Production of (R)-3-hydroxybutyric acid by fermentation and bioconversion processes with Azohydromonas lata

Feasibility of producing (R)-3-hydroxybutyric acid ((R)-3-HB) using wild type Azohydromonas lata and its mutants (derived by UV mutation) was investigated. A. lata mutant (M5) produced 780 mg/l in the culture broth when sucrose was used as the carbon source. M5 was further studied in terms of its specificity with various bioconversion substrates for production of (R)-3-HB. (R)-3-HB concentration produced in the culture broth by M5 mutant was 2.7-fold higher than that of the wild type strain when sucrose (3% w/v) and (R,S)-1,3-butanediol (3% v/v) were used as carbon source and bioconversion substrate, respectively. Bioconversion of resting cells (M5) with glucose (1% v/w), ethylacetoacetate (2% v/v), and (R,S)-1,3-butanediol (3% v/v), resulted in (R)-3-HB concentrations of 6.5 g/l, 7.3 g/l and 8.7 g/l, respectively.     

Biological hydrogen production by the algal biomass Chlorella vulgaris MSU 01 strain isolated from pond sediment

Chlorella vulgaris MSU 01 strain isolated from the sediment of the pond is able to produce molecular hydrogen in a clean way. To relate the dynamic coupling between the cultural conditions and biological responses, an original lab scale set up has been developed for hydrogen production. Different sources like mannitol, glucose, alanine, citric acid, aspartic acid, l-alanine, l-cysteine, sodium succinate and sodium pyruvate were used for algal media optimization. Corn stalk, from 1 to 5 g/L was tested for the effective algal growth and hydrogen production. The cell concentration of 1.6-19 g/L dry cell weight (DCW) was found at the 10th day. The kinetic parameters involved in the hydrogen production at 4 g/L corn stalk using the algal inoculum (50 mL) in the bioreactor volume (500 mL) was found to be with the hydrogen production potential (P_s) of 7.784 mL and production yield of (P_r) 5.534 mL respectively. The growth profile of the algal biomass at the above mentioned condition expressed the logistic model with R² 0.9988. The final pH of the broth was increased from 7.0 to 8.5-8.7. The anaerobic fermentation by C. vulgaris MSU 01 strain involved in the conversion process of complex carbon source has increased the H₂ evolution rate and higher butyrate concentration in the fermentate.     

Enhanced hydrogen production by Rhodopseudomonas palustris CQK 01 with ultra-sonication pretreatment in batch culture

In the present study, the photo-hydrogen production performances by Rhodopseudomonas palustris CQK 01 growing from the inoculated cells with ultra-sonication pretreatment (R. palustris CQK 01-USP) were experimentally investigated in batch culture and compared with that without pretreatment (R. palustris CQK 01-NP). It was found that the ultra-sonication pretreatment modified membrane morphology and broke up part of the cells, resulting in improvement of membrane permeability and bacterial activities and hence, helping the improvement of hydrogen production. The hydrogen production rate, hydrogen yield and energy conversion efficiency with R. palustris CQK 01-USP were increased to be nearly 2 times higher than that with R. palustris CQK 01-NP. The parametric study showed that under the conditions of initial glucose concentration 50 mmol/l, inoculum size 12%, illumination wavelength 590 nm, the photobioreactor with R. palustris CQK 01-USP obtained the optimal hydrogen production rate 0.54 mmol/l/h, hydrogen yield 1.2 mol-H₂/mol-glucose and energy conversion efficiency 9.03%.     

Hydrogen production by Rhodopseudomonas palustris WP 3-5 in a serial photobioreactor fed with hydrogen fermentation effluent

In this study, a lab-scale serial photobioreactor composed of three column reactors was constructed and continuously operated to investigate several parameters influencing photohydrogen production when using the synthetic wastewater and the anaerobic hydrogen fermentation effluents as the influents. The results indicated that better hydrogen production rate was obtained when the serial photobioreactor was operated under cellular recycling at a short HRT of 8 h. The serial photobioreactor maintained high hydrogen content ca. 80% in the produced gas and 0.4× dilution ratio was the suitable ratio for hydrogen production. When the photobioreactor fed with the real wastewater (Effluent 1) containing 100 mg/L NH₄Cl, Column 1 reactor successfully reduced ammonia concentration to about 60 mg/L for cell synthesis, resulting in a steady hydrogen production in the following two column reactors. The average hydrogen production rate was 205 mL-H₂/L/d.     

Enhanced start-up of anaerobic facultatively autotrophic biocathodes in bioelectrochemical systems

Biocathodes in bioelectrochemical systems (BESs) can be used to convert CO₂ into diverse organic compounds through a process called microbial electrosynthesis. Unfortunately, start-up of anaerobic biocathodes in BESs is a difficult and time consuming process. Here, a pre-enrichment method was developed to improve start-up of anaerobic facultatively autotrophic biocathodes capable of using cathodes as the electron donor (electrotrophs) and CO₂ as the electron acceptor. Anaerobic enrichment of bacteria from freshwater bog sediment samples was first performed in batch cultures fed with glucose and then used to inoculate BES cathode chambers set at −0.4 V (versus a standard hydrogen electrode; SHE). After two weeks of heterotrophic operation of BESs, CO₂ was provided as the sole electron acceptor and carbon source. Consumption of electrons from cathodes increased gradually and was sustained for about two months in concert with a significant decrease in cathode chamber headspace CO₂. The maximum current density consumed was −34 ± 4 mA/m². Biosynthesis resulted in organic compounds that included butanol, ethanol, acetate, propionate, butyrate, and hydrogen gas. Bacterial community analyses based on 16S rRNA gene clone libraries revealed Trichococcus palustris DSM 9172 (99% sequence identity) as the prevailing species in biocathode communities, followed by Oscillibacter sp. and Clostridium sp. Isolates from autotrophic cultivation were most closely related to Clostridium propionicum (99% sequence identity; ZZ16), Clostridium celerecrescens (98–99%; ZZ22, ZZ23), Desulfotomaculum sp. (97%; ZZ21), and Tissierella sp. (98%; ZZ25). This pre-enrichment procedure enables simplified start-up of anaerobic biocathodes for applications such as electrofuel production by facultatively autotrophic electrotrophs.     

In silico optimization and low structured kinetic model of poly[(R)-3-hydroxybutyrate] synthesis by Cupriavidus necator DSM 545 by fed-batch cultivation on glycerol

Glycerol was utilized by Cupriavidus necator DSM 545 for production of poly-3-hydroxybutyrate (PHB) in fed-batch fermentation. Maximal specific growth rates (0.12 and 0.3 h⁻¹) and maximal specific non-growth PHB production rate (0.16 g g⁻¹ h⁻¹) were determined from two experiments (inocula from exponential and stationary phase). Saturation constants for nitrogen (0.107 and 0.016 g L⁻¹), glycerol (0.05 g L⁻¹), non-growth related PHB synthesis (0.011 g L⁻¹) and nitrogen/PHB related inhibition constant (0.405 g L⁻¹), were estimated. Five relations for specific growth rate were tested using mathematical models. In silico performed optimization procedures (varied glycerol/nitrogen ratio and feeding) has resulted in a PHB content of 70.9%, shorter cultivation time (23 h) and better PHB yield (0.347 g g⁻¹). Initial concentration of biomass 16.8 g L⁻¹ and glycerol concentration in broth between 3 and 5 g L⁻¹ were decisive factors for increasing of productivity.     

Conversion of squid pen by Pseudomonas aeruginosa K187 fermentation for the production of N-acetyl chitooligosaccharides and biofertilizers

Pseudomonas aeruginosa K187, a protease- and chitinase-producing bacterium, exhibited protease and chitinase activity after three and five days of incubation, respectively. The protease and chitinase were both produced by using 1% squid pen powder (SPP) (w/v) as sole carbon and nitrogen source. After fermentation, the deproteinization rate of the recovered squid pen gradually increased up to 68% on the fourth day. After five days of fermentation, the production of GlcNAc, (GlcNAc)₂, (GlcNAc)₃, (GlcNAc)₄ and (GlcNAc)₅ were 1.18 mg/mL, 0.76 mg/mL, 1.02 mg/mL, 0.93 mg/mL and 0.90 mg/mL, respectively. The culture supernatant of K187 also exhibited activity of enhancing vegetable growth. For Brassica chinensis Linn treated with the fifth day culture supernatant, the total weight and total length increased up to 529% and 148%, respectively, compared to the control group. With this method, the production of protease, chitinase, N-acetyl chitooligosaccharides and biofertilizers may be useful for biological applications.     

Poly-3-hydroxybutyrate synthase from the periplasm of Escherichia coli

This is the first report of a poly-3-hydroxybutyrate (PHB) synthase in Escherichiacoli. The enzyme was isolated from the periplasm using ammonium sulfate fractionation, hydrophobic, and size-exclusion chromatography and identified by LC/MS/MS as YdcS, a component of a putative ABC transporter. Green Fluorescent Protein-tagged ydcS, purified by 2D native gel electrophoresis, also exhibited PHB synthase activity. Optimal conditions for enzyme activity were 37 °C, pH 6.8-7.5, 100 mM KCl. K_m was 0.14 mM and V_max was 18.7 nmol/mg protein/min. The periplasms of deletion mutants displayed <25% of the activity of the parent strain. Deletion mutants exhibited ∼25% less growth in M9 medium, glucose, and contained ∼30% less PHB complexed to proteins (cPHB) in the outer membranes, but the same concentration of chloroform-extractable PHB as wild-type cells. The primary sequence of YdcS suggests it may belong to the α-/β-hydrolase superfamily which includes polyhydroxybutyrate (PHB) synthases, lipases, and esterases.     

Cytotoxic activity of acetogenins and styryl lactones isolated from Goniothalamus undulatus Ridl. root extracts against a lung cancer cell line (COR-L23)

An investigation of the chemical constituents in a dichloromethnae extract of Goniothalamus undulatus root led to the isolation of three known styryl lactones (5-acetoxyisogoniothalamin oxide, O-acetylaltholactone and altholactone), and four known annonaceous acetogenins (annonacin, cis-annonacin, goniothalamicin and cis-goniothalamicin). These compounds were subjected to a sulphorhodamine B (SRB) cytotoxicity assay against human large cell lung carcinoma (COR-L23), and normal human fetal fibroblast (MRC-5), cell lines. The isolated acetogenins showed higher cytotoxic activity against COR-L23 compared to the styryl lactones, with IC₅₀ values in the range of 0.5-1.7 μM and 7.4-15.4 μM, respectively. A similar pattern of cytotoxicity was also observed against the other cell line (MRC-5); acetogenins IC₅₀ values were in the range of 11.8-31.4 μM, and those for styryl lactones were in the range of 48.7-102.8 μM. This is the first report of a bioassay-guided isolation of chemical constituents from G. undulatus and on cytotoxic studies of the isolated compounds using these particular lung cancer cell lines.     

Biofiltration of reduced sulphur compounds and community analysis of sulphur-oxidizing bacteria

The present work aims to use a two-stage biotrickling filters for simultaneous treatment of hydrogen sulphide (H₂S), methyl mercaptan (MM), dimethyl sulphide (DMS) and dimethyl disulphide (DMDS). The first biofilter was inoculated with Acidithiobacillus thiooxidans (BAT) and the second one with Thiobacillus thioparus (BTT). For separate feeds of reduced sulphur compounds (RSC), the elimination capacity (EC) order was DMDS > DMS > MM. The EC values were 9.8 g_MM-S/m³/h (BTT; 78% removal efficiency (RE); empty bed residence time (EBRT) 58 s), 36 g_DMDS-S/m³/h (BTT; 94.4% RE; EBRT 76 s) and 57.5 g_H2S-S/m³/h (BAT; 92% RE; EBRT 59 s). For the simultaneous removal of RSC in BTT, an increase in the H₂S concentration from 23 to 293 ppmv (EBRT of 59 s) inhibited the RE of DMS (97-84% RE), DMDS (86-76% RE) and MM (83-67% RE). In the two-stage biofiltration, the RE did not decrease on increasing the H₂S concentration from 75 to 432 ppmv.     

Isolation and characterization of a fungus able to degrade pyrethroids and 3-phenoxybenzaldehyde

Fungal strain HU, isolated from activated sludge and identified as a member of the genus Cladosporium based on morphology and sequencing of 28S rRNA, was shown to degrade 90% of fenvalerate, fenpropathrin, β-cypermethrin, deltamethrin, bifenthrin, and permethrin (100 mg L⁻¹) within 5 days. Fenvalerate was utilized as sole carbon and energy source and co-metabolized in the presence of sucrose. Degradation of fenvalerate occurred at pH 5-10 at 18-38 °C. The fungus first hydrolyzed the carboxylester linkage to produce α-hydroxy-3-phenoxy-benzeneacetonitrile and 3-phenoxybenzaldehyde, and subsequently degraded these two compounds with a q_max, K_s and K_i of 1.73 d⁻¹, 99.20 mg L⁻¹ and 449.75 mg L⁻¹, respectively. Degradation followed first-order kinetics. These results show that the fungal strain may possess potential to be used in bioremediation of pyrethroid-contaminated environments.     

Involvement of the Reserve Material Poly-β-Hydroxybutyrate in Azospirillum brasilense Stress Endurance and Root Colonization

When grown under suboptimal conditions, rhizobacteria of the genus Azospirillum produce high levels of poly-β-hydroxybutyrate (PHB). Azospirillum brasilense strain Sp7 and a phbC (PHB synthase) mutant strain in which PHB production is impaired were evaluated for metabolic versatility, for the ability to endure various stress conditions, for survival in soil inoculants, and for the potential to promote plant growth. The carbon source utilization data were similar for the wild-type and mutant strains, but the generation time of the wild-type strain was shorter than that of the mutant strain with all carbon sources tested. The ability of the wild type to endure UV irradiation, heat, osmotic pressure, osmotic shock, and desiccation and to grow in the presence of hydrogen peroxide was greater than that of the mutant strain. The motility and cell aggregation of the mutant strain were greater than the motility and cell aggregation of the wild type. However, the wild type exhibited greater chemotactic responses towards attractants than the mutant strain exhibited. The wild-type strain exhibited better survival than the mutant strain in carrier materials used for soil inoculants, but no difference in the ability to promote plant growth was detected between the strains. In soil, the two strains colonized roots to the same extent. It appears that synthesis and utilization of PHB as a carbon and energy source by A. brasilense under stress conditions favor establishment of this bacterium and its survival in competitive environments. However, in A. brasilense, PHB production does not seem to provide an advantage in root colonization under the conditions tested.     

Poly(3-Hydroxybutyrate) Synthesis Genes in Azotobacter sp. Strain FA8

Genes responsible for the synthesis of poly(3-hydroxybutyrate) (PHB) in Azotobacter sp. FA8 were cloned and analyzed. A PHB polymerase gene (phbC) was found downstream from genes coding for β-ketothiolase (phbA) and acetoacetyl-coenzyme A reductase (phbB). A PHB synthase mutant was obtained by gene inactivation and used for genetic studies. The phbC gene from this strain was introduced into Ralstonia eutropha PHB-4 (phbC-negative mutant), and the recombinant accumulated PHB when either glucose or octanoate was used as a source of carbon, indicating that this PHB synthase cannot incorporate medium-chain-length hydroxyalkanoates into PHB.     

Synergistic experimental and computational approach identifies novel strategies for polyhydroxybutyrate overproduction

Polyhydroxybutyrate (PHB) is a sustainable bioplastic produced by bacteria that is a potential replacement for conventional plastics. This study delivers an integrated experimental and computational modeling approach to decipher metabolic factors controlling PHB production and offers engineering design strategies to boost production. In the metabolically robust Rhodopseudomonas palustris CGA009, PHB production significantly increased when grown on the carbon- and electron-rich lignin breakdown product p-coumarate (C₉H₈O₃) compared to virtually no PHB titer from acetate (C₂H₃NaO₂). The maximum yield did not improve further when grown on coniferyl alcohol (C₁₀H₁₂O₃), but comparison of the PHB profiles showed that coniferyl alcohol's higher carbon content resulted in a higher rate of PHB production. Combined experimental results revealed that cytoplasmic space may be a limiting factor for maximum PHB titer. In order to obtain a systems-level understanding of factors driving PHB yield, a model-driven investigation was performed. The model yielded several engineering design strategies including utilizing reduced, high molecular weight substrates that bypass the thiolase reaction (phaA). Based on these strategies, utilization of butyrate was predicted and subsequently validated to produce PHB. Model analysis also explained why nitrogen starvation was not essential for PHB production and revealed that renewable and abundant lignin aromatics are ideal candidates for PHB production. Most importantly, the generality of the derived design rules allows them to be applied to any PHB-producing microbe with similar metabolic features.     

Thermophilic bacterium Caldimonas taiwanensis produces poly(3-hydroxybutyrate-co-3-hydroxyvalerate) from starch and valerate as carbon sources

Caldimonas taiwanensis accumulated polyhydroxybutyrate (PHB) at 55 °C from gluconate, fructose, maltose, and glycerol under nitrogen-limited condition. The PHB content peaked at 14 h after inoculation from gluconate. C. taiwanensis did not grow or accumulate PHA from fatty acids as the sole carbon source; however, it incorporated 3-hydroxyvalerate (3-HV) into PHB polymer from gluconate and valerate as a mixed carbon source. By adjusting the valerate concentration, the molar fraction of 3-HV could be modulated from 10 mol% to 95 mol%. Fatty acid valerate substantially inhibited cell growth and PHA accumulation with the addition of as little as 5 mM to the medium. Supplementing the medium with yeast extract overcame the inhibition, which enhanced not only the yield of biomass but also PHA productivity. The in vivo substrate specificity of PHA synthase ranged from C₄ to C₆. In addition, C. taiwanensis also incorporated a wide range of 3-HV into PHA from soluble starch and valerate as a mixed carbon source. Food-grade starches made from cassava, corn, potato, sweet potato and wheat respectively mixed with valerate were studied for poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] production. In this study, C. taiwanensis exhibited high promise for reducing the production cost of P(3HB-co-3HV).     

Production of poly-β-hydroxybutyric acid from carbon dioxide by Alcaligenes eutrophus ATCC 17697T

The characteristics of PHB production from carbon dioxide by autotrophic culture of Alcaligenes eutrophus ATCC 17697^T using a recycled gas closed circuit culture system under the condition of oxygen limitation were investigated. Cell concentration increased to more than 60 g/l after 60 h of cultivation, while the PHB concentration reached 36 g/l. PHB accumulation in the oxygen-limited culture was superior than that in an ammonium-deficient culture. The PHB produced was identified as a homopolymer of d-3-hydroxybutyrate by ¹H and ¹³C NMR analysis. The stoichiometry for PHB production from CO₂ under the oxygen limitation condition was indicated to be as follows: 33H₂ + 12O₂ + 4CO₂ → C₄H₆O₂ + 30H₂O. This stoichiometry shows that the hydrogen consumption per one mole of CO₂ for PHB production is larger than that for cell formation.     

Efficient mosquitocidal toxin production by Bacillus sphaericus using cheese whey permeate under both submerged and solid state fermentations

Whey permeate (WP) was used efficiently for production of mosquitocidal toxin by Bacillus sphaericus 2362 (B. sphaericus 2362) and the Egyptian isolate, B. sphaericus 14N1 (B. sphaericus 14N1) under both submerged and solid state fermentation conditions. Under submerged fermentation, high mosquitocidal activity was produced by B. sphaericus 2362 and B. sphaericus 14N1 at 50-100% and 25-70% WP, respectively. Initial pH of WP was a critical factor for toxin production by both tested organisms. The highest toxicity was obtained at initial pH 7. Egyptian isolate, B. sphaericus 14N1 was tested for growth and toxin production under solid state fermentation conditions (SSF) by using WP as moistening agent instead of distilled water. The optimum conditions for production of B. sphaericus 14N1 on wheat bran-WP medium were 10 g wheat bran/250 ml flask moistened with 10-70% WP at 50% moisture content, inoculum size ranged between 17.2 × 10⁷ and 34.4 × 10⁷ and 6 days incubation under static conditions at 30 °C. Preliminary pilot-scale production of B. sphaericus 14N1 under SSF conditions in trays proved that wheat bran-WP medium was efficient and economic for industrial production of mosquitocidal toxin by B. sphaericus.     

Evaluation of bioenergy recovery processes treating organic residues from ethanol fermentation process

This study evaluates a two-stage bioprocess for recovering bioenergy in the forms of hydrogen and methane while treating organic residues of ethanol fermentation from tapioca starch. A maximum hydrogen production rate of 0.77 mmol H₂/g VSS/h can be achieved at volumetric loading rate (VLR) of 56 kg COD/m³/day. Batch results indicate that controlling conditions at S₀/X₀ = 12 with X₀ = 4000 mg VSS/L and pH 5.5-6 are important for efficient hydrogen production from fermentation residues. Hydrogen-producing bacteria enriched in the hydrogen bioreactor are likely utilizing lactate and acetate for biohydrogen production from ethanol-fermentation residues. Organic residues remained in the effluent of hydrogen bioreactor can be effectively converted to methane with a rate of 0.37 mmol CH₄/g VSS/h at VLR of 8 kg COD/m³/day. Approximately 90% of COD in ethanol-fermentation residues can be removed and among that 2% and 85.1% of COD can be recovered in the forms of hydrogen and methane, respectively.