Valorisation of date fruits by-products for the production of biopolymer polyhydroxybutyrate (PHB) using the bacterial strain Bacillus paramycoides

Zohra Chekroud1*, Leila Djerrab2, Amer Rouabhia3, Mohamed Abdesselem Dems4, Imane Atailia5, Faiçal Djazy2, Mustapha Adnane Smadi6

aResearch Laboratory of Interactions, Biodiversity, Ecosystems and Biotechnology, LRIBEB University of 20th August 1955–Skikda, Algeria;

bLaboratory of Research on the Physical Chemistry of Surfaces and Interfaces LRPCSI University of 20th August 1955- Skikda, Algeria;

cResearch Laboratory of Biological Treatment of Residues, Department of Chemical Engineering and Food Technology, Institute of Vitivinicultural and Agri-food Research (IVAGRO), University of Cadiz, Cadiz, Spain;

dBioinformatics and Biostatistics Unit (BIBSU), National Center for Biotechnology, Constantine, Algeria;

eSuperior School of Food Sciences and Agro Food Industries, El Harrach, Algeria;

fLaboratory of Animals Biotechnologies, National Center for Biotechnology, Constantine, Algeria


The aim of this research was to study the valorization of the date fruit by-product by conversion of date syrup into biopolymer polyhydroxybutyrate (PHB), based on the metabolic capacity of the bacterial strain Bacillus paramycoides to accumulate PHB from date syrup. Total reducing sugars in date syrup was essayed using 3',5-dinitrosalicylic acid (DNS) and HPLC methods. Bacillus paramycoides was isolated from soil of the botanic garden of Skikda university, Algeria. The accumulated PHB was extracted using chloroform. It was quantified as crotonic acid in concentrated sulfuric acid (H2SO4) by spectrophotometry at 300nm. Date syrup is characterized by high levels of total sugars (79.66 g/L) with 31.86 g/L of total reducing sugars. PHB accumulation reached its maximum (104.3 ug/mL) after 96 h of incubation at pH 7 and temperature 37°C using Tryptophane as the nitrogen source and acid pretreated syrup at a concentration of 8%. HPLC analysis on Aminex HPX-87H showed that the produced PHB from date syrup is characterized by a chromatogram peak with a retention time at 22.5 min.

Key words: Bacillus paramycoides, bioprocess, date syrup, Polyhydroxybutyrate (PHB), valorization

*Corresponding Author: Zohra Chekroud, Research Laboratory of Interactions, Biodiversity, Ecosystems and Biotechnology LRIBEB. University of 20th August 1955-Skikda. B.P.26, El Hadeik road 2100 Skikda, Algeria. Email: [email protected]

Received: 20 May 2022; Accepted: 27 July 2022; Published: 23 August 2022

DOI: 10.15586/ijfs.v34i3.2236

© 2022 Codon Publications
This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International (CC BY-NC-SA 4.0). License (


Date palm Phoenix dactylifera is a tropical and subtropical tree (Chandrasekaran and Bahkali, 2013). It is the main crop of Algerian Saharan agriculture with an average production estimated at 420.290 million tons (Bouguedoura et al., 2015). This production is however accompanied by a substantial loss of large amounts of date during the postharvest processes (Abbés et al., 2011; Nancib et al., 2015). Due to their soft texture, the lost dates known as date by-products are not edible and are often discarded (Chandrasekaran and Bahkali, 2013). They are mainly used for animal feed (Majzoobi et al., 2020). Fermentation processes employing microoganisms is the most common method for biosynthesis of value products using date products and wastes as raw materials. Many eco-friendly products could be derived from date by-products such as organic acids, enzymes, amino acids, biomass (Chandrasekaran and Bahkali, 2013), bioethanol (Ahmad et al., 2021), and biopolymers. Omar et al. (2001) used date molasses for the production of Polyhydroxybutyrate (PHB) using the bacterial strain Bacillus megaterium.

Hence, petrochemical polymers led to severe crisis of the environment with detrimental impact on the ecosystems (Mohapatra et al., 2020), and there has been an increasing demand to produce eco-friendly biopolymers synthesized by microbes and plants from inexpensive and renewable sources (Narayanan et al., 2020) like agro-food wastes. PHB has attracted much attention in recent years due to its varied properties (thermoplastic and elastomeric), bio-compatibility, and biodegradability (Keshavarz and Roy, 2010). It was reported that PHB could be synthesized by many Gram-positive and Gram-negative bacteria as intra-cellular carbon and energy reserve material under nitrogen and phosphorus limiting conditions and surplus of carbon source (Anderson and Dawes, 1990). The main constraint in PHB production is accounted to the cost of raw materials, thus the use of cheap carbon sources like agro food wastes cloud be highly significant (Singh et al., 2013).

The goal of this research was to study a bioprocess technology for the valorization of date fruit by-products to a biodegradable biopolymer and to provide an alternative solution for non renewable fossil resources. The bioprocess is based on the capacity of the bacterial strain Bacillus paramycoides to accumulate PHB from date syrup.

Material and methods

Preparation of date syrup

Date fruits of the variety “Deglet Nour” with poor commercial quality were collected during the month of September 2020, from a private factory in the South of Algeria specialized in the exportation of dates. The fruits were pitted, washed with distilled water, and cut into small pieces. They were added to hot water at a ratio of 1/2.5 (weight/volume) (Chniti et al., 2017). The obtained juice was filtered through a gauze and hand pressed. The juice was then boiled at 70°C for 30 min (El-Nagga and Abd El-tawab, 2012) until obtaining a concentrated thick, dark syrup. The final crude syrup was stored in sterilized dark bottles at room temperature.

Treatment of date syrup

The obtained crude syrup underwent two types of treatment: centrifugation and hydrolytic treatment. A quantity of 15g of syrup was weighed and diluted in 100 mL of sterile distilled water (Ashraf et al., 2015). The obtained solution was centrifuged at 3500 g for 15 min and the supernatant was recovered and filtered using Whatman filter paper no.1. For hydrolytic treatment, the obtained filtered supernatant was hydrolyzed by adding 5 mL of 5M or 1.5 N sulfuric acid or 3M HCl to 100 mL of the solution. The whole concoction was incubated at 90°C in water bath for 1h. Date syrup was then centrifuged at 3000 g for 15 min (Kundu et al., 1984) to eliminate the formed debris. Finally, pH was adjusted to 7 using 1M NaOH. At the end of the treatment two phases were formed; a clear black supernatant and a brown pellet. The supernatant was recovered to be used later. Crude, centrifuged, and acid hydrolyzed syrups were sterilized by tyndallization to avoid thermal degradation of sugars.

Physico-chemical characteristics of date syrup

pH, alkalinity, and total solids of the obtained syrup were determined according to standard methods (Tallon et al., 2005). pH was measured directly using a calibrated pH meter (Crison GLP21) after agitation of the sample. The alkalinity was determined by diluting 1mL of the sample into 50 mL of distilled water. The solution was then titrated by H2SO4 until the pH 4.3. Total solids were measured after drying the syrup at 110 ± 5°C. Total sugars (T Sug) with sucrose and reducing sugars were essayed by an HPLC equipped with a refractive index detector and a Shodex column (SH1011, 8.0 × 300 nm). Total reducing sugars (TRS) after date syrup acid treatment was essayed using 3',5-dinitrosalicylic acid (DNS) method (Gusakov et al., 2011). A volume of 25 uL of DNS was firstly added to 25 uL of the diluted sample (20 uL of sample with 925 uL distilled water). The obtained solution was heated for 10 min at 105ºC and cooled for 5 min. Distilled water (250 uL) was added to the cold solution and absorbance was read at 540nm against a blank of distilled water. The concentration of reducing sugars was determined from a calibration curve previously prepared from different concentrations of glucose (0.25–5 g/L).

Primary screening of the bacterial strains producing PHB

The bacterial strain producing PHB from date syrup was isolated from the soil of the botanic garden of Skikda university, Algeria. Pure bacterial colonies isolated from 1 g of soil using the technique of serial dilutions were inoculated on mineral salt medium (MSM) agar medium (Sharma et al., 2007) added with 2% glucose as carbon source and incubated at 37°C for 48h. Colonies having the capacity to grow on the MSM agar medium with glucose were added to ethanolic solution with 3% Sudan Black B (C29H24N6 224-087 segma) for 30 min and those giving dark blue color were considered as positive for PHB production (Mohd Zahari et al., 2012).

Secondary screening of the bacterial strains producing PHB

To select the best strain producing PHB, the colonies showing positive Sudan black staining were re-cultured in MSM liquid medium (Sharma et al., 2007) with 2% of glucose. A bacterial inoculum of each strain was prepared by inoculating a loop of the bacterial colony into 100 mL of nutrient broth in 250 mL conical flasks. The inoculated flasks were incubated for 24 h at 37°C with an agitation rate of 150rpm. Cells were then collected by centrifugation at 10,000 g for 15 min at 4°C, washed aseptically by sterile distilled water, and resuspended in 250 mL Erlenmeyer flasks containing 100 mL of liquid MSM mineral medium added with 2% glucose and incubated at 37°C for 72h. The strain showing the best yield of PHB accumulation was chosen to test its capacity to accumulate PHB using 2% of centrifuged date syrup instead of 2% glucose in MSM solid medium. Sudan black staining was used to confirm PHB production from date syrup.

The produced PHB bacterial strain from date syrup was then identified on the basis of its 16srRNA partial sequence by comparing consensus sequences to a database library of known 16srRNA gene sequences in GenBank ( by multiple sequence alignment.

Optimization of physico-chemical PHB production conditions

To test the effect of physico-chemical factors on PHB production and bacterial cell growth, a bacterial inoculum was firstly prepared as previously described. Bacterial cells were recovered by centrifugation at 10,000 g for 15 min at 4°C and washed aseptically with sterile distilled water. They were resuspended in MSM liquid medium with 2% of centrifuged date syrup. pH of the medium was adjusted to 7 and the cultures were incubated in a rotary incubator at 37°C and an agitation rate of 150rpm during 24, 48, 72, and 96 h. The effect of aeration was tested by augmenting the agitation rate from 150 rpm to 200 rpm and 300 rpm. The pH was tested at 3, 7, and 8 by adding NaOH (2N) or HCl (2N). The cells growth and bioaccumulation of PHB from date syrup were also tested at 30°C, 37°C, and 44°C. The nitrogen source in the MSM medium (NH4Cl) was substituted by ammonium sulfate, yeast extract, beef extract, and peptone, one at a time. The effect of syrup treatment was tested by replacing centrifuged date syrup with crude syrup and acid hydro-lyzed syrup with 5M and 1.5 N sulfuric acid or with 3M HCl. Finally, the effect of syrup concentration was tested by adding 2, 4, 6, and 8% of 5M H2SO4 hydrolyzed date syrup to the medium, one at a time. The experiments were conducted in triplicates.

Extraction of PHB from bacterial cells

The technique of boiling chloroform was used. The bacterial cells accumulating PHB under different conditions were centrifuged at 4000 g for 10 min. The cells were resuspended in an equivalent amount of 4% NaCl and incubated for 1 h at 37°C. The cells pellet was washed with acetone, ethanol, and distilled sterile water to eliminate undesirable elements. The solution was recentrifuged and the supernatant was discarded. The polymer granules were dissolved in boiling chloroform, which was then allowed to evaporate (Adwitiya et al., 2009) to obtain pure PHB.

Quantification of PHB

The extracted PHB was quantified using an UV spec-trophotometric analysis. The chloroform extracted PHB was converted to Crotonic acid. Ten milliliters of concentrated sulfuric acid (98%) was added to the chloroform dissolved PHB for 15 min. The solution was left for cooling. PHB was determined quantitatively as crotonic acid by measuring the absorbance at 300 nm in a UV spectrophotometer using H2SO4 solution as blank. Standard solution of pure crotonic acid was prepared at different concentrations (0.1–2.0 ug/mL). The quantity of accumulated PHB expressed as microgram per milliliter of bacterial cells (ug/mL) was measured by comparing the absorbance of PHB converted to crotonic acid with the absorbance of the standard pure crotonic acid concentrations (Elsayed et al., 2013).

Cells growth

Ten milliliters of the culture medium containing bacterial cells accumulating PHB was centrifuged at 10,000 g for 10 min. The supernatant was discarded and the bacterial pellet was washed twice with sterile distilled water. The cells pellet was then scrubbed to a weighing pan and dried at 100°C for 48h. The dried cells were diluted to an appropriate concentration and the absorbance was measured at 600nm. Cells’ dry matter was determined according to a standard curve (Naheed et al., 2012) previously generated from cells dry matter concentrations (0.1–1ug/mL). It was expressed as CDW ug/mL. The yield of PHB accumulation was calculated as percentage of PHB content (ug/mL) per cells dry matter (ug/mL).

Identification of PHB using Aminex HPX-85X. HPLC analysis

To confirm the synthesis of PHB polymers, samples containing PHB were analyzed by using an HPLC (LaChrom Elite VWR-Hitachi). Samples were eluted with 0.014 N H2SO4 at a flow rate of 0.7 mL min-1 from an Aminex HPX-87H ion exclusion organic acid analysis column (C18 4.6x250) (Torrance, CA, USA) preceded by an ion exclusion guard column of Aminex HPX-85X. HPLC. The produced PHB was measured as crotonic acid dissolved in concentrated H2SO4. Crotonic acid and pure PHB digested into crotonic acid using concentrated H2SO4 were used as standards.

Statistical analysis

All the experiments were conducted in triplicates. The results expressed as mean ± standard error were analyzed by one way ANOVA analysis of variance (one-way ANOVA), followed by pairwise comparisons using the Fisher’s Least Significant Difference (LSD) post hoc test. The statistical significance was considered at P < 0.05. Data analysis was carried out using Statistica 10 software (StatSoft, Inc.).


Date syrup characterization

Date syrup extracted from “Deglet Nour” was characterized by high levels of total sugars (T Sug) (79.66g/L) and a total reducing sugars (TRS) rate of 31.86 g/L (Table 1). TRS rate increased to 78.86 g/L,72.96g/L, and 72.2 g/L after treatment of date syrup with 5MH2SO4, 3MHCL, and 1.5 M H2SO4, respectively (Table 1).

Table 1. Physico-chemical characteristics of date syrup.

PH TS(g/k) TS% Alkalinity g/L T Sug content Sucrose content TRS content before date syrup acid treatment TRS content after date syrup acid treatment (g/L)
Date syrup (g/L) (g/L) (g/L) 5M H2SO4 3M HCl 1.5 H2SO4
5.63 713.8 79 15.8 79.66 47.8 31.86 78.86 72.96 72.2

TS: total solids; TS%: percentage of total solids; T Sug: total sugars; TRS: total reducing sugars before date syrup acid pretreatment; 5M H2SO4: total reducing sugars after date syrup pretreatment with 5M H2SO4 ; 3M HCL: total reducing sugars after date syrup pretreatment with 3M HCL, 1.5M H2SO4: total reducing sugars after date syrup pretreatment with 1.5 M H2SO4

Screening of the bacterium producing PHB from date syrup

A total of 20 bacterial strains were isolated from the soil of the botanic garden of Skikda university. The preliminary screening of PHB producing strains was further identified by a Sudan black method. Nine isolates (BG1-BG9) showed a black-blue color when stained with Sudan Black B (Figure 1), which indicates that they are positive PHB producing. The quantitative screening in MSM liquid medium with 2% of glucose as carbon source revealed that the highest quantity of PHB was accumulated by the strain BG5 (95.67 ug/mL) followed by the strain BG2 (83.92 ug/mL) after 72h of incubation at 37°C (Figure 2). The strain showing the highest level of PHB accumulation was tested for its ability to accumulate PHB from date syrup instead of glucose. Sudan black staining confirmed the capacity of the strain BG5 to produce PHB from date syrup.

Figure 1. Screening of some bacterial isolates on MSM solid medium with date syrup as carbon source using Sudan Black staining. (A) Before staining, (B) After staining (green-blue colonies). 1:BG1; 2:BG2; 3:BG3; 4:BG4; 5:BG5; 6:BG6; 7:BG7;8:BG8; 9:BG9; BG1-BG9: the nine isolated bacterial strains showing positive PHB production using glucose as carbon source.

Figure 2. PHB content in the nine isolated bacterial strains. Results are expressed as mean of tri-replicates±standard error. BG1, BG2, BG3, BG4, BG5, BG6, BG7, BG8, and BG9: the nine isolated bacterial strains showing positive PHB productionusing glucose as carbon source.

BG5 was identified on the basis of its partial 16srRNA sequencing by comparing consensus sequences to a database library of known 16srRNA gene sequences in GenBank ( Blast.cgi). Analysis showed high identity of BG5 with Bacillus paramycoides MCCC 1A04098, which has a partial 16s rRNA sequence NCBI assessing number NR_157734.1.

Effect of incubation period on PHB production

The results demonstrated in Figure 3 revealed that PHB yield was directly proportional with the incubation period. It increased gradually from 22.18% after 24h of incubation to 26.36% after 72h with a slight increase after 96h (26.65%), where it reached its maximum with a maximum PHB accumulation (64.14 ug/mL) and a maximum cell growth (240.69 ug/mL).

Figure 3. The effect of the incubation period on PHB accumulation. Results are calculated as mean of tri-triplicates ± standard error; different letters indicate significant differences using ANOVA test followed by the post-hoc Fisher test (LSD) at P < 0.05. P = 0.000 for both PHB and CDW.

Effect of aeration rate

The aeration was monitored by agitation of the bacterial cultures. Our results (Figure 4) revealed that the PHB yield increased with the increase in the agitation rate from 150 rpm to 300 rpm. The maximum yield (36.22%) with a maximum PHB production (80.17 ug/mL) and a maximum cell growth (221.35 ug/mL) were recorded at 300 rpm.

Figure 4. Effect of aeration on PHB accumulation. Results are calculated as mean of tri-triplicates ± standard error; different letters indicate significant differences using ANOVA test followed by the post-hoc Fisher test (LSD) at P < 0.05. P = 0.000 for PHB content, P = 0.001 for CDW.

Effect of pH

The rate of PHB production was tested in acid (pH 3), alkaline (pH 8), and neutral (pH 7) media. It was observed that the best rate of PHB accumulation (27.61%) was obtained at pH 7 with a PHB quantity of 75.66 ug/mL and a biomass of 274 ug/mL. It decreased significantly beyond pH 7 (Figure 5).

Figure 5. Effect of pH on PHB accumulation. Results are calculated as mean of tri-triplicates ± standard error; different letters indicate significant differences using ANOVA test followed by the post-hoc Fisher test (LSD) at P < 0.05. P = 0.0001 for both PHB content and CDW.

Effect of temperature

PHB synthesis increased from 21.67% at 30°C to 28.39% at 37°C. It decreased significantly at 44°C (9.40%). Maximum PHB quantity and maximum cell biomass were recorded at 37°C (76.85 ug/mL and 270.67 ug/mL, respectively) (Figure 6).

Figure 6. Effect of temperature on PHB accumulation. Results are calculated as mean of tri-triplicates ± standard error; different letters indicate significant differences using ANOVA test followed by the post-hoc Fisher test (LSD) at P < 0.05. P = 0.0001 for both PHB content and CDW.

Effect of various nitrogen sources

Using tryptophane as a nitrogen source with date syrup gave the best PHB content (63.13 ug/mL) and cells biomass (226.33 ug/mL) followed by peptone (50.61 ug/mL PHB and 175.33 ug/mL CDW). There was, however, a decrease in cells biomass growth and PHB accumulation when we used inorganic nitrogen source (NH4)2SO4 (44.25 ug/mL PHB and 153.67 ug/mL CDW) (Figure 7).

Figure 7. Effect of nitrogen sources on PHB accumulation. Results are calculated as mean of tri-triplicates ± standard error; different letters indicate significant differences using ANOVA test followed by the post-hoc Fisher test (LSD) at P < 0.05. P = 0.000 for both PHB content and CDW.

Effect of date syrup treatment

The effect of crude, centrifuged, and hydrolyzed date syrup was tested. It was noted that hydrolyzed date syrup with 5MH2SO4 gave the best yield of PHB production (34.32%) with maximum PHB content (89.35 ug/mL) and maximum cells growth (260.33 ug/mL). There was however a significant decrease in PHB yield with crude syrup (11.06%) where the PHB content decreased to 27.68 ug/mL and the cells growth to 250.33 ug/mL (Figure 8).

Figure 8. Effect of date syrup treatment on PHB accumulation. Results are calculated as mean of tri-triplicates ± standard error; different letters indicate significant differences using ANOVA test followed by the post-hoc Fisher test (LSD) at P < 0.05. P = 0.000 for both PHB content and CDW.

Effect of date syrup concentration

The effect of syrup concentration on PHB accumulation by Bacillus paramycoides is demonstrated in Figure 9. The PHB yield was directly proportional with the concentration of date syrup. It increased from 26.06% at 2% of date syrup to 32.62% at 8%. The increase of PHB yield was accompanied with an increase of the PHB content and cell growth. We obtained 104.3 ug/mL of PHB and 319.73 ug/mL of cells’ dry matter at 8% of syrup.

Figure 9. Effect of date syrup concentration on PHB accumulation. Results are calculated as mean of tri-triplicates ± standard error; different letters indicate significant differences using ANOVA test followed by the post-hoc Fisher test (LSD) at P < 0.05. P = 0.000 for both PHB content and CDW.

The physico-chemical conditions were monitored in triplicates. Results were expressed as mean± standard error. Variance analysis (ANOVA) showed that there was a very high significant difference between all the conditions (Pr < 0.05). Statistically, the multiple comparisons of the means performed with the LSD post hoc test indicated that the highest amount of PHB production was obtained after 96h of incubation at 37°C and pH 7, using tryptophane as nitrogen source and 8% of hydrolyzed syrup by 5M H2SO4 as the carbon source.

HPLC analysis of produced PHB

The PHB produced by Bacillus paramycoides using date syrup was identified by an HPLC on Aminex HPX-87H technique. The obtained chromatograms of re-crystallized crotonic acid (Figure 10A), pure PHB converted to crotonic acid (Figure 10B), and PHB produced by Bacillus paramycoides from date syrup converted to crotonic acid (Figure 10C) revealed that the three chromatograms share the same peak with a retention time of 22.5 min. This confirms the synthesis of PHB by Bacillus paramycoides using date syrup as the carbon source.

Figure 10. Aminex-HPLC analysis of PHB. (A) Standard crotonic acid (with retention time of 22.5min), (B) Standard pure PHB (with retention time of 22.5 min), (C) produced PHB from date syrup (with retention time of 22.5min).


The agro-food industry produces large amounts of wastes. Many strategies have been established in Europe and other countries of the world for the valorization of food waste streams and the recovery of biomolecules (Baiano, 2014). In this study, an isolated bacterial strain from the botanic garden of Skikda university (BG5) showing the best PHB accumulation using 2% of glucose (95.67 ug/mL) (Figure 2) was confirmed by Sudan black staining to accumulate PHB using 2% of date syrup, and it was identified as Bacillus paramycoides. Many bacterial strains have been confirmed to accumulate PHB from agro wastes like date syrup (Mostafa et al., 2020), banana peel, sugarcane bagasse, corn cob, and teff (Eragrostistef) straw (Getachew and Woldesenbet, 2016). A wide range of PHB producer Bacillus species are recorded in the literature (Mohapatra et al., 2017). Bacillus pumilus, Bacillus megaterium, and Bacillus subtilis have been reported to produce PHB from agro-food wastes (Singh et al., 2013; Vu et al., 2021; Werlang et al., 2021). The bacterial aptitude for utilizing complex carbon sources like agro-food wastes varies according to the material biochemical composition and the enzymes involved by the bacterium (Belal, 2013). The date syrup used in our approach contains high levels of sugars (79.66 g/L) with 31.86 g/L of reducing sugars (Table 1), which makes it a promising source for PHB production since bacteria accumulates PHB under nutrient stress conditions and surplus of carbon source (Blunt et al., 2018).

The optimization of fermentation parameters is among the strategies now used to improve PHB production (Gurieff and Lant, 2007). The yield of PHB accumulation reached its maximum after 96h of incubation (26.65%). The best incubation period for PHB accumulation depends on the bacterial strain. The increase in the PHB content (64.14 ug/mL) and cells growth (240.7 ug/mL) at this incubation period (Figure 3) is in accordance with the results recorded by Gomaa (2014) and Mostafa et al. (2020). Some authors however reported that the best incubation periods are 48h (Thapa et al., 2018) and 72 h (Singh et al., 2013).

Agitation is an important factor for PHB production. It helps in mixing the oxygen, the heat and the nutrients and facilitates the distribution of air in the nutrient broth so that it increases the liquid-gaz contact area (Mantzouridou et al., 2002). This research revealed that increasing the agitation rate to 300 rpm increased the cells growth (221.35 ug/mL) and the PHB content (80.17 ug/mL) (Figure 4). Slow agitation rate leads to an increase in the viscosity of the culture medium and a reduction of the mass transfer (Bandaiphet and Prasertsan, 2006).

pH of the medium is a crucial factor for the activity of the polymerase enzyme responsible of PHB production (Bhagowati et al., 2015). It is clear that pH 7 is more favorable for PHB production where optimum PHB accumulation (75.66 ug/mL) and optimum cells growth (274 ug/mL) were obtained (Figure 5). Many authors have reported that pH 7 is the best pH for PHB accumulation using different bacterial strains (Mostafa et al., 2020; Singh et al., 2013). PHB synthesis decreased at acidic and alkaline pH.

The incubation temperature had a significant effect on PHB synthesis; this is due to the polymerase enzyme involved in the PHB polymerization, highly affected by the temperature changes—mainly the high temperatures (Getachew and Woldesenbet, 2016). The maximum PHB yield with a maximum cell growth and PHB accumulation were achieved at 37°C (Figure 6). Aly et al. (2013) reported the same results, using the bacterial strain Bacillus cereus. Singh et al. (2013), however, recorded that maximum PHB production by Bacillus subtilis was obtained at 40°C.

The nitrogen source affects PHB accumulation and bacterial cells growth. Bacteria accumulate PHB under stress conditions of nitrogen (Hungund et al., 2013). In our study, organic sources mainly tryptophane enhanced PHB accumulation by Bacillus paramycoidesand cells growth (63.31 ug/mL and 226.33 ug/mL, respectively) in comparison with inorganic sources (NH4)2SO4 (Figure 7). This may be explained by the fact that organic sources are considered as precursors of amino acids and bacterial growth factors (Patel et al., 2017). Ammonium sulfate was reported by Kritika et al. (2016) and Singh et al. (2013) to be the best nitrogen source for PHB accumulation

High contents of PHB (89.35 ug/mL) and cells biomass (260.33 ug/mL) were achieved using acid hydrolyzed date syrup in comparison with centrifuged and crude syrup (Figure 8). This is due to the low levels of simple sugars in non treated syrup in comparison with acid-treated syrup (McAdam et al., 2020). Sucrose hydrolysis increased with the increase in sulfuric acid concentration (Sen et al., 2019). The total rate of reducing sugars increased from 31.86 g/L to 78.86 g/L, 72.96 g/L, and 72.2 g/L after treatment of date syrup with 5MH2SO4, 3MHCL, and 1.5 M H2SO4, respectively (Table 1). The same results were reported by Gomaa (2014) where acid preatreated molasses enhanced PHB accumulation in comparison with untreated molasses.

The carbon source is the major factor affecting PHB production costs (Aljuraifani et al., 2019; Gomaa, 2014). PHB production and cells growth are directly proportional to the syrup concentration (Figure 9). They reached their maximums at 8% (104.3 ug/mL and 319.73 ug/mL, respectively). This is due to the high content of sugar in concentrated date syrup. The use of the renewable resources depends on the nature of the complex material and the hydrolytic capacity of the bacterium enzymes (Belal, 2013). Gabr (2018) revealed that the best levels of PHB were accumulated by Bacillus sp. at 8% of date syrup. Therefore, the selection of an economically cost-effective carbon source is the key determining the final product market costs (McAdam et al., 2020). Date syrup by-products are abundant and inexpensive sources for PHB production that may decrease the costs of bioplastic production.

PHB is traditionally identified using HPLC on Aminex HPX-87H technique (Karr et al., 1983). The technique is based on the conversion of PHB into crotonic acid by concentrated sulfiric acid. Crotonic acid is measured in samples containing from 0.01 to 14 ug of PHB. Crotonic acid and pure PHB were used as standards. Chromatograms with the same peaks at the retention time 22.5 min were obtained (Figure 10), with cro-tonic acid, pure PHB, and produced PHB using Bacillus paramycoides and date syrup as carbon source which confirms the conversion of date syrup into PHB by Bacillus paramycoides.


Date syrup, which is an inexpensive by-product, is a promising source for the production of PHB biopolymer. The latest is successefully produced using a bacterial strain isolated from soil, Bacillus paramycoides, and date syrup as carbon source. The best contents of PHB were reached after 96 h of incubation at 37°C, an agitation rate of 300 rpm, and pH 7 using acid preatreted date syrup at a concentration of 8%. Furthemore, more investigations are required to characterize the PHB chemical structure to determine the possibility of producing PHB at industrial levels from date syrup.


The authors are grateful to Mr Nasser ZAYDI, the chief of the hygiene laboratory of the state of Skikda, the engineers of the laboratory of animals biotechnologies, National Center for Biotechnology, Constantine, Algeria, Pr. Luis Isidoro Romero GARCIA, Dr. Kaoutar ABOUDI, and miss Hind ZINE EL ABIDINE from the faculty of Sciences, the university of Cadiz , Spain.

Authors contribution

All the authors contributed in the conception and design of the study. Bacterial isolation and PHB optimization conditions were performed by Leila Djerrab and Zohra CHEKROUD. Amer Rouabhia conducted HPLC analysis. Mohamed Abdesselem DEMS realized the statistical analysis. Mustapha Adnane SMADI provided necessary material and helped in realizing spectrophotometric analysis. Imane Attailia helped in the manuscript redaction. Faiçal Djazy provided some chemical products. The first draft of the manuscript was written by Zohra CHEKROUD and Leila DJERRAB. All the authors read and approved the final manuscript.

Conflict of interest

There is no conflict of interest to declare.


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