Current literature makes as familiar with the tremendously aggravating global plastic situation, which is reflected by an enormous current annual production of highly recalcitrant plastics of petrochemical origin, which is approaching the tremendous quantity of 0.4 Gt. A possible way out of this predicament is switching to biologically produced, biodegradable polymeric materials with plastic-like properties (1); especially microbial polyhydroxyalkanoates (PHAs) are considered auspicious candidates to fulfill this task (2,3). PHAs are biopolyesters accumulated by various prokaryotic organisms starting from renewable feedstocks; both heterotrophic and autotrophic PHA production is reported (4,5). PHAs can be processed to plastic items by various techniques established in polymer industry, such as melt extrusion, injection molding, compression molding, wet spinning, electrospinning, or 3D-printing (6). Selected examples for PHA application involve (food) packaging (7) or the pharmaceutical and biomedical field (8). Among heterotrophic PHA producers, Gram-negative strains currently outperform their Gram-positive counterparts in terms of scientific reports and (semi)industrial application for PHA production (9,10). Beside extremophilic Gram-negative PHA producers thriving in challenging hot, hyper-saline, alkaline, or heavy metal-contaminated habitats (11,12), also various mesophilic bacteria are described as proficient PHA production factories. Among the myriad of investigated strains, especially those utilizing inexpensive feedstocks, growing fast, and accumulating high intracellular PHA fraction attract most attention for bio-economic industrial-scale PHA production (4,5).
As an example for microbial strains accumulating significant quantities of PHAs, the yellow-pigmented betaproteobacterium
However, achieved results for volumetric PHA productivity and intracellular PHA fraction for
Regarding whey retentate, the successful use of enzymatically hydrolyzed whole whey as substrate in PHA production processes was reported some years ago for
The effect of adding whey retentate was compared with cultivation batches using ammonia sulfate as sole, inorganic nitrogen source, and with setups supplied with additional complex nitrogen sources, namely yeast extract, malt extract, meat extract, corn steep liqueur (CSL), hydrolyzed casein, and pressed juice of grass silage. Whey retentate was particularly selected for this study to complement the excellent performance of the strain in producing PHAs from whey permeate as carbon source (16,17); using both the permeate and the retentate fraction in a PHA production process would pave the way towards a complete utilization of the surplus material whey, which, due to its high (bio)chemical oxygen demand, currently poses environmental threats when disposed as waste into aquatic environments (29). In addition, this new application of whey retentate for PHA biosynthesis can be considered a complementary strategy to other value-added applications of this material in the bioplastic field, such as coating of films made of non-degradable petrochemical plastics to enhance oxygen barrier properties and reduce the plastic´s ecological footprint (31), or to fine tune gas barrier properties of films made of other biodegradable polymers (32).
Chemical composition of milk and different types of whey (based on (33))
Compound | Milk | Sweet | Dry Sweet | Fermented | Dry sweet | Whey | Whey |
---|---|---|---|---|---|---|---|
ca. 4.8 | 4.7–4.9 | 73.5 | 4.5–4.9 | 65.6 | 23 | 14 | |
- | traces | traces | 0.5 | 7 | n.d. | n.d. | |
3.4-3.5 | 0.75–1.1 | 12.9 | 0.45 | 12.3 | 0.75 | 13 | |
>4.2 | 0.15–0.2 | 1.1 | traces | 1.0 | n.d. | 3-4 | |
~0.7 | ca. 0.7 | 8 | 0.6-0.7 | 11 | ca. 2.7 | ca. 0.7 |
n.d.: not detected
Processing of whey towards a feedstock for PHA production (based on (34)).
After this basic experiment, the application of different concentrations of whole whey retentate and acidically hydrolyzed whey retentate were compared in terms of impact on growth kinetics and biomass formation of
Pre-culture: 100 mL of H3 containing hydrolyzed whey permeate (total sugar concentration 10 g/L) was inoculated with 5 mL of the LB cultures (
Sweet whey (composition see Table 1), a by-product of the Asiago DOC cheese production, was provided by the Italian dairy company Latterie Vincentine S.c.a., Italy (LAVI). Separation of whey into whey permeate and whey retentate was accomplished directly at LAVI via ultrafiltration. Lactose in whey permeate was hydrolyzed enzymatically by adding the enzyme formulation Maxilact LG 2000TM (DSM Food Specialities, UK; 2.5 mL per L whey permeate) at a pH of 6.5-7 and a temperature of 37ºC and stirring for 24 hours according to a previously published protocol (35). The whey retentate fraction was subjected towards acidic hydrolysis by adding 6 M HCl (1 mL per g retentate) and continuous magnetic stirring at 90ºC for 24 hours. After cooling and monitoring completeness of hydrolysis, the pH-value of the hydrolysis cocktail was adjusted to 7.0 by adding an adequate quantity of aqueous NaOH solution.
Microbial growth of
Ammonium was measured electrochemically with an ion-sensitive Orion electrode using ammonium sulfate standard solutions (30-3000 ppm) (27).
5 mL of liquid sample broth was centrifuged (4°C, 12,000 g; Megafuge 1.0R Hereus Sepatech) in pre-weighed glass tubes. The filtered supernatant was used for determination of carbon sources (glucose and galactose) and ammonium sulfate. The remaining pellet was frozen and lyophilized (freeze-drying apparatus Christ Alpha 1-4 B) until mass constancy. After weighing, the difference between tubes containing dry cell pellets and corresponding empty tube weights was defined as the cell dry mass (CDM) in 5 mL fermentation broth.
The concentrations of glucose, galactose, and lactose were measured with an HPLC equipment consisting of a thermostated Aminex HPX 87H column, an HP 7673 controller, a JASCO 880-PU intelligent HPLC pump, and a BISCHOFF RI-Detector 8110. The substrates to be analyzed were eluted in isocratic mode with 0.005 M H2SO4 at a rate of 0.60 mL/min. Solutions of pure sugars (glucose, galactose, lactose) and lactic acid were used for external calibration.
The frozen and lyophilized biomass pellets from the cell dry mass determination were used for measuring the PHA concentration in the biomass and PHA´s monomeric composition according to Braunegg´s method (36). Briefly, PHA in the lyophilized biomass pellets was simultaneously extracted by chloroform and transesterificated by acidic methanolysis; hexanoic acid acted as internal standard. The methanolysis cocktail was analyzed using an HP 5890 Series II gas chromatograph (GC) equipped with a 5 m HP 1 capillary pre-column and 30 m HP5 column and a flame ionization detector (FID) for detecting the volatile methyl esters of the PHA building blocks. Helium was used as carrier gas, H2 and synthetic air (oxygen source) as detector gases, and nitrogen as auxiliary gas. The injection volume amounted to 1 μL, the split ratio was 1/10. Pure Biopol™ poly(3HB-
Time patterns of OD (a), CDM (b), and inorganic nitrogen source ammonium sulfate (c).
Increase of both OD and CDM with time demonstrate that most of the complex nitrogen sources tested definitely enhanced growth rate and biomass formation of
Growth rates (
Time | Only inorg. N-source | Yeast extract | Malt extract | Meat extract | CSL | Casein Hydrolysate | Pressed grass silage juice | Whey retentate | |
---|---|---|---|---|---|---|---|---|---|
5-8 h | 0.52 | 1.05 | 0.70 | 0.94 | 0.63 | 0.60 | 0.31 | 0.44 | |
8-11 h | 1.11 | 1.88 | 1.64 | 1.84 | 1.74 | 1.58 | 1.53 | 1.68 | |
5-8 h | 0.22 | 0.34 | 0.26 | 0.31 | 0.20 | 0.24 | 0.09 | 0.16 | |
8-11 h | 0.28 | 0.30 | 0.34 | 0.31 | 0.34 | 0.37 | 0.35 | 0.42 |
Hydrolyzed and not hydrolyzed whey retentate (HWR and NHWR, respectively) were added in different concentrations to a H3 medium containing hydrolyzed whey permeate as carbon source, and ammonium sulfate as inorganic nitrogen source. Hydrolysis was carried out in order to generate amino acids and smaller peptides, which might me more easily accessible by the cells than the whole whey proteins. Hence, the effect of HWR and NHWR on the growth phase of
(a) times curves of OD (420 nm) during the phase of microbial growth (t = 0-15 h); (b): CDM at time 0 h (black bars), 11.75 h (grey bars), and 26.25 h (white bars); A: only inorganic N-source, B: 0.5 g/L HWR, C: 2 g/L HWR, D: 5 g/L HWR, E: 0.5 g/L NHWR, F: 2 g/L NHWR, G: 5 g/L NHWR.
The same conclusion can be made from
Remaining concentrations of ammonium sulfate (a), total sugars (sum glucose + galactose, b), glucose (c), and galactose at time 0 h (black bars), 11.75 h (grey bars), and 26.25 h (white bars). A: only inorganic N-source, B: 0.5 g/L HWR, C: 2 g/L HWR, D: 5 g/L HWR, E: 0.5 g/L NHWR, F: 2 g/L NHWR, G: 5 g/L NHWR.
Concentrations of PHA (a), residual biomass (CDM minus PHA, b), mass fraction
Based on measured OD values,
Growth rates
Mathematical modelling of both growth and product formation phase PHA production process has recently become an emerging topic, helping to better understand and optimize such bioprocesses, which ultimately shortens the way towards industrial realization and helps avoiding technological deadlocks already at a very early stage of process development (39,40). In this context, it is well visible from all time curves illustrated in
a) Dixon plot to determine inhibition constant
For the setups containing NHWR as complex nitrogen source, Haldane and Monod kinetics, which reflect the dependence of μ on substrate concentration, were modelled using the solver function in Microsoft excel program (
μ: specific growth rate; μ
Based on the Haldane model, a
Kinetic constants for whey retentate for
Equation | μ | ||
---|---|---|---|
Haldane | 0.18 | 0.46 | 14.98 |
Monod | 0.11 | 0.42 | - |
With all complex nitrogen sources (except malt extract, which may contain inhibiting compounds (37))
As discussed above, when whey permeate is used as carbon source, it would be convenient to use also the whey retentate fraction as nitrogen source. In an optimized scenario, PHA will be produced in the same factory as cheese whey, which means that both the permeate and the retentate would be available for the PHA production in house without generating any transportation costs (29). That way, reduction of a waste material stream (whey) from the cheese production process would conveniently be combined with enhanced growth of the PHA producing bacteria. For this reason, the subsequent experiment was carried out for a more in depth analysis of whey retentate as biotechnological nitrogen source.
Growth and growth rate of the auspicious PHA production strain
In contrast, the experimental results clearly show that addition of HWR results in lower
However, it appears logical that, after hydrolysis of whey retentate, its amino acids and small peptides should be better available for the strain if compared to whole proteins, which have to hydrolyzed by the strain before being accessible for utilization. With acidically hydrolyzed retentate, this was however not the case. Therefore, it should also be tested in follow-up experiments if enzymatically (proteases) hydrolyzed whey retentate would be advantageous compared to NHWR. In addition, desalination of acidic hydrolysates and simple methods for detoxification to remove inhibitors should be tested as subsequent steps. Such detoxifications, based on overliming, active carbon, and lignite, performed successfully in the past when hydrolyzing, e.g., lignocellulose materials to generate inexpensive substrates for other PHA production strains, as described in various literature reports (43, 44, 45).