Optimization of Chlorella biomass harvesting by flocculation and its potential for biofuel production

Optimization of microalgal biomass harvesting is essential to produce effective and optimum outcomes that can contribute towards a feasible and economical harvesting technique. Two Chlorella species were used, namely, C. vulgaris and C. sorokiniana UKM3. Two essential factors affecting microalgal biomass harvesting via flocculation, namely, the initial pH of the microalgal broth and flocculant (chitosan) concentration were studied. The optimization process was conducted by using a response surface methodology (RSM) based on the model of face-centered-central composite design (FC-CCD). The potential for biofuel application of the harvested biomass was evaluated based on the production of fatty acid methyl esters (FAMEs) by transesterification. The quadratic models obtained from the RSM significantly fitted the experiment data as the p-values were less than 0.05. The initial pH of the microalgal suspension was found to have a more significant effect on the flocculation process than flocculant concentration. For C. vulgaris, the highest flocculant efficiency of 98.7% was obtained at a chitosan concentration of 0.2 g L^?1 and an initial pH of 12.0, whereas for C. sorokiniana UKM3, at 0.15 g L^?1 of chitosan and initial pH of 12.0 produced the highest efficiency of 97.1%. The harvested biomass of both species exhibited a high content of palmitic acid (C16:0) with 29.74 wt% and 11.81 wt% of dry biomass for C. vulgaris and C. sorokiniana UKM3, respectively. This study showed that Chlorella species can be harvested efficiently using the flocculation process and manifested an excellent potential for biodiesel production where palmitic acid (C16:0) is one of the main compounds for high-acid oil-biodiesel.

     

Microbial flocculation, a potentially low-cost harvesting technique for marine microalgae for the production of biodiesel

Microbial flocculation is investigated as a separation technique for harvesting marine microalgae for the production of biodiesel. Organic carbon (acetate, glucose or glycerine) was used as substrate for the growth of flocculating microbes in situ. Under stress, due to nutrient depletion, these microbes produced extracellular polymeric substances that promote flocculation of the coccolithophorid alga, Pleurochrysis carterae. Maximum recovery efficiency was achieved at low concentration of organic substrate (0.1 g L⁻¹) and with a long mixing time (24 h); an average recovery efficiency of over 90% and a concentration factor of 226 were achieved. The recovery efficiency is positively correlated with mixing time (R ² = 0.90). The concentration factor is negatively correlated to the product of substrate concentration and mixing time (R ² = 0.73). The microalgae cells were not under stress and remained viable, thus potentially allowing media to be reused in large-scale processes without further treatment. Other advantages of the process are that no metallic flocculants were required and the organic substrates are readily available, e.g. glycerine is a by-product of biodiesel production and acetic acid may be produced by anaerobic digestion of the biomass residue after lipid extraction. Further research is required to optimise the process.     

Evaluation of electro‐coagulation–flocculation for harvesting marine and freshwater microalgae

Although microalgae are considered as a promising feedstock for biofuels, the energy efficiency of the production process needs to be significantly improved. Due to their small size and low concentration in the culture medium, cost‐efficient harvesting of microalgae is a major challenge. In this study, the use of electro‐coagulation–flocculation (ECF) as a method for harvesting a freshwater (Chlorella vulgaris) and a marine (Phaeodactylum tricornutum) microalgal species is evaluated. ECF was shown to be more efficient using an aluminum anode than using an iron anode. Furthermore, it could be concluded that the efficiency of the ECF process can be substantially improved by reducing the initial pH and by increasing the turbulence in the microalgal suspension. Although higher current densities resulted in a more rapid flocculation of the microalgal suspension, power consumption, expressed per kg of microalgae harvested, and release of aluminum were lower when a lower current density was used. The aluminum content of the harvested microalgal biomass was less than 1% while the aluminum concentration in the process water was below 2 mg L⁻¹. Under optimal conditions, power consumption of the ECF process was around 2 kWh kg⁻¹ of microalgal biomass harvested for Chlorella vulgaris and ca. 0.3 kWh kg⁻¹ for Phaeodactylum tricornutum. Compared to centrifugation, ECF is thus more energy efficient. Because of the lower power consumption of ECF in seawater, ECF is a particularly attractive method for harvesting marine microalgae. Biotechnol. Bioeng. 2011;108: 2320–2329. © 2011 Wiley Periodicals, Inc.     

Cold preservation of rat osteochondral tissues in two types of solid organ preservation solution, culture medium and saline

We compared Dulbecco’s modified Eagle’s medium (DMEM), saline, Euro-Collins (EC) solution and University of Wisconsin (UW) solution to determine which was best for cold preservation of rat osteochondral tissues (OCTs). After 7 days’ cold preservation, OCTs kept in UW solution had the highest relative viable cell number by the tetrazolium assay and the lowest activity of lactate dehydrogenase released from damaged cells. Histological evaluation revealed chondrocyte deformity, such as shrunken cytoplasm and pyknotic nuclei, particularly in the deeper layer of articular cartilage after preservation in saline and EC solution and predominantly in all layers if preserved in DMEM. In contrast, chondrocyte morphology in all layers of the articular cartilage preserved in UW solution was relatively unchanged and remained similar to fresh OCTs. It is therefore concluded that UW solution is the most suitable for cold preservation of rat OCTs as well as solid organs.     

Effect of different CO2 concentrations on biomass,pigment content,and lipid production of the marine diatom Thalassiosira pseudonana

The marine diatom Thalassiosira pseudonana grown under air (0.04% CO₂) and 1 and 5% CO₂ concentrations was evaluated to determine its potential for CO₂ mitigation coupled with biodiesel production. Results indicated that the diatom cultures grown at 1 and 5% CO₂ showed higher growth rates (1.14 and 1.29 div day⁻¹, respectively) and biomass productivities (44 and 48 mg_AFDWL⁻¹ day⁻¹) than air grown cultures (with 1.13 div day⁻¹ and 26 mg_AFDWL⁻¹ day⁻¹). The increase of CO₂ resulted in higher cell volume and pigment content per cell of T. pseudonana. Interestingly, lipid content doubled when air was enriched with 1–5% CO₂. Moreover, the analysis of the fatty acid composition of T. pseudonana revealed the predominance of monounsaturated acids (palmitoleic-16:1 and oleic-18:1) and a decrease of the saturated myristic acid-14:0 and polyunsaturated fatty acids under high CO₂ levels. These results suggested that T. pseudonana seems to be an ideal candidate for biodiesel production using flue gases.

     

Induction of flocculation of brewer's yeast strains of Saccharomyces cerevisiae by changing the calcium concentration and pH of culture medium

Strains of Saccharomyces cerevisiae (NCYC 1190, 1214 and 1364) behaved as nonflocculent in defined culture medium, as well as the strain NCYC 1214 in rich medium. Flocculation was induced by Ca²⁺ addition and/or by correcting the pH to a suitable value. Since the free Ca²⁺ concentration is pH-dependent, these two factors (total Ca²⁺ concentration and pH) cannot be dissociated and are the critical parameters governing flocculation, in culture medium, of the brewing strains studied.     

Cultivation and biomass production of the diatom Thalassiosira weissflogii as a live feed for white-leg shrimp in hatcheries and commercial farms in Vietnam

This study investigated the biomass production process from the laboratory to the pilot scale in order to use the nutrient-rich biomass of the diatom Thalassiosira weissflogii as live feed for white-leg shrimp (Litopenaeus vannamei) at larval stages (zoeal, mysis, and postlarval) and in commercial production in hatcheries in Vietnam. Our results showed that T. weissflogii was successfully cultured in 1–2 L Erlenmeyer flasks, 0.2–3.5 m³ composite tanks, and 6.5 m³ tubular photobioreactors, with the highest cell density of 1.6 × 10⁶ cells mL^?1 reached after 6 days of culture. Under optimal culture conditions, the protein, lipid, and carbohydrate contents in this algal biomass were 13.2%, 20.0%, and 10.0% of dry cell weight, respectively. The fatty acid composition contains high amount of palmitic acid (C16:0, 43.11% of total fatty acid), and polyunsaturated fatty acids (PUFAs), such as eicosapentaenoic acid (EPA, C20:5ω-3), approximated 16.5% of total fatty acid. In a 50 L larval rearing tank, at the optimal stocking density of 125 nauplii L^?1, the survival percentage (75.55%), the total body length (from 5.376 ± 0.007 to 10.860 ± 0.030 mm), and weight (at from PL₁ to PL₁₂ stages) (from 0.145 ± 0.002 to 1.158 ± 0.005 g) of the white-leg shrimp larvae reached the highest values but the metamorphosis time (234 h) was shortest compared with the other stocking densities. Further, adding living T. weissflogii biomass to the diet of white-leg shrimp larvae at the nauplii 6 stage led to an increase in the body length, weight, and survival percentage of white-leg shrimp larvae of 21.17%, 35.7%, and 33% higher compared with those of larvae fed the control diet (without the addition of T. weissflogii), respectively. At the same time, the metamorphosis time of larvae (from Z₁ to PL₁) decreased by 4 h compared to the control group. In intensive ponds (area of 6400 m² pond^?1), using seed stocks at the postlarvae 12 stage that had been fed T. weissflogii, the final weight, yield, and survival percentage of the shrimp were increased by 7.3%, 14.2%, and 16.3%, respectively, compared with those of the control group. There were no statistically significant differences in the protein and carbohydrate contents in the shrimp flesh among the experimental and control group (p > 0.05). The lipid, omega-3, omega-6, and omega-9 fatty acid contents of shrimp flesh in experiment formula (per 100 g shrimp) were 1.21 g, 72.9 mg, 114 mg, and 86.1 mg, 11%, 29%, 21.6%, and 17.7% higher than that those in control, respectively. The obtained results show the great potential of using T. weissflogii as live feed on white-leg shrimp farms in Vietnam.

     

Optimization of culture conditions and medium components for the production of mycelial biomass and exo-polysaccharides with Cordyceps militaris in liquid culture

Both crude exo-biopolymers and mycelial biomass, produced by liquid culture of Cordyceps species, are believed to possess several potential health benefits. As a result of its known biological activities, Cordyceps militaris has been extensively characterized in regards to potential medicinal applications. However, optimized liquid culture conditions for enhanced polysaccharide productivity have yet to be developed, which is a necessary step for industrial applications. Therefore, in this study, the liquid culture conditions were optimized for maximal production of mycelial biomass and exo-polysaccharide (EPS) by C. militaris. The effects of medium composition, environmental factors, and C/N ratio were investigated. Among these variables 80 g, glucose; 10 g, yeast extract; 0.5 g, MgSO₄·7H₂O; and 0.5 g, KH₂PO₄ in 1 L distilled water were found to be the most suitable carbon, nitrogen, and mineral sources, respectively. The optimal temperature, initial pH, agitation, and aeration were determined to be 24°C, uncontrolled pH, 200 rpm, and 1.5 vvm, respectively. Under these optimal conditions, mycelial growth in shake flask cultures and 5 L jar bioreactors was 29.43 and 40.60 g/L, respectively, and polysaccharide production in shake flask cultures and 5 L jar bioreactors was 2.53 and 6.74 g/L, respectively.     

Promotion of growth and flocculation of a marine photosynthetic bacterium, Rhodovulum sp. by metal cations

A marine photosynthetic bacterium, Rhodovulum sp., (PS88) isolated from a sea sediment mud self-flocculated during aerobic cultivation in the dark. Flocculation and growth of PS88 were especially by Mg2 up to 10 mM in culture medium. Ca2 had a lesser stimulating effect. © Rapid Science Ltd. 1998     

Regulation of carbonic anhydrase expression by zinc, cobalt, and carbon dioxide in the marine diatom Thalassiosira weissflogii

TWCA1 is the major Zn-requiring isoform of carbonic anhydrase (CA) in the marine diatom Thalassiosira weissflogii. We have examined the roles that trace metals and CO(2) play in the regulation of TWCA1 expression over ranges of concentrations that bracket those encountered in the marine environment. Both steady-state levels of TWCA1 and the kinetics of induction were measured by western analysis. TWCA1 levels correlated well with cellular CA activity levels. TWCA1 was induced at a low CO(2) concentration but the level of induction, as determined by western analysis, was dependent on the availability of Zn. Co effectively substituted for Zn in regulating TWCA1 expression and promoting TWCA1 activity. Upon shift from low to high CO(2), the concentration of TWCA1 decreased. The expression of TWCA1 is diel cycle regulated, and cellular TWCA1 decreased during the dark phase. These results provide the basis for studying the expression of CA in field populations and, taken together with previous radiolabeling studies, provide strong evidence of in vivo metal substitution of Co for Zn in a CA. Our data also support the conclusion that TWCA1 plays a central role in carbon acquisition in T. weissflogii.     

Production of carbohydrates by the marine diatom Chaetoceros affinis var. willei (Gran) Hustedt. I. Effect of the concentration of nutrients in the culture medium

The chemical composition of cells of Chaetoceros affinis var. willei (Gran) Hustedt grown in batch culture was markedly influenced by the concentration of nutrients in the medium. In the logarithmic growth phase the content of cellular glucan was relatively low, but in the stationary phase the glucan content showed a rapid increase which seemed to coincide with the depletion of nitrate from the medium. This led to very pronounced variations in the ratio of protein to carbohydrate. This ratio can easily be determined and seems to be a sensitive and convenient parameter for characterizing the physiological state of the diatom cells.     

The nitrogen status of Austrian forest ecosystems as influenced by atmospheric deposition,biomass harvesting and lateral organomass exchange

Measurements of the deposition rates of atmospheric trace constituents to forest ecosystems in Austria have shown that the deposition of plant utilizable nitrogen compounds is in the range from 12 kg N to more than 30 kg N ha^-1 a^-1. Locally, even higher deposition rates are encountered as a consequence of point sources or special deposition mechanisms such as fog interception, hoar frost formation, and accumulation in snow drifts. In order to place these values into perspective, they are compared with the nitrogen demand of past and present forest land use and with natural processes of nitrogen depletion and accumulation in forest ecosystems. During wind erosion of forest litter, woody material with a wide C/N-ratio remains on the windward side of ridges, while nutrient-rich material with a narrow C/N-ratio is deposited on the leeward side. As a result, total nitrogen storage in the forest soil as well as overall C/N-ratios change dramatically along a transect over a ridge, thus indicating a strong influence of litter C/N ratio on nitrogen retention in the forest soil. A study of nitrogen stores in the soil of beech ecosystems of the same yield class in the Vienna Woods showed a significant correlation of total N-content with base saturation. These results suggest that nitrogen storage capacity of forest soils may be managed by liming and tree species selection. As knowledge is still meagre, a special study on factors which determine nitrogen storage in forest soils is proposed within the FERN-programme.     

In vitro culture and conservation of microalgae: Applications for aquaculture, biotechnology and environmental research

Summary Microalgae are a highly diverse group of unicellular organisms comprising the eukaryotic protists and the prokaryotic cyanobacteria or blue-green algae. The microalgae have a unique environmental status; being virtually ubiquitous in euphotic aquatic niches, they can occupy extreme habitats ranging from tropical coral reefs to the polar regions, and they contribute to half of the globe’s photosynthetic activity. Furthermore, they form the basis of the food chain for more than 70% of the world’s biomass. Microalgae are a valuable environmental and biotechnological resource, and the aim of this review is to explore the use of in vitro technologies in the conservation and sustainable exploitation of this remarkable group of organisms. The first part of the review evaluates the importance of in vitro methods in the maintenance and conservation of microalgae and describes the central role of culture collections in applied algal research. The second part explores the application of microalgal in vitro technologies, particularly in the context of the aquaculture and biotechnology industries. Emphasis is placed upon the exploitation of economically important algal products including aquaculture feed, biomass production for the health care sector, green fertilizers, pigments, vitamins, antioxidants, and antimicrobial agents. The contribution that microalgae can make to environmental research is also appraised; for example, they have an important role as indicator organisms in environmental impact assessments. Similarly, designated culture collection strains of microalgae are used for ecotoxicity testing. Throughout the review, emphasis is placed on the application of in vitro techniques for the continued advancement of microalgal research. The paper concludes by assessing future perspectives for the novel application of microalgae and their products.     

Influence of culture conditions and medium composition on the production of antibacterial compounds by marine Serratia sp. WPRA3

This study was undertaken to investigate the influence of culture conditions and medium components on production of antibacterial compounds by Serratia sp. WPRA3 (JX020764) which was isolated from marine water of Port Dickson, Malaysia. Biochemical, morphological, and molecular characteristics suggested that the isolate is a new candidate of the Serratia sp. The isolate showed strong antimicrobial activity against fungi, Gram-negative and Gram-positive bacteria. This bacterium exhibited optimum antibacterial compounds production at 28°C, pH 7 and 200 rev/min aeration during 72 h of incubation period. Highest antibacterial activity was obtained when sodium chloride (2%), yeast extract (0.5%), and glucose concentration (0.75%) were used as salt, nitrogen, and carbon sources respectively. Different active fractions were obtained by Thin-Layer Chromatography (TLC) and Flash Column Chromatography (FCC) from ethyl acetate crude extracts namely OCE and RCE in different culture conditions, OCE (pH 5, 200 rev/min) and RCE (pH 7/without aeration). In conclusion, the results suggested different culture conditions have a significant impact on the types of secondary metabolites produced by the bacterium.     

Bioreactor culture of oil palm (Elaeis guineensis) and effects of nitrogen source, inoculum size, and conditioned medium on biomass production

We report the successful culture of oil palm (Elaeis guineensis Jacq.) suspension cells in a bioreactor. In vitro propagation of this perennial monocotyledonous tree is an important part of the oil palm industry's approach to clonal propagation of high-yielding accessions. During culture of oil palm cells in a batch bioreactor, nutrients and extracellular metabolites were monitored, and kinetic parameters and nutrient-to-biomass conversion yields were calculated. The biomass increased approximately 3.5-fold per month, consistent with values reported for shake flask cultures. Although the carbon source was completely depleted by the end of the run, nitrogen sources remained in large excess and the sugar-to-biomass conversion yield remained low. Linear growth indicated that the cells were limited. The results obtained from the bioreactor runs indicated that we should be able to improve biomass production by carrying out optimization studies. Therefore, we initiated multi-factorial analyses using response surface experimental designs to investigate the effects of different nitrogen sources, as well as inoculum size and conditioned medium, on biomass production in flask cultures. Whereas glutamine does not have a significant effect on biomass production, ammonia has a positive effect up to an optimum concentration. Both inoculum density and conditioned medium have positive, synergistic effects on biomass production.     

Statistical optimization of a culture medium for biomass and poly(3-hydroxybutyrate) production by a recombinant Escherichia coli strain using agroindustrial byproducts.

A statistically based Plackett-Burman screening design identified milk whey and corn steep liquor concentrations as well as ionic strength (based on phosphate buffer concentration) as the three main independent components of the culture medium that significantly (p < 0.05) influenced biomass and poly(3-hydroxybutyrate) (PHB) production in recombinant cells of Escherichia coli. This strain carries a plasmid encoding phb genes from a natural isolate of Azotobacter sp. Response surface methodology, using a central composite rotatable design, demonstrated that the optimal concentrations of the three components, defined as those yielding maximal biomass and PHB production in shaken flasks, were 37.96 g deproteinated milk whey powder/l, 29.39 g corn steep liquor/l, and 23.76 g phosphates/l (r2 = 0.957). The model was validated by culturing the recombinant cells in medium containing these optimal concentrations, which yielded 9.41 g biomass/l and 6.12 g PHB/l in the culture broth. Similar amounts of PHB were obtained following batch fermentations in a bioreactor. These results show that PHB can be produced efficiently by culturing the recombinant strain in medium containing cheap carbon and nitrogen sources.     

Determination and quantification of alpha,beta,gamma,delta-unsaturated aldehydes as pentafluorobenzyl-oxime derivates in diatom cultures and natural phytoplankton populations: application in marine field studies

Reactive alpha,beta,gamma,delta-unsaturated aldehydes and oxo-acids produced by marine diatoms upon cell damage interfere negatively with the reproduction success of their grazers. A simple, sensitive and specific method based on gas-chromatography coupled to mass spectrometry (EI or CI/EC) was developed for the quantification of these deleterious substances in laboratory diatom cultures and in natural phytoplankton populations. For aldehyde quantification, diatom containing samples are damaged in the presence of O-(2,3,4,5,6-pentafluorobenzyl) hydroxylamine hydrochloride (PFBHA.HCl) which leads to an in situ derivatisation without inhibition of the biosynthesis of the aldehydes. The oxime derivates of oxo-acids were in addition reacted with N-tert-butyldimethylsilyl-N-methyl-trifluoracetamide (MTBSTFA).     

Multiple use of aquatic green biomass for food/feed protein concentrate, bioenergy and microbial fermentation products

Fractionation of aquatic green biomass of three water weeds for multiple use through yield of protein concentrate, fibrous residue and whey (deproteinised juice) has been studied. The potential of protein concentrate for use as food/feed supplement, that of fibrous residue as ensilaged fodder/substrate for mushroom growth/production of bioenergy and of whey as substrate for microbial fermentation has been studied and is discussed.