Biomedical applications of microbially engineered polyhydroxyalkanoates: an insight into recent advances, bottlenecks, and solutions.

Biopolymeric polyhydroxyalkanoates (PHAs) are fabricated and accumulated by microbes under unbalanced growth conditions, primarily by diverse genera of bacteria. Over the last two decades, microbially engineered PHAs gained substantial interest worldwide owing to their promising wide-range uses in biomedical field as biopolymeric biomaterials. Because of non-hazardous disintegration products, preferred surface alterations, inherent biocompatibility, modifiable mechanical properties, cultivation support for cells, adhesion devoid of carcinogenic impacts, and controllable biodegradability, the PHAs like poly-3-hydroxybutyrate, 3-hydroxybutyrate and 3-hydroxyvalerate co-polymers, 3-hydroxybutyrate and 4-hydroxybutyrate co-polymers, etc., are available for various medical applications. These PHAs have been exploited to design in vivo implants like sutures as well as valves for direct tissue repairing as well as in regeneration devices like bone graft substitutes, nerve guides as well as cardiovascular patches, etc. Furthermore, they are also emerged as attractive candidates for developing effective/novel drug delivery systems because of their biocompatibility and biodegradability with the ability to deliver and release the drugs at a specific site in a controllable manner and, therefore widen the therapeutic window with reduced side effects. However, there still remain some bottlenecks related to PHA purity, mechanical properties, biodegradability, etc., that are need to be addressed so as to make PHAs a realistic biomaterial. In addition, innovative approaches like PHAs co-production with other value-added products, etc., must be developed currently for economical PHA production. This review provides an insight toward the recent advances, bottlenecks, and potential solutions for prospective biomedical applications of PHAs with conclusion that relatively little research/study has been performed presently toward the viability of PHAs as realistic biopolymeric biomaterials.

Role of cultivation parameters in carbohydrate accretion for production of bioethanol and C-phycocyanin from a marine cyanobacterium Leptolyngbya valderiana BDU 41001: A sustainable approach.

The investigation aimed to augment carbohydrate accumulation in the marine cyanobacterium Leptolyngbya valderiana BDU 41001 to facilitate bioethanol production. Under the standardised physiochemical condition (SPC), i.e. 90 µmol photon m-2 s-1 light intensity, initial culture pH 8.5, 35 °C temperature and mixing at 150 rpm increased the carbohydrate productivity ∼70 % than the control, while a 47 % rise in content was obtained under the nitrate (N)-starved condition. Therefore, a two-stage cultivation strategy was implemented, combining SPC at the 1st stage and N starvation at the 2nd stage, resulting in 80 % augmentation of carbohydrate yield, which enhanced the bioethanol yield by ∼86 % as compared to the control employing immobilised yeast fermentation. Moreover, biomass utilisation was maximised by extracting C-phycocyanin, where a ∼77 % rise in productivity was recorded under the SPC. This study highlights the potential of L. valderiana for pilot-scale biorefinery applications, advancing the understanding of sustainable biofuel production.

Effects of calcium, magnesium and sodium chloride in enhancing lipid accumulation in two green microalgae.

Biodiesel from microalgae has the potential as a sustainable fuel, since some species show exceptionally high lipid accumulation potential under various stresses. Effects of different concentrations of Ca, Mg and NaCl in the growth medium on biomass yield and lipid accumulation of Chlorella vulgaris and Scenedesmus obliquus grown under batch culture mode were investigated. Starvation of Mg showed a marginal rise in lipid content for a short period of time. Ca-starved cultures, however, demonstrated a profound rise in lipid content, i.e. 40% of dry cell wt. (dcw) was recorded against 11.9% control for C. vulgaris and 37% (dcw) against 11.3% for S. obliquus. Under increased concentration of Mg, significant rise in biomass and lipid yield was recorded. Effect of NaCl-induced osmotic stress showed lipid accumulation of approximately 40% (dcw) in both the test algae, whereas the biomass yield was severely affected. The fatty acid profiles under the above stresses were analysed and discussed.

Comparative assessment of various lipid extraction protocols and optimization of transesterification process for microalgal biodiesel production.

Biodiesel, using microalgae as feedstocks, is being explored as the most potent form of alternative diesel fuel for sustainable economic development. A comparative assessment of various protocols for microalgal lipid extraction was carried out using five green algae, six blue-green algae and two diatom species treated with different single and binary solvents both at room temperature and using a soxhlet. Lipid recovery was maximum with chloroform-methanol in the soxhlet extractor. Pretreatments ofbiomass, such as sonication, homogenization, bead-beating, lyophilization, autoclaving, microwave treatment and osmotic shock did not register any significant rise in lipid recovery. As lipid recovery using chloroform-methanol at room temperature demonstrated a marginally lower value than that obtained under the soxhlet extractor, on economical point of view, the former is recommended for microalgal total lipid extraction. Transesterification process enhances the quality of biodiesel. Experiments were designed to determine the effects of catalyst type and quantity, methanol to oil ratio, reaction temperature and time on the transesterification process using response surface methodology. Fatty acid methyl ester yield reached up to 91% with methanol:HCl:oil molar ratio of 82:4:1 at 65 degrees C for 6.4h reaction time. The biodiesel yield relative to the weight of the oil was found to be 69%.

Biodiesel production by the green microalga Scenedesmus obliquus in a recirculatory aquaculture system.

Biodiesel production was examined with Scenedesmus obliquus in a recirculatory aquaculture system with fish pond discharge and poultry litter to couple with waste treatment. Lipid productivity of 14,400 liter ha(-1) year(-1) was projected with 11 cultivation cycles per year. The fuel properties of the biodiesel produced adhered to Indian and international standards.

A waste-to-wealth initiative exploiting the potential of Anabaena variabilis for designing an integrated biorefinery.

The current research work was an innovative approach providing dual advantages of waste bioremediation and an effective biorefinery. The study attempted to exploit wastewater like aqua discharge and solid wastes like poultry litter/cow dung for cyanobacterial cultivation. Aqua discharge appended with 7.5 g L-1 poultry litter turned out as the best combination generating 46% higher carbohydrate yield than BG-11 control. A. variabilis cultivation in this waste-utilized medium also revealed its excellent bioremediation ability. While 100% removal was observed for nitrite, nitrate, and orthophosphate, a respective 74% and 81% reduction was noted for ammonium and total organic carbon. Chemical and biological oxygen demands were also reduced by 90%. This work was also novel in developing a sequential design for the production of bioethanol and co-products like exopolysaccharides, sodium copper chlorophyllin, C-phycocyanin, and poly-ß-hydroxybutyrate from the same cyanobacterial biomass. The developed biorefinery implementing the waste-utilized medium was one of its kind, enabling biomass valorization of 61%. Therefore, the present study would provide a leading-edge for tackling the high production costs that limit the practical viability of biorefinery projects. The recyclability of the bioremediated wastewater would not only curtail freshwater usage, the waste disposal concerns would also be mitigated to a great extent.

Waste utilization and biodiesel production by the green microalga Scenedesmus obliquus.

Scenedesmus obliquus was cultivated in three types of waste discharges to couple waste treatment with biodiesel production. The lipid pool accumulation was boosted to 1.0 g liter(-1) against 0.1 g liter(-1) for the control. The waste-grown S. obliquus showed an increase in the content of the saturated fatty acid pool, which is desirable for good-quality biodiesel.

Wastewater utilization for poly-ß-hydroxybutyrate production by the cyanobacterium Aulosira fertilissima in a recirculatory aquaculture system.

Intensive aquaculture releases large quantities of nutrients into aquatic bodies, which can lead to eutrophication. The objective of this study was the development of a biological recirculatory wastewater treatment system with a diazotrophic cyanobacterium, Aulosira fertilissima, and simultaneous production of valuable product in the form of poly-ß-hydroxybutyrate (PHB). To investigate this possible synergy, batch scale tests were conducted under a recirculatory aquaculture system in fiber-reinforced plastic tanks enhanced by several manageable parameters (e.g., sedimentation, inoculum size, depth, turbulence, and light intensity), an adequate combination of which showed better productivity. The dissolved-oxygen level increased in the range of 3.2 to 6.9 mg liter⁻¹ during the culture period. Nutrients such as ammonia, nitrite, and phosphate decreased to as low as zero within 15 days of incubation, indicating the system's bioremediation capability while yielding valuable cyanobacterial biomass for PHB production. Maximum PHB accumulation in A. fertilissima was found in sedimented fish pond discharge at 20-cm culture depth with stirring and an initial inoculum size of 80 mg dry cell weight (dcw) liter⁻¹. Under optimized conditions, the PHB yield was boosted to 92, 89, and 80 g m⁻², respectively for the summer, rainy, and winter seasons. Extrapolation of the result showed that a hectare of A. fertilissima cultivation in fish pond discharge would give an annual harvest of ∼17 tons dry biomass, consisting of 14 tons of PHB with material properties comparable to those of the bacterial polymer, with simultaneous treatment of 32,640 m³ water discharge.

Microalgal Biodiesel Production: Realizing the Sustainability Index.

Search for new and renewable sources of energy has made research reach the tiny little tots, microalgae for the production of biodiesel. But despite years of research on the topic, a definitive statement, declaring microalgae as an economically, environmentally, and socially sustainable resource is yet to be seen or heard of. With technological and scientific glitches being blamed for this delay in the progress of the production system, an assessment of the sustainability indices achieved so far by the microalgal biodiesel is important to be done so as to direct future research efforts in a more coordinated manner to achieve the sustainability mark. This article provides a review of the current economic, environmental, and social status of microalgal biodiesel and the strategies adopted to achieve them, with suggestions to address the challenges faced by the microalgal biodiesel production system.

Immobilization of Microalgae.

Several microalgae synthesize metabolites of great commercial interest. Microalgae also act as filters for wastewater N and P, heavy metals, and xenobiotic compounds. However, the cost-effective harvesting of microalgae is one of the major bottlenecks limiting the microalgal biomass applications. In this context, immobilization of algal cells has been proposed for circumventing the harvest problem as well as retaining the high-value algal biomass for further processing. In recent years, innovative approaches have been employed in the field of coimmobilization and microencapsulation, which have proved the superiority of immobilized cells over the free cells. Further, the development in the field of biosensor technology with immobilized microalgae presents an early warning device to monitor pollutants in natural waters. This chapter reviews the various applications of immobilized microalgae and addresses the specific methods concerning the production of coimmobilized beads and the protocol for construction of optical algal biosensors.

Microalga Scenedesmus obliquus as a potential source for biodiesel production.

Biodiesel from microalgae seems to be the only renewable biofuel that has the potential to completely replace the petroleum-derived transport fuels. Therefore, improving lipid content of microalgal strains could be a cost-effective second generation feedstock for biodiesel production. Lipid accumulation in Scenedesmus obliquus was studied under various culture conditions. The most significant increase in lipid reached 43% of dry cell weight (dcw), which was recorded under N-deficiency (against 12.7% under control condition). Under P-deficiency and thiosulphate supplementation the lipid content also increased up to 30% (dcw). Application of response surface methodology in combination with central composite rotary design (CCRD) resulted in a lipid yield of 61.3% (against 58.3% obtained experimentally) at 0.04, 0.03, and 1.0 g l(-1) of nitrate, phosphate, and sodium thiosulphate, respectively for time culture of 8 days. Scenedesmus cells pre-grown in glucose (1.5%)-supplemented N 11 medium when subjected to the above optimized condition, the lipid accumulation was boosted up to 2.16 g l(-1), the value approximately 40-fold higher with respect to the control condition. The presence of palmitate and oleate as the major constituents makes S. obliquus biomass a suitable feedstock for biodiesel production.

Exploitation of inexpensive substrates for production of a novel SCL-LCL-PHA co-polymer by Pseudomonas aeruginosa MTCC 7925.

Studies conducted with various inexpensive carbon sources such as whey, vegetable oils (palm, mustard, soybean and coconut), a low-cost source of glucose-D, rice and wheat bran, and mustard and palm oil cakes demonstrated palm oil as the best substrate for accumulation of a novel short-chain-length-long-chain-length polyhydroxyalkanoate (SCL-LCL-PHA) co-polymer containing SCL 3HAs [3-hydroxybutyric acid (3HB) and 3-hydroxyvaleric acid (3HV)] and LCL 3HAs of 3-hydroxyhexadecanoic acid (3HHD) and 3-hydroxyoctadecanoic acid (3HOD) units as constituents by a sludge-isolated Pseudomonas aeruginosa MTCC 7925. The co-polymer content reached up to 60% of dry cell weight (dcw) at 48 h of incubation in 0.5% (v/v) palm oil and the extract of 0.5% (v/v) palm oil cake supplemented vessels. The PHAs pool was further enhanced up to 69 and 75% (dcw), when the above culture was subjected to P- and N-limitation, respectively. The mol fraction of 3HB:3HV:3HHD:3HOD units were, respectively, 83.1:7.7:3.8:5.4 and 87.3:5.1:3.6:4.0 in P- and N-limited cultures. Consequently, a co-polymer yield of 5 g l(-1) (approx.) was achieved, which was about 80-fold higher as compared to 69 mg l(-1) of the control culture. On substrate basis, the accumulation reached up to 0.62 g PHAs per g substrate, which was significantly higher as compared to the yield obtained from starch by Haloferax mediterranei and Azotobacter chroococum, from molasses by A. vinelandii UWD, and from lactose and xylose by Pseudomonas cepacia. This novel P(3HB-co-3HV-co-3HHD-co-3HOD) co-polymer exhibited better thermal and mechanical properties as revealed from the differential scanning calorimetry and mechanical property studies, thus opens up new possibilities for various industrial applications.

Utilization of Scenedesmus obliquus Protein as a Replacement of the Commercially Available Fish Meal Under an Algal Refinery Approach.

The approach of algal refinery as a method to reduce the cost of algal biodiesel by co-production of various value-added chemicals is the most up-coming strategy suggested for the economic viability of microalgal biodiesel. This concept being relatively new and novel, abundant literature on the subject is not available although fragmented data on some feedstocks are present. The main objective of this research paper is to propose an algal refinery design through utilization of Scenedesmus obliquus biomass for production of various industrially important products. For this purpose, first a protocol was standardized for maximum extraction of protein from S. obliquus biomass. Then, different experiments were conducted for 90 days each to find out the optimum concentration of microalgal protein that can be substituted in the diets of freshwater fishes for their maximum growth. During these experiments eight different growth parameters and seven water quality parameters were tested. Results showed that the standard + whole microalgal biomass + extracted microalgal protein diet (25:25:50) was the best diet for maximum growth of the freshwater fishes. After conducting these experiments, a detailed sequential extraction process for maximum valorization of the S. obliquus biomass or in other words an algal refinery was designed. The detailed sequential process developed, yielded 0.06 g of ß-carotene, 10 g of protein, 38 g (43 mL) of biodiesel, 2 g of omega-3 fatty acid, 3 g (2.4 mL) of glycerol and 18 g (23 mL) of bioethanol from 1 Kg wet (≈100 g dry) S. obliquus biomass thus converting 70% of the test microalgal biomass into biodiesel and other value-added products by using an algal refinery approach.

Poly-beta-hydroxybutyrate accumulation in Nostoc muscorum: effects of metabolic inhibitors.

Poly-beta-hydroxybutyrate (PHB) accumulation in Nostoc muscorum was studied in presence of various metabolic inhibitors. Supplementation of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) was found to suppress PHB accumulation in phosphate-limited N. muscorum under photoautotrophic growth condition. PHB accumulation increased up to 21% and 17% from an initial PHB content of 8.5% of dry weight, respectively, under carbonylcyanide m-chlorophenylhydrazone (CCCP) and dicyclohexylcarbodiimide (DCCD) treatment, whereas 2,4 -dinitrophenol (DNP) supplementation depicted insignificant effect on PHB pool of the test cyanobacterium. Supplementation of l-methionine-dl-sulfoximine (MSX) and azaserine was also found to increase PHB accumulation in N(2) -fixing and NH(4)(+) -grown N. muscorum, but not in NO(3)(-) -grown cells. The stimulatory action of monofluoroacetate on PHB accumulation was suppressed in presence of alpha-ketoglutarate and DCMU. Interestingly, 2,3 -butanedione supplementation was not only found inhibitory for accumulation of PHB in P-deficient, N-deficient and chemoheterotrophically grown N. muscorum but suppression of PHB synthesis was also evident in control cultures in presence of 2,3 -butanedione. The possible mechanisms are discussed.

Process optimization for poly-beta-hydroxybutyrate production in a nitrogen fixing cyanobacterium, Nostoc muscorum using response surface methodology.

A five-level-four-factor central composite rotary design was employed to find out the interactive effects of four variables, viz. concentrations of acetate, glucose and K2HPO4, and dark incubation period on poly-beta-hydroxybutyrate (PHB) production in a N2-fixing cyanobacterium, Nostoc muscorum. Acetate, glucose and dark incubation period exhibited positive impacts on PHB yield. Using response surface methodology (RSM), a second order polynomial equation was obtained by multiple regression analysis. A yield of 45.6% of dry cell weight (dcw) was achieved at reduced level of nutrients, i.e. 0.17% acetate, 0.16% glucose and 5 mg l(-1) K2HPO4 at a dark incubation period of 95 h as compared to 41.6% PHB yield in 0.4% acetate, 0.4% glucose and 40 mg l(-1) K2HPO4 at a dark incubation period of 168 h under single factor optimization strategy.

Advances in cyanobacterial polyhydroxyalkanoates production.

Polyhydroxyalkanoates (PHAs) have received much attention in the current scenario due to their attractive material properties, namely biodegradability, biocompatibility, thermoplasticity, hydrophobicity, piezoelectricity and stereospecificity. All these properties make them highly competitive for various industrial applications similar to non-degradable conventional plastics. In PHA biosynthesis, PHA synthase acts as a natural catalyst for PHA polymerization process using the (R)-hydroxyacyl-CoA as substrate. Cyanobacteria can accumulate PHAs under photoautotrophic and/or mixotrophic growth conditions with organic substrates such as acetate, glucose, propionate, valerate, and so on. The natural incidence of PHA accumulation by the cyanobacteria is known since 1966. Nevertheless, PHA accumulation in cyanobacteria based on the cell biomass and volumetric productivity is critically lower than the heterotrophic bacteria. Consequently, cyanobacteria are nowadays not considered for commercial production of PHAs. Thus, strain improvements by genetic modification, new cultivation and harvesting techniques, advanced photobioreactor development, efficient and sustainable downstream processes, alternate economical carbon sources and usage of various metabolic inhibitors are suggested for enhancing cyanobacterial PHA accumulation. In addition, identification of transcriptional regulators like RNA polymerase sigma factor (SigE) and a response regulator (Rre37) together with the recent major scientific breakthrough on the existence of complete Krebs cycle in cyanobacteria would be helpful in taking PHA production from cyanobacteria to a new-fangled height in near future.

Optimization of cultural and nutritional conditions for accumulation of poly-beta-hydroxybutyrate in Synechocystis sp. PCC 6803.

Poly-beta-hydroxybutyrate (PHB) accumulation in the unicellular cyanobacterium, Synechocystis sp. PCC 6803, was studied under various cultural and nutritional conditions. Under controlled condition, cells harvested at the stationary phase of growth depicted maximum accumulation of PHB, i.e., 4.5% (w/w of dry cells) as compared to lag (1.8%) or logarithmic (2.9%) phases of cultures. A temperature range of 28-32 degrees C and pH between 7.5 and 8.5 were preferred for PHB accumulation. Cells cultivated under regular light-dark cycles accumulated more PHB (4.5%) than those grown under continuous illumination (2.4%). Nitrogen and phosphorus starvation stimulated PHB accumulation up to the tune of 9.5 and 11% (w/w of dry cells), respectively. Synechocystis cells pre-grown in glucose (0.1%)-supplemented BG-11 medium when subjected to P-deficiency in presence of acetate (0.4%), PHB accumulation was boosted up to 29% (w/w of dry cells), the value almost 6-fold higher with respect to photoautotrophic condition. Fishpond discharges were found as suitable media for PHB accumulation in the test cyanobacterium.

Studies on poly-beta-hydroxybutyrate synthase activity of Nostoc muscorum.

This study compares the PHB synthase activity of Nostoc muscorum, a N(2)-fixing cyanobacterium under control (grown in usual BG-11 medium), nitrogen (N) and phosphorus (P) deprivation and chemoheterotrophic conditions. Specific activity of PHB synthase did not depict significant variations in the latter three types of cultures, except for the control one, where a significantly lower activity was recorded. PHB synthase activity was detected only in the soluble fractions of both the control as well as cells incubated under chemoheterotrophic conditions. A K(m) of 80.2 microM DL-beta-hydroxybutyryl-CoA and V(max) of 197.5 nmol thiobenzoate (TNB) mg protein(-1)min(-1) were observed for the enzyme. PHB synthase remained insensitive to acetyl-CoA, ATP, NADP, NADPH supplementation under in vitro condition. Addition of acetyl phosphate was found to activate the enzyme and the level of activation was dependent on the concentration of acetyl phosphate supplementation. Inhibition of PHB synthase in 2,3-butanedione supplemented cultures and reactivation following acetyl phosphate addition proved the post-translational control of acetyl phosphate over PHB synthase.

Progress and Challenges in Microalgal Biodiesel Production.

The last decade has witnessed a tremendous impetus on biofuel research due to the irreversible diminution of fossil fuel reserves for enormous demands of transportation vis-a-vis escalating emissions of green house gasses (GHGs) into the atmosphere. With an imperative need of CO2 reduction and considering the declining status of crude oil, governments in various countries have not only diverted substantial funds for biofuel projects but also have introduced incentives to vendors that produce biofuels. Currently, biodiesel production from microalgal biomass has drawn an immense importance with the potential to exclude high-quality agricultural land use and food safe-keeping issues. Moreover, microalgae can grow in seawater or wastewater and microalgal oil can exceed 50-60% (dry cell weight) as compared with some best agricultural oil crops of only 5-10% oil content. Globally, microalgae are the highest biomass producers and neutral lipid accumulators contending any other terrestrial oil crops. However, there remain many hurdles in each and every step, starting from strain selection and lipid accumulation/yield, algae mass cultivation followed by the downstream processes such as harvesting, drying, oil extraction, and biodiesel conversion (transesterification), and overall, the cost of production. Isolation and screening of oleaginous microalgae is one pivotal important upstream factor which should be addressed according to the need of freshwater or marine algae with a consideration that wild-type indigenous isolate can be the best suited for the laboratory to large scale exploitation. Nowadays, a large number of literature on microalgal biodiesel production are available, but none of those illustrate a detailed step-wise description with the pros and cons of the upstream and downstream processes of biodiesel production from microalgae. Specifically, harvesting and drying constitute more than 50% of the total production costs; however, there are quite a less number of detailed study reports available. In this review, a pragmatic and critical analysis was tried to put forward with the on-going researches on isolation and screening of oleaginous microalgae, microalgal large scale cultivation, biomass harvesting, drying, lipid extraction and finally biodiesel production.

Poly-beta-hydroxybutyrate accumulation in Nostoc muscorum and Spirulina platensis under phosphate limitation.

Nostoc muscorum and Spirulina platensis were grown under phosphate deficiency in order to investigate the role of internal phosphate pool and activity of alkaline phosphatase on poly-beta-hydroxybutyrate (PHB) accumulation. PHB accumulation in N. muscorum increased to 22.7% of dry weight (dw) after 4 day of phosphate deficiency, while the internal phosphate pool reduced to the level of 0.02 microM mg dw(-1) at a maximum APase activity of 2.57nM PNP mg dw(-1) h(-1). In contrary, S. platensis depicted maxima of 1.39nM PNP mg dw(-1) h(-1) on day 30 of incubation, which was about 2 fold lower than the observed value of N. muscorum. PHB content in S. platensis remained low even after prolonged phosphate starvation, and a rise only up to 3.5% of dw was recorded on day 60 of phosphate deficiency. Supplementation of NADPH exogenously to S. platensis cultures grown under phosphate deficiency favoured PHB accumulation in 10, 20 and 30 days old cultures, but not in the cultures grown under phosphate deficiency for 60 days. The possible role of phosphate limitation on PHB accumulation is discussed.