Cheese whey to biohydrogen and useful organic acids: A non-pathogenic microbial treatment by L. acidophilus

The burgeoning organic waste and continuously increasing energy demands have resulted in significant environmental pollution concerns. To address this issue, the potential of different bacteria to produce biogas/biohydrogen from organic waste can be utilized as a source of renewable energy, however these pathogenic bacteria are not safe to use without strict contact isolation. In this study the role of safe food grade lactic acid bacteria (Lactobacillus spp.) was investigated for production of biogas from cheese waste with starting hexose concentration 32 g/L. The bacterium Lactobacillus acidophilus was identified as one of the major biogas producers at optimum pH of 6.5. Further the optimum inoculum conditions were found to be 12.5% at inoculum age of 18 h. During the investigation the maximum biogas production was observed to be 1665 mL after 72 hours of incubation at pH 6.5. The biogas production was accompanied with production of other valuable metabolites in the form of organic acids including pyruvate, propionate, acetate, lactate, formate and butyrate. Thus this research is paving way for nonpathogenic production of biohydrogen from food waste.

by means of fossil fuels. Several microorganisms have the enzyme hydrogenases which help to release molecular hydrogen by oxidizing hydrogen to electrons and protons. The common way to produce hydrogen biologically is microbial fermentation i.e. by decomposition of organic substrates to hydrogen and carbon dioxide 15 . The bacteria which are able to produce hydrogen have the capability to grow autotrophically by utilizing the hydrogen gas as an energy substrate and oxygen as a terminal electron acceptor for production of water as end product 16 . Variety of biological species are involved in production of hydrogen viz. cyanobacteria, fermentative and photosynthetic bacteria etc 17 .
The estimated yearly production of lignocellulosic biomass is nearly 2.20 × 10 12 Kg (dry weight) from agriculture and forestry residuals, energy crops, aquatic plants and algae 18 which have potential to produce H 2 at very low costs by hydrolysis and fermentation 19 . Translating the energy from these wastes into valuable energy offers two concurrent benefits: the production of energy and reduction in environmental pollution. Diverse groups of anaerobic microorganisms possess the potential to break complex organic matter into three valuable energy outputs, which can be garnered proficiently: methane gas (CH 4 ), hydrogen gas (H 2 ) and electrons produced by a microbial fuel cell (MFC) [6][7][8] .
The different reactor conditions reported use of different bacteria capable of bioconversion of organic wastes into hydrogen and most of the seed cultures used include pathogenic strains of bacteria and required specific growth conditions. The application of food grade bacteria including Lactobacillus and Lactococcus can help in overcoming these problems by introducing non-pathogenic way of waste predisposal treatment and energy production. Hence, in this study first time the effects of different food grade bacteria on biohydrogen production have been analysed, along with the effects of different parameters such as inoculum conditions and pH. Based on the previous reports it can be hypothesized that food grade bacteria will produce the biohydrogen comparable to that from pathogenic bacteria. The proposed hypothesis was studied by analysis of biohydrogen production from food wastes by using non-pathogenic bacteria. Different parameters such as pH, inoculum age and size have been optimized to get the maximum biohydrogen production. Cheese whey has been used as a complex substrate for biohydrogen production by Lactobacillus acidophilus in a batch reactor of capacity 2 litres. Comparison of microbes for evaluation of biohydrogen production potential. All the microbes were screened for hydrogen producing capability by conducting batch experiments in triplicates under anaerobic conditions. The medium composition remains the same as mentioned above. All the experiments were conducted using polypropylene bottles (capacity 150 mL) with working volume of 100 mL. These bottles were tightly capped with butyl rubber stopper to make it air tight and the whole setup was made anaerobic by sparging of argon gas. For the collection of gas evolved during the fermentation, the set up was equipped with disposable air tight syringes. The sealed bottles were carefully placed in incubator shaker (100 rpm) maintaining temperature at 37 °C. The gas collected was measured by water displacement method. optimization experiments of growth conditions for hydrogen production.

Materials and Methods
www.nature.com/scientificreports www.nature.com/scientificreports/ The fermentation effluent collected at different time during experiment were analysed by using HPLC (Shimadzu) fitted with column Animex HPX-87 H (S. N0. 31262 and diameter 300 × 7.8 mm) using 0.018 M H 2 SO 4 with a flow rate of 0.7 mL/min at 65 °C for 30 minutes. statistical analysis. The one way ANOVA was performed to test the significance of the experimental data using Microsoft Excel. During the ANOVA analysis the results of test samples were compared with the control samples and analysed for significance level of 95% (p < 0.05).

Result and Discussion
evaluation of food grade bacterial strains for biogas production. The selected strains were analysed for biogas production and the results are represented in Fig. (1). From the data it can be observed that L. acidophilus was most efficient in biogas production with a value of 85 mL/100 mL of culture media used, whereas L. casei was observed to be least efficient with a production value of 36 mL/100 mL. The other three strains used viz. L. paracasei, Lactococcus lactis and E. coli were moderate producers of biogas with production values of 42 mL/100 mL, 65 mL/100 mL and 50 mL/100 mL respectively. In a study, the waste bread hydrolysate was used as substrate for biohydrogen production by Biohydrogenbacterium R3. The maximum H 2 yield of 103 mL H 2 /g waste bread was observed 20 . In another experiment, effective hydrogen production from food waste hydrolysate in different continuous mixed immobilized sludge reactors (CMISRs) at packing ratios of 10%, 15% and 20% have been reported. The biohydrogen production at a packing ratio of 15% was highest with a hydrogen production rate of 353.9 mL/h/L and at high organic loading rate of 40 kg/m 3 /d 21 . Anaerobic digestion of food waste by using Escherichia cloacae and Enterobacter aerogenes had produced biohydrogen of 155.2 mL/g of volatile solids (VS) 22 . In a continuous stirred tank reactor (CSTR) provided with sugarcane, 3.38 mmol H 2 /L/h hydrogen were produced by using Clostridium butyricum 23 . Configurations including membrane filtration enhanced H2 yield by over 300% compared to a more conventional stirred reactor 24 . Effect of pH on biogas production. The effect of pH on biogas production by L. acidophilus is represented in Fig. 2. The biogas production was observed at different starting pH values of 5.5, 6.0, 6.5, 7.0 and 7.5. From the results it has been observed that the biogas production initially increased up to pH 6.5 with a production value of 135 ml/100 ml. Above this pH, biogas production started decreasing. Therefore, it can be concluded that pH  www.nature.com/scientificreports www.nature.com/scientificreports/ 6.5 is optimal for biogas production under these experimental conditions. By using coconut milk wastewater as the substrate, maximum biogas production i.e. 0.28 L H 2 /L has been reported at pH 6.5 indicating that such parameter is crucial for dark fermentative hydrogen production 25 . Activity of enzymes varied according to different pH conditions that become a very important factor for stimulating the biohydrogen producing ability of microrganisms.
Among the studied fermentation pH values, relatively high biogas yield was observed at pH 6.5 (135 mL/ 100 mL). Next higher values of biogas produced was observed at pH 7.0 (110 mL/100 mL), whereas biogas production in fermentation at acidic pH values decreased more substantially. This decrease in biogas production at lower pH values can be attributed to decline in the system pH values below 5.0 due to acid production, that results in shifting of metabolic pathway to solventogenesis from acidogenesis leading to H 2 production suppression. The data obtained in this study is in accordance with other reports, showing maximum biogas/H 2 production at pH values of 6.0 by use of anaerobic mixed microflora 26 , pH 6.5 by use of sewage microflora 25 . The slight variation in optimum pH values in different studies can be attributed to change in seed culture composition, inoculum conditions and culture conditions. Effect of inoculum age and volume. Further the effect of inoculum conditions viz. inoculum age and inoculum volume were investigated. To study the effect of inoculum age the inoculum from cultures incubated for different time intervals were used. Figure 3 represents the effect of inoculum age on biogas production. The results show that initially the biogas production increased with increase in inoculum up to 18 h, attaining a maximum value of 155 ml. The next sample was at 20 h, where showed the biogas production decreasing again. Therefore, it can be inferred from the results that 18 h is the optimum inoculum age for biogas production.
The experiment was performed to examine the inoculum age effect on hydrogen production by using the inoculum grown for 10, 12, 14, 16, 18 and 20 h.
The results showed the strong relationship among inoculum age and healthy growth of the culture alongside H 2 production. The inoculum with 18 h age exhibited the highest percentage of biogas (155 mL/L) production. The large retention times might be responsible for the bacterial metabolism to drive in the synthesis of other metabolites such as Poly-Beta-Hydroxybutyrate (PHB) alongside the H 2 production 27 . The loss of H 2 production activity in batch cultures of R. sphaeroides S was associated with the declined activity of the electron carrier ferredoxin in time dependant manner 28,29 . For PNS bacteria, an inoculum from the exponential growth phase is most suitable for elevated yield of H 2 30 . All the studies are highlighting the effect of inoculum age on different strains with a common conclusion of declined hydrogen yield with increased time span and this study also demonstrates that H 2 production by food grade bacteria can be achieved maximum at the inoculum age of 18 h and lowered with increase in inoculum age.
After that the effect of inoculum volume on biogas production was analysed by varying the volumes of inoculum used, from the results shown in Fig. 4, it can be observed that initially the biogas production increased with increase in inoculum volume and attained a maximum value of 190 mL at 12.5% inoculum volume and then further started to decrease in concentration dependent manner with minimum biogas production of 80 mL at 25% inoculum volume thus indicating that 12.5% inoculum volume is optimum for biogas production.
The inoculum volume also plays an imperative role in biogas/H 2 production. 10%, 12.5%, 15%, 20%, 25% were studied as different inoculum volumes. The results exhibit that the most productive inoculum volume was 12.5% to produce 190 mL of biogas but at lower and higher inoculum volumes the biogas production decreased. In a study, it has been observed that the H 2 yield improved from 2 mmol h −1 at 1% inoculum size to 2.36 mmol h −1 with 10% inoculum size but the highest yield was achieved with 1% inoculum 31 signifies a high substrate to cell ratio would extend the growth phase and provide a longer duration of high rates of H 2 production. However, in another study it has been reported that a low substrate (cellulose) to cell density enabled higher H 2 yield by using a mixed H 2 producing culture 32 . The decline in total gas or H 2 at the end of the metabolic process can be credited to the consumption of gas by bacteria as the collected gas was directly in contact with the culture without any trap system. www.nature.com/scientificreports www.nature.com/scientificreports/ Biogas production in batch fermentation and sugar utilization. The kinetics of biogas production with sugar utilization was observed during the batch fermentation (in two phases, I and II) for 72 h and the data was plotted between total sugar concentration vs biogas produced. Biogas production was monitored with initial sugar (hexose) concentration being 32 g/L at pH 7.0 in phase I (from 0 h to 48 h) with 20% inoculum (18 h grown). At the start of phase II i.e. 48 h after start of the experiment, 100 mL of inoculum (12.5% v/v) (18 h grown) was introduced into the spent media (800 mL) and pH value was set at 6.5 in the same batch reactor. From the graph (Fig. 5) it can be inferred that at the time of inoculation the total sugar concentration was maximum with a value of 32 g/L and then it started declining gradually with increase in biogas production after adjusting the pH from 7.0 to pH 6.5 in the second phase until completion of the experiment (72 h). From the graph it can be observed that the biogas production increased with decrease in total sugar concentration.

Biomass concentration.
The biomass concentration/cell growth was measured by taking absorbance at 600 nm after regular intervals of time and graph between time (h) and absorbance was plotted and is represented in Fig. (6). From the plot it can be inferred that the biomass concentration exhibited diauxic pattern that kept on increasing at a lower rate and reached maximum at 48 h and start growing further in Phase II after the reintroduction of inoculum at 48 h, the biomass concentration reached maximum at 64 h. Biogas production. The culture was observed for production of biogas from the time of initiation of the experiment and a graph between volume of biogas produced vs time was plotted as represented in Fig. 5. From the graph it can be observed that the biogas produced was minimum with a value of 40 mL just after the inoculation  www.nature.com/scientificreports www.nature.com/scientificreports/ and then it increased during the bacterial growth and attained a value of 1665 mL at the end of the phase II indicating that the amount of biogas produced is proportional to the cell biomass concentration in the culture and varies in concentration dependent manner.
production of end metabolites. The end metabolites produced during bacterial growth which is directly related to the microbial metabolism influencing the fermentation pathways, were analysed for their concentrations using HPLC after sampling at different time intervals under the optimized conditions in batch experiment and metabolite concentrations were plotted against time as represented in Fig. (6). During sugar fermentation by Lactobacillus, the produced metabolites include lactate in homofermentation, lactate, acetate 33 and propionate 34 in heterofermentation. The other metabolites detected during the heterofermentation by Lactobacillus bacteria include pyruvate, formate and butyrate 35 during the biogas production. The amounts of these metabolites varied in different studies owing to experiment conditions viz. inoculum age, inoculum amount, type of substrate used, pH, type of fermentation used [33][34][35][36] . In the present study the metabolites produced included pyruvic acid (PA), propionic acid (PrA), formic acid (FA), lactic acid (LA), butyric acid (BA) and acetic acid (AA) having maximum production at 60 h, 52 h, 24 h, 52 h, 64 h and 52 h respectively along with biogas production. During anaerobic fermentation pyruvate is further oxidized to acetyl-CoA by pyruvate: ferredoxin oxidoreductase (PFOR) complex as represented in the equation below.

Pyruvate CoA Fd
Acetyl CoA FdH CO 2 Further reduced ferredoxin is also generated in the reaction with NADH, with the reaction being catalysed by NADH: ferredoxin oxidoreductase (NFOR) as given below.

NADH Fd NAD FdH
In this metabolic pathway, hydrogen is released by hydrogenases responsible for catalysing proton reduction utilizing electrons from ferredoxin where the activity of both the involved enzymes is controlled by hydrogen concentration. The hydrogen partial pressure >60 Pa are reported to inhibit NFOR activity and result in production of non-gaseous end-products from acetyl-CoA including acetate, butyrate, ethanol, butanol and lactate. Similarly, PFOR is active at hydrogen concentrations lower than 3 × 10 4 Pa. The theoretical maximum hydrogen yield during this type of fermentation is 4 ces of hydrogen per mole of hexose utilized. Whereas, during conversion of hexose into butyrate, the hydrogen yield drops to 2 moles per mole of hexose 34,35 . The results obtained in present study are in accordance with the previous reports of metabolite production during sugar fermentation using lactic acid bacteria.
The calculated values of the molar amounts of the end metabolites from the experiment were, AA (0.12 moles), PA (0.031 moles), BA (0.01 moles), LA (0.56 moles), FA (0.125 moles) and PrA (0.165 moles) from the 0.114 moles of hexose which are in accordance with the theoretical values. There is an increasing trend for formic acid synthesis as it is clear from graph (6) from 4-8 h corroborating the significant increase in biogas production through FHL pathway. However, which further increased with time from 8-12 hours. The flux of formic acid remains the same with reduction in lactic acid production where we achieved maximum biogas. However, production rate decreases after 8 hours of fermentation. Results are clearly showing that there is no significant rise after 10 hours of fermentation. However, accumulation of lactic acid increased (32.1%) from 10 h-48 h. From the graph it can be inferred that with time as lactic acid concentration increased, the production of biogas had also increased, which is in accordance with the results previously reported in other studies supporting that lactic acid produced in Phase I inhibited more production of lactic acid and supported more production of biogas in Phase II of fermentation 37 . www.nature.com/scientificreports www.nature.com/scientificreports/ This finding demonstrated that L. acidophilus can be used for biohydrogen production as well as lactic acid, pyruvic acid, formic acid, acetic acid, butyric acid and propionic acid confirming the mixed acid fermentation under controlled anaerobic environment. Further the molar yield of biohydrogen produced was calculated with a value of 1 mol of H 2 (67%) produced/mol of the hexose used. In a study, the maximum hydrogen yield obtained corresponds to 180 mL H 2 /gVS at 5% GLC (glycerol) with the maximum specific production rate value of 13 mL H 2 /(gVS.h) 38 . Different microorganisms yield H 2 under specific conditions, such as use of light as energy source by microalgae to split water in production of H 2 and cyanobacteria which utilize carbohydrates to store energy from photosynthesis and to produce H 2 from water. In various studies different reactor configurations and reaction conditions have been analysed for bio-hydrogen production from food and other organic waste. In a study, waste paper has been utilized for biohydrogen gas production through dark fermentation. The highest amount of hydrogen gas obtained corresponds to 18.9 g/L of initial sugar concentration whereas sugar concentrations higher than 18.9 g/L resulted in inhibition of product formation 39 .
In another study anaerobic batch fermenter has been used in production of biohydrogen from cow solid waste under the specific hydrolysis conditions. The results exhibited a hydrogen production yield of 97 mL H 2 /g cow solid waste 40 . In a study where anaerobic fermentation of glucose by Clostridium butyricum yielded 2.02 mol H 2 /mol glucose have been reported 13 . Food waste hydrolysate has been used as a substrate with the production 1.97 mol H 2 /mol glucose in the batch system 2 . The comparison of biohydrogen produced utilizing various biowastes are shown in Table 1.
Hydrogen production from cheese processing wastewater by using mixed microbial cultures in anaerobic fermentation reached to a value of 10 mM/gCOD 41 while using human wastes and activated sludge 2.186-3.999 mmol H 2 /g of waste has been obtained by thermophilic bacteria 42 . Production of 2.74 mol H 2 /mol lactose has been reported by E. coli using cheese whey as substrate 43 .
In another study by using cheese whey and glucose as substrate, 1.33 mol hydrogen/mol lactose 44 and 7.8 mol H 2 /mol glucose have been achieved respectively 45 . Using cheese whey powder (CWP) 1.03 mol H 2 /mol glucose has been obtained by thermophilic dark fermentation 46 . From the comparison studies, it can be inferred that biohydrogen produced in our designed experiment using non-pathogenic food grade bacteria with different parameters is within values reported by others.

Conclusion
In comparison to the lipid, protein and cellulose components, the carbohydrate fraction in food waste plays a significant role in the hydrolysis step during anaerobic degradation. In present study the role of Lactobacillus was observed for biogas production in fed batch reactor using anaerobic fermentation. Among all the bacterial cultures under study, L. acidophilus was observed to have maximum biogas production value. For L. acidophilus, it was observed that under optimized inoculum conditions such as inoculum of 18 h age, and 12.5 mL of inoculum per 100 mL (12.5% v/v) of culture at pH value of 6.5, maximum biogas (1665 mL) was produced after 72 hours of incubation. Along with the biogas other valuable organic acids including pyruvate, propionate, acetate, lactate, formate and butyrate were also produced during anaerobic metabolism. Dark fermentation of food waste for biogas generation exhibits the potential to create an impact on the global energy market for the production of energy from a cheap and renewable carbon source. The isolation of viable and nonpathogenic LAB (lactic acid bacteria) cells for fermentative biogas production from cheese whey indicated that the LAB are capable of surviving in reactor conditions and can influence biogas production.  Table 1. Comparison of hydrogen production found in different studies by using wastes as the substrate.