Motakatla Venkateswar Reddy

With the growing interest in bioplastics, there is an urgent need to develop rapid analysis methods linked to production technology development. This study focused on the production of a commercially non-available homopolymer, poly(3-hydroxyvalerate) (P(3HV)), and a commercially available copolymer, poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (P(3HB-co-3HV)), through fermentation using two different bacterial strains. The bacteria Chromobacterium violaceum and Bacillus sp. CYR1 were used to produce P(3HV) and P(3HB-co-3HV), respectively. The bacterium Bacillus sp. CYR1 produced 415 mg/L of P(3HB-co-3HV) when incubated with acetic acid and valeric acid as the carbon sources, whereas the bacterium C. violaceum produced 0.198 g of P(3HV)/g dry biomass when incubated with sodium valerate as the carbon source. Additionally, we developed a fast, simple, and inexpensive method to quantify P(3HV) and P(3HB-co-3HV) using high-performance liquid chromatography (HPLC). As the alkaline decomposition of P(3HB-co-3HV) releases 2-butenoic acid (2BE) and 2-pentenoic acid (2PE), we were able to determine the concentration using HPLC. Moreover, calibration curves were prepared using standard 2BE and 2PE, along with sample 2BE and 2PE produced by the alkaline decomposition of poly(3-hydroxybutyrate) and P(3HV), respectively. Finally, the HPLC results obtained by our new method were compared using gas chromatography (GC) analysis.

The world’s rising energy needs, and the depletion of fossil resources demand a shift from fossil-based feedstocks to organic waste to develop a competitive, resource-efficient, and low-carbon sustainable economy in the long run. It is well known that the production of fuels and chemicals via chemical routes is advantageous because it is a well-established technology with low production costs. However, the use of toxic/environmentally harmful and expensive catalysts generates toxic intermediates, making the process unsustainable. Alternatively, utilization of renewable resources for bioprocessing with a multi-product approach that aligns novel integration improves resource utilization and contributes to the “green economy”. The present review discusses organic waste bioprocessing through the anaerobic fermentation (AF) process to produce biohydrogen (H₂), biomethane (CH₄), volatile fatty acids (VFAs) and medium chain fatty acids (MCFA). Furthermore, the roles of photosynthetic bacteria and microalgae for biofuel production are discussed. In addition, a roadmap to create a fermentative biorefinery approach in the framework of an AF-integrated bioprocessing format is deliberated, along with limitations and future scope. This novel bioprocessing approach significantly contributes to promoting the circular bioeconomy by launching complete carbon turnover practices in accordance with sustainable development goals.

Synthetic plastics derived from fossil fuels—such as polyethylene, polypropylene, polyvinyl chloride, and polystyrene—are non-degradable. A large amount of plastic waste enters landfills and pollutes the environment. Hence, there is an urgent need to produce biodegradable plastics such as polyhydroxyalkanoates (PHAs). PHAs have garnered increasing interest as replaceable materials to conventional plastics due to their broad applicability in various purposes such as food packaging, agriculture, tissue-engineering scaffolds, and drug delivery. Based on the chain length of 3-hydroxyalkanoate repeat units, there are three types PHAs, i.e., short-chain-length (scl-PHAs, 4 to 5 carbon atoms), medium-chain-length (mcl-PHAs, 6 to 14 carbon atoms), and long-chain-length (lcl-PHAs, more than 14 carbon atoms). Previous reviews discussed the recent developments in scl-PHAs, but there are limited reviews specifically focused on the developments of mcl-PHAs. Hence, this review focused on the mcl-PHA production, using various carbon (organic/inorganic) sources and at different operation modes (continuous, batch, fed-batch, and high-cell density). This review also focused on recent developments on extraction methods of mcl-PHAs (solvent, non-solvent, enzymatic, ultrasound); physical/thermal properties (Mw, Mn, PDI, Tm, Tg, and crystallinity); applications in various fields; and their production at pilot and industrial scales in Asia, Europe, North America, and South America.

The bacterium Rhodococcus erythropolis MI2 uses 4,4´-dithiodibutyric acid (DTDB) as carbon source to synthesize polythioesters (PTE). The first step for the production of PTE using DTDB is catalyzed by an NADH:flavin oxidoreductase (nox) as it was previously shown in our laboratory, and the second step is catabolized by a putative luciferase-like monooxygenase (Llm). In the current study, experiments were carried out to identify the function of Llm. Hence, the llm gene, which encodes for the Llm protein, was amplified from the genomic DNA of MI2 using polymerase chain reaction and expressed in Escherichia coli BL21 cells. Protein purification was done using His Spin Trap affinity columns. Enzyme assay was carried out using the purified protein and p-coumaric acid as substrate giving a specific activity of 1.6 U/mg protein for the purified Llm. The responsible gene (llm) was deleted in the genome of MI2, and a single deletion mutant was subsequently generated. Finally, growth of the wild-type strain (MI2) and the mutant strain (MI2Δllm) were compared using DTDB or succinate as carbon sources. Whereas the wild type was successfully grown with DTDB or succinate, the llm-negative mutant exhibited low grow with DTDB although it grows very well with succinate.

Cheese whey (CW) can be an excellent carbon source for polyhydroxyalkanoates (PHA)-producing bacteria. Most studies have used CW, which contains high amounts of lactose, however, there are no reports using raw CW, which has a relatively low amount of lactose. Therefore, in the present study, PHA production was evaluated in a two-stage process using the CW that contains low amounts of lactose. In first stage, the carbon source existing in CW was converted into acetic acid using the bacteria, Acetobacter pasteurianus C1, which was isolated from food waste. In the second stage, acetic acid produced in the first stage was converted into PHA using the bacteria, Bacillus sp. CYR-1. Under the condition of without the pretreatment of CW, acetic acid produced from CW was diluted at different folds and used for the production of PHA. Strain CYR-1 incubated with 10-fold diluted CW containing 5.7 g/L of acetic acid showed the higher PHA production (240.6 mg/L), whereas strain CYR-1 incubated with four-fold diluted CW containing 12.3 g/L of acetic acid showed 126 mg/L of PHA. After removing the excess protein present in CW, PHA production was further enhanced by 3.26 times (411 mg/L) at a four-fold dilution containing 11.3 g/L of acetic acid. Based on Fourier transform infrared spectroscopy (FT-IR), and ¹H and ¹³C nuclear magnetic resonance (NMR) analyses, it was confirmed that the PHA produced from the two-stage process is poly-β-hydroxybutyrate (PHB). All bands appearing in the FT-IR spectrum and the chemical shifts of NMR nearly matched with those of standard PHB. Based on these studies, we concluded that a two-stage process using Acetobacter pasteurianus C1 and Bacillus sp. CYR-1 would be applicable for the production of PHB using CW containing a low amount of lactose.

Medium-chain fatty acids (MCFA) such as caproic, heptanoic, and caprylic acids are monocarboxylic acids, which can be used as precursor molecules to synthesize biodiesel, bioplastics, antimicrobials, and corrosion inhibitors. Anaerobic bacteria transform ethanol and short-chain fatty acids (SCFA) such as acetate, butyrate, and valerate into MCFA through chain elongation. SCFA and MCFA production were evaluated in this study using two types of waste: apple waste (AW) and pumpkin waste (PW). Two kinds of PW, pumpkin pulp waste (PPW) and pumpkin skin waste (PSW), were used. AW and PW were first converted into a paste using a blender, and then into a leachate (liquid extract obtained from the paste) by adding an anaerobic mixed bacterial culture to the paste in a separate anaerobic chamber. The leachate containing SCFA was further transformed to MCFA using a Clostridium kluyveri augmented, especially enriched anaerobic mixed bacterial culture. The production of SCFA, MCFA and biogas was established for 75 days and analyzed throughout the experiment using high-pressure liquid chromatography (HPLC) and gas chromatography (GC), respectively. Bacteria incubated with AW produced significant amounts of SCFA (7.2 g l⁻¹ of butyric acid) and MCFA (1.2 g l⁻¹ of caproic acid). Bacteria incubated with PPW produced 4.4 g l⁻¹ of butyric acid and 6.1 g l⁻¹ of caproic acid, whereas, bacteria incubated with PSW showed lower amounts of SCFA (1.7 g l⁻¹ of butyric acid) and MCFA (2.5 g l⁻¹ of caproic acid). Clean energy in the form of biohydrogen (H₂) constituted the highest proportion (75%) of the biogas composition.

Trichloroethene (TCE) and cis-1,2-dichloroethene (cDCE) are volatile organic compounds (VOCs) that are ubiquitous in the environment and cause health concerns. In addition, the non-degradable nature of conventional plastics causes many environmental problems. Therefore, it is necessary to identify the bacteria that can degrade the toxic VOCs and convert them into biodegradable plastics. In this study, we used the bacteria Cupriavidus sp. CY-1 to degrade VOCs and produce biodegradable plastics, i.e., poly-β-hydroxybutyrate (PHB). The strain CY-1 showed growth when it was supplemented with TCE or cDCE along with the co-substrates phenol or Tween 80. TCE was co-oxidized and degraded into CO₂, and the phenol degradation produced two metabolites, catechol and the fission products of catechol. TCE or cDCE was converted into PHB by CY-1 when supplemented with phenol. CY-1 showed the highest cell dry mass (CDM) when it was grown with cDCE (0.74 ± 0.09 g L⁻¹) or TCE (0.68 ± 0.04 g L⁻¹) along with phenol and Tween 80. CY-1 showed the highest PHB accumulation (350 ± 15 mg g⁻¹ CDM) when it was incubated with cDCE, phenol and Tween 80. PHB production was calculated based on the bacterial CDM and high-pressure liquid chromatography results. FT-IR, ¹H and ¹³C NMR analyses were conducted to identify PHB. The thermal properties of PHB were confirmed, using thermogravimetric and differential scanning calorimetry analysis. The chemical shifts of PHB produced from VOCs nearly matched with standard PHB; therefore, CY-1 could convert toxic VOCs into valuable chemicals such as PHB.