Edited by: Guadalupe Virginia Nevárez-Moorillón, Autonomous University of Chihuahua, Mexico
Reviewed by: Nicolas Oscar Soto-Cruz, Durango Institute of Technology, Mexico; Yejun Han, University of Chinese Academy of Sciences, China
This article was submitted to Sustainable Food Processing, a section of the journal Frontiers in Sustainable Food Systems
This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
Pollution resulting from the persistence of plastics in the environment has driven the development of substitutes for these materials through fermentation processes using agro-industrial wastes. Polyhydroxybutyrate (PHB) is a rapidly biodegradable material with chemical and mechanical properties comparable to those of some petroleum-derived plastics. PHB accumulates intracellularly as an energy reserve in a wide variety of microorganisms exposed to nutritionally imbalanced media. The objective of this study was to evaluate the use of a banana waste product as a carbon source for PHB production. PHB was extracted by acid methanolysis and detected by gas chromatography-mass spectrometry. Eleven bacterial strains with potential for PHB production were evaluated by
Biotechnological alternatives to conventional plastics are increasingly important in industry worldwide (Sirohi et al.,
Polyhydroxybutyrate (PHB), the most common biodegradable homopolymer of polyhydroxyalcanoates (PHA) (Maity et al.,
The major limitation for commercial use of biopolymers is the high cost of production (Li et al.,
In 2019, Costa Rica exported 120 million boxes of bananas (18.14 kg/box), which reached markets such as the European Union and the United States. The banana industry estimated 328,303 tons of fruit are rejected annually in packing plants (CORBANA,
The objective of this research was to use banana pulp juice, an agroindustrial by-product as the carbon source for PHB production in a submerged fed-batch fermentation.
The banana by-product, obtained from a pulp production process, was supplied by Compañía Mundimar (Limón, Costa Rica) and consisted of a puree with a high seed content. The material was subjected to enzymatic maceration with Crystalzyme® PMLX (White Labs) to increase the content of fermentable sugars. A pneumatic press was then used to obtain a juice from the banana purée (JBP) with a final concentration of 20° Brix and pH 3.6. Sugar content in JBP was measured by HPLC (Agilent 1260 Infinity). The instrument was equipped with a refractive index detector (RID) (Sullivan and Carpenter,
Eleven native isolates were provided by the Center for Research in Cellular and Molecular Biology of the Universidad de Costa Rica (CIBCM-UCR). These strains are part of a collection generated from samplings in different places in Costa Rica and have been obtained for various research projects of the CIBCM. The strains used in this study were selected from the positive reaction to Nile blue staining, a lipophilic fluorescent dye used to visualize hydrophobic cell structures such as membranes or lipid-like inclusions, like PHB (Juengert et al.,
Two mL of cultures were centrifuged at 10,600 rcf for 10 min. The cell pellets were resuspended in 2 mL of distilled water and transferred to 10 mL headspace vials. A volume of 6 mL of an 85:15 mixture of methanol:sulfuric acid (Dalal et al.,
Methyl (R)-3 hydroxybutyrate (3-MHB) (Sigma®, CAS: 3976-69-0) was used as a standard for PHB quantification (Cavalheiro et al.,
Fermentations were carried out in two stages: a growth stage and a PHB production stage with limited nitrogen (Amirul et al.,
Nitrogen (NH4Cl) and carbon (JBP) concentrations in culture media for
JBP percentages and NH4Cl concentrations evaluated by a central composite design for
1 | 0 | 12.5 | 3.5 |
2 | –+ | 5 | 5 |
3 | A0 | 23 | 3.5 |
4 | 0a | 12.5 | 1.4 |
5 | –– | 5 | 2 |
6 | a0 | 1.9 | 3.5 |
7 | 0A | 12.5 | 5.6 |
8 | +– | 20 | 2 |
9 | 0 | 12.5 | 3.5 |
10 | ++ | 20 | 5 |
11 | 0 | 12.5 | 3.5 |
12 | 0 | 12.5 | 3.5 |
For each experiment, 50 mL of culture broth supplemented with the test concentrations of JBP and NH4Cl were prepared in 250 mL erlenmeyer flasks. Besides the test concentrations of NH4Cl and JBP, the media contained 5 g/L NaCl and 2.5 g/L KH2PO4. For each broth formulation, JSB, salts solution and water were combined. pH was adjusted to 7.1 with 1 M NaOH. NH4Cl solution was autoclaved separately and aseptically added to each flask.
Each flask was inoculated with 1 mL of cryopreserved bacterial suspension and incubated at 30°C with 150 rpm agitation for 24 h. The optical density of the culture was measured at the end of the incubation.
A fed-batch fermentation process was performed in triplicate using the
Fermentation parameters were controlled with the ez-Control console and BioXpert® XP software (Applikon® Biotechnology). Culture conditions were: temperature 30°C, agitation 350 rpm, pH 7 adjusted with 2 M NaOH and 100% air saturation at a flow of 1 vvm. The biomass production stage was carried out over 24 h, and was followed by the PHB production stage, initiated by the addition of a fructose pulse (500 g/L), at an agitation speed of 500 rpm. In this stage, the effect of the initial concentration of fructose (30, 40 or 50 g/L) on PHB production was evaluated. Data were analyzed by one-way ANOVA using the program JMP 8 (SAS Institute Inc.). Samples were collected every 24 h; the concentration of PHB was determined by GC-MS, and sugar content was measured by HPLC (Agilent 1260 Infinity). The instrument was equipped with a refractive index detector (RID). A Zorbax Carbohydrate column with dimensions: 5 μm × 150 mm × 4.6 mm was used for the analysis. The following analytical conditions were applied: flow: 1.2 mL/min; oven and detector temperature; 30°C and injection volume: 5 μL Nitrogen concentration was determined by Rapid N- Exceed® Combustion Method according to AOAC International (
Enzymatic treatment of banana agroindustrial by-product produced a viscous concentrated juice. Chemical characterization showed values for fermentable sugars and minerals that can be used by the microorganism during fermentation. The ratio of sucrose, glucose and fructose in the JBP was 1:3.7:3.6, respectively. The nitrogen content was 0.056/100 g (
Chemical composition of banana by-product juice (JBP).
Sucrose (g/100 g) | 1.88 ± 0.04 |
Glucose (g/100 g) | 6.97 ± 0.45 |
Fructose (g/100 g) | 6.75 ± 0.43 |
Nitrogen (g/100 g) | 0.056 ± 0.001 |
Calcium (mg/100 g) | 11.62 ± 0.39 |
Sodium (mg/100 g) | 0.64 ± 0.04 |
Potassium (mg/100 g) | 214.48 ± 6.3 |
Iron (mg/100 g) | 0.07 ± 0.002 |
Magnesium (mg/kg) | 305.76 ± 2.02 |
Phosphorous (mg/kg) | 258.85 ± 20.95 |
Sulfur (g/100 g) | 1.38 ± 0.01 |
PHB production in native strains was not quantifiable (Measured PHB concentrations stood between detection and quantification limits defined for the method), except for CR-12 which showed a concentration of 0.5 g PHB/L culture broth. The control strain produced 2.82 g/L of culture broth. According to ANOVA, significant differences (
The effects of JBP (
Sorted parameter estimates for the response surface analysis: effect of JBP and NH4Cl concentrations in culture medium and their interaction in
Effect of different JBP and NH4Cl supplementation levels in growth medium on of
Optical Density at 425 nm of
+– | 20 | 2 | 0.579 |
0 | 12.5 | 3.5 | 1.731 |
– – | 5 | 2 | 4.300 |
++ | 20 | 5 | 0.254 |
– – | 5 | 2 | 4.158 |
0 | 12.5 | 3.5 | 2.078 |
–+ | 5 | 5 | 3.515 |
0 | 12.5 | 3.5 | 1.896 |
+– | 20 | 2 | 0.600 |
++ | 20 | 5 | 0.273 |
0 | 12.5 | 3.5 | 2.072 |
– – | 5 | 2 | 3.983 |
–+ | 5 | 5 | 3.568 |
++ | 20 | 5 | 0.304 |
+– | 20 | 2 | 0.572 |
–+ | 5 | 5 | 3.403 |
0 | 12.5 | 3.5 | 2.135 |
0 | 12.5 | 3.5 | 2.195 |
Dissolved oxygen (DO) saturation was below 15% during the first stage of exponential growth, while levels of 20–40% were observed in the second stage of the process. Oxygen levels remained near zero during the first hours of fermentation. At the beginning of the fermentations, the fructose concentration was 2 g/L. After 25 h of culture, this value reached 50 g/L due to the fructose pulse added to initiate the PHB production stage. The sugar content then decreased and was completely depleted by the time the process reached 72 h. Little change in nitrogen concentration was observed at the end of the growth phase of the microorganism (
Fructose and nitrogen levels during fermentation supplemented with 50 g/L fructose
A biomass concentration of 24.42 ± 2.25 g/L was obtained at the end of the first fermentation stage. For fermentations where 30 and 40 g/L fructose were tested, PHB production was not detectable at any of the sampling times (24, 48, 72, and 96 h). A maximum PHB production of 1.3 g/L was reached after 96 h of fermentation for the 50 g/L fructose assay. In such experiments, C/N ratio was roughly 10, while C/N ratio obtained in fermentations with 30 and 40 g/L fructose was lower than 10.
In addition to methyl (S)-(+)-3-hydroxybutyrate (PHB) signal, various fatty acids and carboxylic acids were also identified (
Compounds present in bioreactor fermentation samples, identified by GC-MS.
Methyl (S)-(+)-3-hydroxybutyrate (PHB) | 5.08 |
Methyl ester of levulinic acid | 6.06 |
Dimethyl ester of succinic acid | 6.25 |
Methyl ester of lauric acid | 8.01 |
Methyl ester of myristic acid | 10.03 |
Glutaric acid, 2-oxo-, dimethyl ester | 10.92 |
Agro-industrial by-products can contain internal structures such as starches, cellulose and complex sugars that are difficult to transform directly by microorganisms into products of interest (Kulkarni et al.,
Regarding the native strains PHB production evaluation, the absence of this polymer in the majority such strains may be related to the individual nutritional requirements of the isolates. The optimization of production media is specific for the microorganism used (Pereira et al.,
The concentrations of 2 g/L NH4Cl and 5% JBP were optimum within the experimental range. However, results of the study indicate that higher biomass production may be obtained by widening the experimental range to include lower concentrations of these two nutrients. For
During fermentation processes implementation for PHB production, oxygen is an important parameter.
In general, fructose is the most utilized carbon source for
In this research, a roughly constant level of nitrogen was maintained; this may have prevented the C/N imbalance necessary for high PHB yields, or PHB synthesis may have been inhibited by the presence of nitrogen (Wang and Yu,
Signals of other compounds have been detected during PHB analysis by GC-MS. Werker et al. (
Biosynthesis of PHAs and fatty acids share the same metabolic route with acetyl-CoA and propionyl-CoA as common intermediates. Thus, both routes compete for precursors, especially in nutrient-limited conditions. The mechanisms to explain this competition have not yet been elucidated, and further research is needed to be able to direct the synthesis process toward the component of interest, obtain high PHA productivities and control precisely the composition of these polymers (Magdouli et al.,
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.
CV-C contributed to obtaining the funds for the study. CV-C and MA-R did the conception and design of the study. MA-R and VM-V executed all the bacterial culture experiments to produce PHB, both by shaker flask and bioreactor, they extracted and determined the concentration of PHB by GC. All authors contributed to manuscript revision, read, and approved the submitted version.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
The authors are thankful to the project collaborators (M. Mora, E. Thompsom, N. Guzman), F. Granados (CINA-UCR), the CITA-UCR chemistry laboratory work team and N. Barboza for her contribution in the revision of this document.