a large scale at low costs. Many of these new vaccine technologies are based on oral delivery systems and avoid the risks of disease transmission associated with the use of syringes for injectable vaccines. In addition, many of these novel systems have extended shelf life, often not requiring refrigeration and thus are applicable in developing countries or remote locations. Here we describe the development of microalgal-based immunization systems. Antigens expressed in the chloroplast or anchored to the surface of plasma membrane are shown to effectively immunize fish and rabbits. The effective oral delivery of antigens by microalgae provides a safe and inexpensive mechanism to immunize animals. The applications of microalgal vaccines are currently being investigated.

Molecular mechanisms of proline-mediated tolerance to toxic heavy metals in transgenic microalgae.

Pro has been shown to play an important role in ameliorating environmental stress in plants and microorganisms, including heavy metal stress. Here, we describe the effects of the expression of a mothbean delta(1)-pyrroline-5-carboxylate synthetase (P5CS) gene in the green microalga Chlamydomonas reinhardtii. We show that transgenic algae expressing the mothbean P5CS gene have 80% higher free-Pro levels than wild-type cells, grow more rapidly in toxic Cd concentrations (100 microM), and bind fourfold more Cd than wild-type cells. In addition, Cd-K edge extended x-ray absorption fine structure studies indicated that Cd does not bind to free Pro in transgenic algae with increased Pro levels but is coordinated tetrahedrally by sulfur of phytochelatin. In contrast to P5CS-expressing cells, Cd is coordinated tetrahedrally by two oxygen and two sulfur atoms in wild-type cells. Measurements of reduced/oxidized GSH ratios and analyses of levels of malondialdehyde, a product of the free radical damage of lipids, indicate that free Pro levels are correlated with the GSH redox state and malondialdehyde levels in heavy metal-treated algae. These results suggest that the free Pro likely acts as an antioxidant in Cd-stressed cells. The resulting increased GSH levels facilitate increased phytochelatin synthesis and sequestration of Cd, because GSH-heavy metal adducts are the substrates for phytochelatin synthase.

Cadmium- and iron-stress-inducible gene expression in the green alga Chlamydomonas reinhardtii: evidence for H43 protein function in iron assimilation.

Early transcriptional responses of a cell wall-deficient mutant of the green alga Chlamydomonas reinhardtii to heavy-metal stress have been investigated using the method of mRNA differential display. We have identified, sequenced, and quantified the induction of a number of transcripts that are up-regulated by a brief (2-h) exposure to 25 microm cadmium chloride, including one transcript which is also highly responsive to iron (Fe) deficiency. These transcripts represent both nuclear- and chloroplast-encoded genes, and include both novel genes and genes with known or suspected functions. Among these is a gene with significant homology to HCR1, a high-CO(2)- and Fe-deficiency-inducible gene from Chlorococcum littorale. We further characterized the regulation of the HCR1-like gene ( H43) and found that this transcript is also induced by Fe-depletion of the medium. Heterologous expression of H43 in the Fe-uptake mutant fet3fet4 of Saccharomyces cerevisiae resulted in partial suppression of the slow-growth phenotype of this mutant in minimal medium, and resulted in a 2-fold increase in Fe accumulation per cell. Our results demonstrate the utility of Chlamydomonas cw(-) strains for functional genomics studies of metal stress. The magnitudes of induction and functional analyses suggest possible utility for these genes in the study of metal stress sensing in green plants and development of novel Fe acquisition and phytoremediation strategies.