Bioresource Technology 387 (2023) 129594

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Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Transforming acid whey into a resource by selective removal of lactic acid

and galactose using optimized food-grade microorganisms
Ge Zhao a, Shuangqing Zhao a, Line Hagner Nielsen b, Fa Zhou a, Liuyan Gu a,
Belay Tilahun Tadesse a, Christian Solem a, *
a
National Food Institute, Technical University of Denmark, DK-2800 Kgs. Lyngby, Denmark
b
DTU Health Tech, Department of Health Technology, Technical University of Denmark, DK-2800 Kgs. Lyngby, Denmark

H I G H L I G H T S G R A P H I C A L A B S T R A C T

• It is the first time to use C.glutamicum to

remove lactic acid from AW.
• C.glutamicum mutants with enhanced
lactate metabolic rate were isolated
using ALE.
• Best adapted strain reduced time
required for lactic acid removal from
AW by 50%.
• The removal of lactic acid increased
lactose yield of powder after spray
drying.

A R T I C L E I N F O A B S T R A C T

Keywords: The presence of lactic acid and galactose makes spray drying of acid whey (AW) a significant challenge for the
Acid whey dairy industry. In this study, a novel approach is explored to remove these compounds, utilizing food-grade
Corynebacterium glutamicum microorganisms. For removing lactic acid, Corynebacterium glutamicum was selected, which has an inherent
Adaptive laboratory evolution
ability to metabolize lactic acid but does so slowly. To accelerate lactic acid metabolism, a mutant strain G6006
Lactic acid
was isolated through adaptive laboratory evolution, which metabolized all lactic acid from AW two times faster
Galactose
than its parent strain. To eliminate galactose, a lactose-negative mutant of Lactococcus lactis that cannot produce
lactate was generated. This strain was then co-cultured with G6006 to maximize the removal of both lactic acid
and galactose. The microbially “filtered” AW could readily be spray dried into a stable lactose powder. This study
highlights the potential of utilizing food-grade microorganisms to process AW, which currently constitutes a
global challenge.

* Corresponding author.
E-mail address: chso@food.dtu.dk (C. Solem).

https://doi.org/10.1016/j.biortech.2023.129594
Received 22 June 2023; Received in revised form 26 July 2023; Accepted 30 July 2023
Available online 1 August 2023
0960-8524/© 2023 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
G. Zhao et al.
1. Introduction
Acid whey (AW) is a liquid leftover from acid-coagulated dairy
products, including cream, ricotta and cottage cheese, and strained
(Greek) yogurt (Rocha-Mendoza et al., 2021). This dairy side stream has
a pH of 4.2–4.5 and a solid content of about 5–6.4 %, with lactose
making up the majority (3.9–4.9%), followed by lactate/lactic acid
(~0.8%) and smaller amounts of galactose and protein (Chandrapala
et al., 2016). The projected global Greek yogurt market size is expected
to reach $11.2 billion by 2027, driven by the increasing awareness of
consumer health and dietary preferences (https://www.alliedmarket
research.com/greek-yogurt-market-A06295). This growing demand for
Greek yogurt will consequently lead to the generation of very large
quantities of AW as a byproduct. Despite massive profits generated from
Greek yogurt production, manufacturers have been struggling to find a
more economical and environmentally sustainable way to dispose of the
AW, driven by environmental policies that have become increasingly
stringent (Chandrapala et al., 2016; Rocha-Mendoza et al., 2021). The
biological oxygen demand (BOD) for degrading AW is high
(BOD>35,000 ppm) and costly treatment facilities are needed (Bolwig
et al., 2019). In general, AW has been used as animal feed in raw form or
for biogas production (Prazeres et al., 2012; Chen et al., 2016). How­
ever, these applications are not sufficiently attractive economically.
Processing AW to make lactose powder is an alternative and poten­
tial value-added application as most of the solid content of AW is lactose.
One of the indispensable steps in this process is spray drying (Chen et al.,
2016). However, spray drying AW is not an easy task since the high
concentrations of lactic acid and low pH of AW interfere with spray
drying, causing the powder to stick to the dryer walls, thereby reducing
product quality and yield, and furthermore, there is a risk of damaging
the spray dryer during the operation (Boonyai et al., 2004, Saffari &
Langrish, 2014). Moreover, it has been found that increased thermo­
plasticity and hygroscopicity caused by an elevated content of lactic acid
promotes inter-particle adhesion, resulting in the generation of large
particles during storage and a significant decrease in the shelf life of the
powders (Chandrapala et al., 2016; Chandrapala & Vasiljevic, 2017).
Therefore, it is necessary to maximize the removal of this compound
from AW in order to decrease operational problems in downstream spray
drying and obtain higher-value powders from AW. To address these
challenges, there have been attempts at using ultrafiltration, nano­
filtration, and electrodialysis or their combination to remove lactic acid
(Chandrapala et al., 2016c; Chen et al., 2016; Chandrapala et al., 2017;
Dufton et al., 2019; Talebi et al., 2020). However, none of these stra­
tegies have proven to be sufficiently efficient. Moreover, the environ­
mental impact of the above-mentioned processes is also tremendous
(Dufton et al., 2019). Apart from lactic acid, the small amount of
galactose present in AW could decrease the storage stability of spray-
dried powders, as galactose has been reported to increase the ten­
dency to caking (Rao et al., 2004; Thorakkattu, 2020). Therefore, there
is a need for developing new cost-efficient and environmentally friendly
approaches for removing lactic acid and galactose from AW for its
valorization.
Corynebacterium glutamicum, a Gram-positive bacterium, is widely
used for the production of food and feed amino acids (Neuner and
Heinzle, 2011). It has been granted “Generally Recognized As Safe”
(GRAS) status, has relatively few nutritional requirements and grows
well in defined medium to high cell densities (Lee et al., 2016). More­
over, C. glutamicum is unable to metabolize lactose or galactose but
grows well on lactate/lactic acid as the sole carbon and energy source
(Kato et al., 2010). These characteristics make C. glutamicum a good
candidate for removing lactic acid in AW while leaving its valuable
constituents untouched.
Lactococcus lactis is a food-grade lactic acid bacterium (LAB), most
commonly known for its involvement in cheesemaking (Ainsworth et al.,
2014; Zhao et al., 2021). For this microorganism, it is well-known that
lactose metabolism is plasmid-encoded (Siezen et al., 2005). The curing
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Bioresource Technology 387 (2023) 129594
of this plasmid can be accomplished through protoplast formation or by
using mutagens such as ethidium bromide and acriflavine, and such
strains are unable to metabolize lactose but still retain the ability to
metabolize galactose (Gasson and Davies, 1980; Gasson, 1983). An
L. lactis strain RD1M5, obtained through classical chemical mutagenesis,
was previously isolated, which has almost completely lost its lactic acid-
producing ability, and this strain grows quite well in dairy waste (Liu
et al., 2020). A derivative of RD1M5 lacking its lactose plasmid has great
potential to be used as a whole-cell catalyst to selectively remove
galactose, especially since the main fermentation product of RD1M5 is
acetoin, a volatile compound, that will be lost during spray drying.
The primary goal of the present study is to investigate whether
C. glutamicum and L. lactis can be used as efficient whole-cell catalysts to
selectively remove lactate and galactose in AW. First, the wild-type
C. glutamicum ATCC 13032 strain is tested, revealing its potential for
lactate removal, and afterwards, adaptive laboratory evolution (ALE) is
used to obtain strains with improved performance. Candidates with
improved performance are isolated and one of these is characterized in
detail. Second, an effort is made to isolate an L. lactis strain deficient in
lactate dehydrogenase, which is unable to metabolize lactose and this
strain is subsequently tested for its galactose removing ability. Finally,
spray-dried powders of microbially treated AW are prepared, and their
quality compared to powders obtained from non-treated AW.
2. Materials & methods
2.1. Microorganisms and growth condition
C. glutamicum ATCC 13032 was cultivated in Brain Heart Infusion
(BHI) broth or CGXII minimal medium containing 107 mM lactate
(CGXIIL, pH 6.5) at 30 ◦ C and 200 rpm. RD1M5, a natural mutant of
L. lactis subsp. lactis biovar diacetylactis SD96, has almost completely
lost its lactic acid-producing ability (Liu et al., 2021), was grown aero­
bically in M17 medium with 1% lactose (LM17) or 1% glucose (GM17),
or defined SAL medium (Solem et al., 2013) with 1% lactose (SALL), 1%
glucose (SALG) or 1% galactose (SALGa).
2.2. Optimization of initial pH and initial OD600 of AW
AW with pH 4.2 was obtained from Arla Foods, and contained about
117 mM, 45 mM, and 107 mM of lactose, galactose, and lactate,
respectively, which was sterilized at 95 ◦ C for 30 min before use. An
overnight BHI broth culture of wild-type C. glutamicum (WT) was
centrifuged at 5000 × g for 5 min to collect cell pellets which were
washed twice with sterile 0.85% NaCl solution and re-suspended in the
same solution to a final OD600 of 5, 10, 20, 40, 80 and 160, respectively.
The optimization experiments were conducted in 100 mL conical flasks,
each containing 19 mL of AW. To compare the effects of pH on lactate
metabolism rate in AW, the pH of AW was adjusted using ammonia
solution to obtain different pH levels of 5, 5.5, 6, 6.5, 7, 7.5, and 8.
Subsequently, 1 mL of cell suspension with OD600 of 20 was inoculated
into AW at an initial OD600 of 1. To investigate the effects of initial OD600
on lactate metabolic rate, 1 mL of suspension with OD600 of 5, 10, 20, 40,
80, and 160 was transferred to 19 mL of AW with pH 6.5 to initial OD600
of 0.25, 0.5, 1, 2,4, and 8. Each experiment was performed in triplicates.
2.3. Adaptive evolution of C. glutamicum in CGXIIL
Adaptation of C. glutamicum in CGXIIL was carried out using a serial-
transfer regime as illustrated in Fig. 2a. Cells from a frozen glycerol stock
were streaked on the CGXIIL plates with pH 6.5. Single colony was
picked and inoculated in a glass tube containing 5 mL of BHI to obtain
the preculture. Then an inoculum comprising 1% (v/v) of preculture was
inoculated into 5 mL of CGXIIL medium. After the culture had entered
the growth phase, the inoculation was conducted again and this growth-
and-dilution process was continued for approximately 3 months. For
G. Zhao et al.
every fifth passage, 800 μL of culture was taken and preserved through
storage at − 80 ◦ C for future analysis. Frozen stocks from passages 10, 35,
and 60 were streaked on CGXIIL agar plates with a pH of 6.5, and ten
isolates forming larger colonies were picked and saved individually at
-80 ◦ C.
2.4. Initial performance comparison of WT and its adapted mutants
2.4.1. Growth in high-throughput microbioreactor
Single colonies of both the WT and its adapted mutants were inoc­
ulated into 24-well plates filled with 1 mL of CGXllL medium to prepare
precultures, which were aerobically incubated at 30 ◦ C. After 24 h of
incubation, 50 μL of preculture was transferred into 48-well plates filled
with 1 mL of CGXIIL medium and then incubated at 30 ◦ C using a Bio­
lector (M2p-labs, Germany) with constant shaking at 800 rpm for 24 h.
Subsequently, 500 μL of culture from each well was withdrawn to
measure OD600.
2.4.2. Lactate removal in AW
Lactate removal using the adapted derivatives was carried out in a
100 mL conical flask containing 20 mL of AW (pH 6.5) with an initial
OD600 of 2. Samples were withdrawn at 0, 12, 18, and 24 h to determine
lactate concentration. Each experiment was performed in triplicates.
2.4.3. Comparison of growth and lactate metabolism kinetics of WT and its
adapted derivative G6006 in the flask
A single colony of both WT and G6006 strains was separately inoc­
ulated into a glass tube containing 10 mL of BHI broth. These cultures,
along with their 10-fold serial dilutions, were incubated overnight at
30 ◦ C. When the culture entered the exponential phase at an OD600
around 0.5, the cells were collected by centrifugation and washed twice
with 0.85% NaCl solution. The collected pellets were then resuspended
in CGXIIL medium at an OD600 of 5, and 0.5 mL of this suspension was
immediately transferred into 50 mL of CGXIIL medium in a 300 mL flask.
Each experiment was performed in triplicates.
2.5. Whole genome sequencing
Whole genome sequencing of WT, G3507, and G6006 was conducted
on the DNBseq platform by BGI China. The resulting raw paired-end
150-bp reads corresponding to these three strains were initially pro­
cessed and then mapped to the reference genome (C. glutamicum ATCC
13032, Genbank Accession number: NZ_CP115148) using Geneious
Prime 2023. Subsequently, single nucleotide variants (SNVs) were
identified in both the parental strain and adapted strains using the same
software. SNVs were deemed to be valid if they had a mutant frequency
of >90% and a strand bias of <75%. Any SNVs that were found in both
the parental strain and adapted strains were excluded from the final
analysis.
2.6. Transcriptomics analysis
Exponentially growing cells of both the WT and G6006 in CGXIIL (pH
6.5) were harvested (at an OD600 around 1) and centrifuged at 4 ◦ C at
7000 × g for 5 min.Then total RNA was extracted from the harvested
cells using Qiagen RNeasy kit, and further purified using Qiagen RNase-
Free DNase Set, respectively, according to the protocols of manufac­
turer. Each sample was analyzed in duplicates. RNA sequencing was
performed by BGI-Tech (Hong Kong) using BGISEQ-500 sequencing
platforms and about 2 GB of clean data with trimmed reads was obtained
for each sample. The quality control of clean reads was performed using
FastQC. Afterwards, the RNA sequence data were mapped to trans­
lational region of the reference genome (ASM284740v1) using Geneious
Prime 2023. DEseq2 algorithm was used to compare the gene-level
expression of two groups. Differentially expressed genes (DEGs) were
identified using cutoff thresholds for false discovery rate (FDR) < 0.05
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Bioresource Technology 387 (2023) 129594
and a log2 |fold change (FC)| > 1. To evaluate the global expression
categories of G6006, Clusters of Orthologous Groups (COG) analysis was
performed using eggNOG-mapper with an adjusted p-value (Padj) < 0.05
as a threshold for statistical significance, excluding the nondescriptive
category Function Unknown (category S).
2.7. Isolation of derivatives of RD1M5 cured of the lactose plasmid
Curing of the lactose plasmid was accomplished by growing the cells
at 30 ◦ C in the presence of ethidium bromide (EB). RD1M5 was grown in
GM17 broth overnight. Subsequently, 50 μL of preculture was trans­
ferred to 5 mL of fresh GM17 broth containing different concentrations
of EB (0.5, 1, 2, 3, 4, and 5 μg/mL) and cultivated for 12 h. After the
curing treatment, serially diluted cultures were plated on 2,3,5-tri­
phenyl tetrazolium (TTC) indicator agar and incubated for 48 h. TTC
indicator agar is composed of (g/L): glucose (5), casein peptone (15),
yeast extract (5), MgSO4 (0.5), ascorbic acid (0.5), agar (15), and TTC
(0.1)(Liu et al., 2020). Then the dark red colonies were simultaneously
inoculated into SALG, SALL, and SALGa medium. The isolates that only
grew up in SALG and SALGa medium and produced almost no lactate
were selected for experiments where their galactose-removing ability in
AW was tested.
2.8. Co-cultivation of G6006 and LAC1
In order to simultaneously remove galactose and lactate from AW, an
optimization experiment was carried out to determine the appropriate
amount of LAC1 to be inoculated. In brief, cell suspensions of G6006 and
LAC1 were simultaneously inoculated into 100 mL conical flasks con­
taining 20 mL of AW (pH 6.5). The initial OD600 of G6006 was set at 2,
while the initial OD600 values of LAC1 were set at 0, 0.25, 0.5,1, 2, and 4,
respectively. Samples were withdrawn at 0, 12, 18, and 36 h to deter­
mine lactate and galactose concentrations. Each experiment was per­
formed in triplicates.
2.9. Spray drying of raw AW and processed AW
A Buchi-B290 laboratory mini spray dryer was employed to spray dry
three distinct feed solutions: raw AW (RWL), lactate-removed AW, also
known as de-lactate AW (AWL), and AW with both lactate and partial
galactose removed (AWLG). Before the drying process, all feed solutions
underwent pre-filtration using a sterile membrane (0.22 μm). During the
experiment, the nozzle air flow rate and feed solution flow rate were
maintained at 8 kg/h and 0.6 L/h, respectively, while the inlet and outlet
temperatures were carefully controlled at 180 ◦ C ± 5 ◦ C and 75 ± 5 ◦ C,
respectively. After allowing sufficient time for the system to reach a
steady-state condition (approximately 30 min), the spray dryer operated
for 1 h. Following the drying process, the resulting powder was promptly
collected, sealed, and stored in a desiccator containing silica gel beads
until further analysis.
2.10. Lactose powder yield and powder properties
The effects of the presence of lactate and galactose on powder
properties were assessed, focusing on lactose powder yield, hygroscop­
icity, and particle size distribution.
Lactose powder yield was determined as the percentage weight
fraction of the initial lactose content present in the AW that was suc­
cessfully introduced into the spray dryer. Each experiment was per­
formed in triplicates.
Hygroscopicity was assessed by measuring the final moisture content
(g/100 g dry weight) after exposing the samples to an atmosphere with
40% and 80% relative humidity at 25◦ . The water content was then
measured by weighing, following the method described by Thorakkattu
(2020). Each experiment was performed in triplicates.
The particle size distribution of powders was analyzed using
G. Zhao et al.
Mastersizer 2000 laser particle size analyzer (Malvern Instruments Ltd,
Malvern, UK). Powders were dispersed using a dry sampling system, and
the particle refractive index was set to 1.45. The median diameter d (0.5)
and De Brouckere mean D [4,3] were calculated and selected to evaluate
the particle size distribution.
2.11. Analytic methods
Optical density (OD600) was measured with a UV-1600PC spectro­
photometer (VWR, Denmark) at 600 nm, and pH was measured with a
Lab 845 pH Meter (SI Analytics, Denmark). The lactate, lactose, and
galactose were quantified on an HPLC system (Dionex, Sunnyvale, USA)
equipped with a refractive index detector (Shodex RI-101;Showa Denko
K.K., Tokyo, Japan) and an Aminex HPX-87H column (Bio-Rad, Her­
cules, USA) (Zhao et al., 2022).
3. Results and discussion
3.1. Effects of initial pH and initial OD600 on lactate removal by WT
It has previously been shown that lactate can serve as the sole carbon
and energy source to support the growth of C. glutamicum ATCC 13032
(Kato et al., 2010). Therefore, it was investigated if this strain could be
used to remove lactate from raw AW with a pH of 4.2. The WT could
hardly metabolize lactate in this low pH food waste (Fig. 1a), even with
an initial OD600 of 20 (data not shown), which may be due to an inability
to maintain the proton motive force (PMF) and thus a high intracellular
pH at the low pH (Follmann et al., 2009). To allow C. glutamicum to
Fig. 1. Effect of pH and initial OD600 on lactate removal rate of the WT strain. (a) Lactate concentration and (b) pH change in AW with different initial pH; (c) Lactate
concentration and (d) pH change in AW (pH 6.5) with different initial OD600.
4
Bioresource Technology 387 (2023) 129594
metabolize lactate in AW, the pH of the AW was adjusted to 5–8 with
ammonia solution. As shown in Fig. 1a, when the initial pH of AW was
adjusted to 5.5 and above, this enabled the WT to metabolize all lactate
within 36 h and the final pH consistently increased to around 9.1, except
when the initial pH was 5.5. However, the lactate metabolic rate began
to decrease at a pH below 5.5. Among the different pH values tested, raw
AW with a pH of 6.5 showed the lowest residual lactate concentration
(9.98 mM) after 24 h of incubation. Therefore, this pH value was chosen
as fixed pH when determining the optimal initial cell density.
In Fig. 1c lactate removal over time by the WT at different initial
OD600 can be seen. At the lowest initial OD600 (OD600 = 0.25), lactate
was not completely metabolized in 36 h. Using a higher initial OD600
accelerated the rate at which lactate disappeared, and the fastest lactate
removal occurred at an initial OD600 of 2, where the lactate was almost
eliminated within 24 h. However, when the initial OD600 was increased
to 8, lactate disappeared more slowly. It is tempting to speculate that
nutrient competition at an initial OD600 of 8 between the bacteria could
hamper their performance, eventually disrupting bacterial growth and
lactate metabolism (Jeff Sumardee et al., 2020).
There have been no previous attempts at removing lactate from AW
using C. glutamicum. The findings in this study indicate that
C. glutamicum has great potential to do so, and by optimizing the initial
pH and inoculum, almost complete elimination of the lactate could be
accomplished in 24 h. It should be stressed that no other method or
technology capable of eliminating lactate from AW, that also leaves
galactose and lactose untouched, has been described in the past. It has
been reported that C. glutamicum grows well in cheap defined media
(Wang et al., 2018) and da Luz et al. (2017) achieved a maximum OD600
G. Zhao et al. Bioresource Technology 387 (2023) 129594

of 40.5 using their modified minimal medium, suggesting that there is a
great potential for processing large volumes of AW using C. glutamicum.
3.2. Isolation of C. glutamicum mutants with improved ability to remove
lactate from AW
Although lactate in AW can be almost completely removed within 24
h after optimization of extracellular process parameters such as pH and
inoculum size, it is still relevant to improve the removal efficiency
further as shorter processing time can decrease production costs. ALE
has been widely investigated in recent years as a non-genetic engi­
neering technique to improve substrate utilization rates in host organ­
isms, which is particularly relevant due to strict regulations on
genetically modified organisms (GMOs) in food production (Long and
Antoniewicz, 2018). Therefore, to generate natural C. glutamicum strains
with superior lactate metabolic rate, ALE was performed in a lactate
minimal medium (CGXIIL). This strategy was founded on the premise
that beneficial mutations might emerge over hundreds to thousands of
generations, leading to the optimization of lactate utilization properties
in C. glutamicum. It should be emphasized that the decision to directly

Fig. 2. Isolation of C. glutamicum mutants with higher lactate metabolic rates. (a) Outline of the ALE experiment. For the adaptation process, the WT strain was sub-
cultured in CGXIIL medium (pH 6.5) at 30 ◦ C and 200 rpm. After 10, 35, and 60 passages, cells were streaked on CGXIIL agar, and ten larger colonies from each plate
were randomly isolated, resulting in three sets of ten isolates each. These sets were designated as G10, G35, and G60. (b) Growth monitoring of the WT and its
mutants using a BioLector cultivation system, where growth curves for the WT and mutants are colored in cyan and purple , respectively. (c) Cell density of isolated
colonies after 24 h cultivation in BioLector. (d) Lactate removal from AW (pH 6.5) by WT and strains of the G35 set at an initial OD600 of 2. (e) Lactate removal from
AW (pH 6.5) by WT and strains of the G60 set at an initial OD600 of 2.

5
G. Zhao et al.
evolve C. glutamicum in the lactate minimal medium, rather than in the
AW, was made due to the ease of monitoring strain growth in the former
since the latter has a milky and cloudy appearance at pH above 5.0.
First, culture samples obtained after 10, 35 and 60 passages were
plated on CGXIIL, and ten randomly picked colonies from each passage,
designated as the G10, G35, and G60 sets, were selected for character­
ization. From Fig. 2b and c, it is evident that the majority of the isolates
grew faster and had a higher final OD600 than the WT, especially isolates
from the 35 and 60 passages. Subsequently, the lactate metabolization
ability of G35 and G60 sets was determined in AW. As shown in Fig. 2
d and e, all the isolates from these two passages metabolize lactate in AW
faster than the WT. Especially isolates from the 60 passage culture (G60
sets) stood out and a total of four strains named G6001, G6005, G6003,
and G6006 could remove lactate within 18 h. However, only one isolate,
named G3507, from the 35 passage culture (G35 sets) was also able to
remove lactate within 18 h. The most efficient isolate/strain, G6006,
was capable of almost eliminating lactate in AW after 12 h, nearly saving
12 h as compared to the WT. Additionally, this strain exhibited stable
traits both during process use and storage.
3.3. The adapted strain G6006 grows better in defined CGXIIL and
metabolizes lactate faster
As mentioned, the growth of the evolved strains was monitored in
CGXIIL medium using a BioLector and then a robust strain G6006 was
identified from the last passage culture, which could eliminate lactate in
AW twice as fast as the WT. However, due to the relatively small scale of
the Biolector experiments, it was challenging to perform a detailed
characterization and comparison of the WT and the adapted G6006
strains, particularly regarding their maximum specific growth rate and
lactate consumption rate. These properties are crucial for understanding
the lactate removal ability of G6006 in AW. Characterization of the WT
and G6006 using shake flask experiments (Fig. 3) confirmed that growth
and lactate metabolism of G6006 was significantly faster than that of the
WT at pH 6.5. It should be noted that both strains entered the stationary
phase more slowly than they did when cultivated in a microtiter plate in
the Biolector, which may be ascribed to more effective aeration in the
Biolector microbioreactor and the reduced fermentation volume (Wang
et al., 2018). Characterization is summarized in Table 1 and revealed
that G6006 grew faster than the WT by 146%, and its lactate con­
sumption rate (18.29 ±0.14 mmol/h/g DW) correspondingly increased
by 56%. In addition, the biomass yield (26.22±0.04) of G6006 and final
OD600 was increased by around 32%, which indicated that less lactate is
used for the same biomass production in G6006. It has been reported
Fig. 3. Time course of OD600 (a) and lactate consumption (b) for WT and G6006 grown in CGXII medium with 0.67 % lactate (pH 6.5).
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Bioresource Technology 387 (2023) 129594
Table 1
Characterization of WT and G6006 grown in CGXII medium with 0.67 % lactate
(pH 6.5) in flasks.
Strains μmax(h− 1) Lactate consumption rate Biomass yield (g DW/
(mmol/h/g DW) mol of lactate)
WT 0.19 ± 11.70 ± 0.81 19.93 ± 0.34
0.01
G6006 0.46 ± 18.29 ± 0.14 26.22 ± 0.04
0.00
that biomass of C. glutamicum can be used as a biosorption agent to
remove and/or recover heavy metals from industrial wastewater (Choi
and Yun, 2004), which provides a sustainable route for the reuse of
G6006 biomass waste after it has been used to remove lactate from AW.
3.4. Scrutinizing the genomes of strains adapted to growth on lactate
To identify the mutations responsible for the faster growth pheno­
type of G6006, its genome as well as that of the parent strain were
sequenced. The genome of G3507 from the G35 set of strains was also
sequenced, as this strain required 25% less time to eliminate lactate in
AW than the WT. In addition, the specific growth rate, lactate con­
sumption rate, and biomass yield for this strain in lactate minimal me­
dium were much greater than for the WT, however, the improvement
was less than for G6006 (Table 1 and see supplementary material). As
shown in Table 2, in total, 4 SNVs were identified in the genomes of
G3507 and G6006, including 2 SNVs in intergenic regions and 2 SNVs in
protein-coding regions. Interestingly, both G3507 and G6006 had an
SNV between divergently oriented genes encoding an Acetyl-CoA
carboxylase β-subunit (accD1) and a DUF485 domain-containing pro­
tein, respectively, which could potentially affect transcription of both
genes by altering the promoter region. The remaining two mutations
were in genes encoding regulators. One SNV detected in G3507 was a T
to G change in sigM, encoding a sigma-70 family RNA polymerase sigma
factor (SigM), leading to amino acid replacement of F by L. It has been
reported that SigM is involved in the regulation of the heat and oxidative
stress response in C. glutamicum (Nakunst et al., 2007). It is tempting to
hypothesize that this mutation changed the target range/activity of
SigM, thereby affecting the growth rate of G3507 under aerobic condi­
tions. The other SNV detected in G6006 was an insertion of a G in the
whcA gene, encoding a WhiB family transcriptional regulator, leading to
a frameshift mutation. WhcA is known to play a negative role in the
oxidative stress response (Bush, 2018). Park et al,(2012) found that
overexpressing whcA resulted in slow cell growth and increased
G. Zhao et al. Bioresource Technology 387 (2023) 129594

Table 2 phenotypes, the transcriptomes of the WT and the adapted G6006 strain
Mutations identified in the mutants G3507 and G6006 adapted to growth on were compared. Principal-component analysis (PCA) confirmed the
CGXIIL medium. eligibility of data for analysis (see supplementary material). Total RNA
Strains RPa Region NCb AACc product or (distance from both C. glutamicum strains cultivated in CGXIIL during the expo­
from CDS) neared nential phase was prepared, processed, and analyzed. Differentially
product expressed genes (DEGs) revealed a total of 221 down-regulated genes
G3507 882,896 intergenic (G)6 None (-344 bp) Acetyl-CoA and 192 up-regulated genes in the G6006 compared to WT (Fig. 4a).
-> carboxylase β-subunit COG analysis revealed that most of the DEGs were in the category
(G)5 AccD4 and (-270 bp)
inorganic ion transport and metabolism (category P), where G6006 had
DUF485 domain-
containing protein 34 downregulated and 28 upregulated genes (Fig. 4b). Subsequently,
Cg0952 amino acid metabolism and transport (category E) was strongly repre­
3,272,666 gene CDS T -> F -> L Sigma-70 family RNA sented in G6006, with 21 downregulated genes and 13 upregulated
G polymerase sigma genes, respectively. It was remarkable that the category involved energy
factor SigM
G6006 298,846 gene CDS (G)6 Frameshift WhiB family
production and conversion (category C) and carbohydrate transport and
-> transcriptional metabolism (category G) were also significantly enriched, with 20 and
(G)7 regulator WhcA 10 downregulated and 11 and 15 upregulated genes, respectively.
882,893 intergenic G -> None (-341 bp) Acetyl-CoA Changes in these two categories were expected since they contain most
A carboxylase β-subunit
of the genes involved in cell-associated cell processes such as glycolysis,
AccD1and (-273 bp)
DUF485 domain- pentose phosphate pathway (PPP), and citrate cycle (TCA cycle). It has
containing protein been reported that pyc (encoding pyruvate carboxylase) and malE
Cg0952 (encoding malic enzyme) belonging to category C, are essential for
a
Reference position refers to the genome sequence of C. glutamicum ATCC C. glutamicum growing on lactate (Peters-wendisch et al., 1998;Gourdon
13032 (Accession number: NZ_CP115148). et al., 2000). Transcript analysis found that pyc and malE were signifi­
b
Nucleotide change. cantly upregulated in G6006, approximately 4.2-fold and 2.6-fold in­
c
Amino acid change. creases were observed (see supplementary material), which is consistent
with previous studies showing that overexpression of pyc or malE
susceptibility to oxidants. Based on this available evidence, it can be enhanced the lactate metabolic rate (Peters-wendisch et al., 1998;
inferred that the frameshift mutation in the whcA gene in G6006 is likely Gourdon et al., 2000).
to decrease its activity, leading to decreased oxidative stress. This may Previously, it was hypothesized that a mutation that lies in the
explain why the G6006 has a faster growth rate and higher biomass promoter region of accD4 and cg0952 could affect their expression.
yield. However, transcriptomic analysis revealed that only the expression of
cg0952 was significantly changed (see supplementary material). cg0952
3.5. RNA sequencing revealed strong changes in inorganic ion transport is followed by and cotranscribed with mctC gene, encoding a mono­
and metabolism in G6006 carboxylate transporter that is involved in the uptake of pyruvate, ace­
tate and propionate, but it has not been indicated that MctC is involved
To reveal more about the underlying mechanism for the improved in lactate transport in C. glutamicum (Jolkver et al., 2009). These

Fig. 4. Overall transcriptomics profile obtained in an analysis using DESeq2. (a) The volcano plot for DEGs between the strain WT and G6006. Gray, blue, and red
dots represent genes with no significant differences, significantly down-regulated genes, and significantly upregulated genes, respectively; (b) COG analysis
comparing expression between WT and G6006. Categories are as follows: C, energy production and conversion; D, cell cycle control and mitosis; E, amino acid
metabolism and transport; F, nucleotide metabolism and transport; G, carbohydrate metabolism and transport; H, coenzyme metabolism and transport; I, lipid
metabolism and transport; J, translation; K, transcription; L, replication and repair; M, cell wall/membrane/envelope biogenesis; O, post-translational modification,
protein turnover, and chaperone functions; P, inorganic ion transport and metabolism;Q, secondary structure; T, signal transduction; U, intracellular trafficking and
secretion;V, defense.

7
G. Zhao et al. Bioresource Technology 387 (2023) 129594

findings lead to speculation that pyruvate derived from lactate could be
more rapidly utilized intracellularly in G6006 as pyc was significantly
upregulated, and thus less pyruvate excretion occurred, which resulted
in significant downregulation of the cg0952-mctC operon in G6006
compare to WT as mctC expression is induced by pyruvate. However, this
hypothesis needs to be experimentally verified. The other mutation
caused a frameshift mutation in whcA, which might lead to enhanced
tolerance to oxidative stress and thus positively affect the growth of cells
(Choi et al., 2009; Park et al., 2012). Interestingly, a significant increase
in the expression of whcA-regulated genes involved in the oxidative
stress response was not detected in G6006 (Choi et al., 2009).
galactose was removed, suggesting that the remaining nutrients from
AW or the high pH caused by lactate removal were inadequate to sustain
LAC1′s growth. It is noteworthy that the addition of LAC1 prolonged the
time needed for depleting lactate, indicating that LAC1 negatively in­
fluences lactate removal by G6006. Taking into account the economic
cost of time, an initial OD600 of 1 for LAC1 and an 18-hour metabolic
period were selected for the complete elimination of lactate and partial
removal of galactose. To completely remove galactose while removing
lactate, it is necessary to screen for LAC1 mutants that rapidly metab­
olize galactose by non-genetic engineering methods, such as physical
and chemical mutagenesis and adaptive evolution.

3.6. Isolation of lactose plasmid-cured derivatives of L. lactis RD1M5

3.8. AW powder without lactate contains more lactose and is less
hygroscopic
In a previous study, RD1M5 was isolated after exposing RD01 to a
chemical mutagen (Liu et al., 2020), a strain with an almost eliminated
When lactate and galactose in AW are eliminated by microorgan­
ability to produce lactic acid. RD1M5 can metabolize lactose and a
isms, other metabolites such as amino acid and acetoin are inevitably
lactose utilization gene cluster was found to be present on one plasmid
generated. To assess the effectiveness of the microbial treatment, a
(Dorau et al., 2020). To obtain a lactose-negative derivative of RD1M5, a
spray-dried powder was prepared using a laboratory mini spray dryer
plasmid curing experiment was carried out with ethidium bromide (EB).
(Buchi- B290). According to Table 3, AWL powder was similar to AWLG
Since exposure of RD1M5 to 3 μg/mL EB resulted in a 99% reduction in
powder in terms of lactose yield, hygroscopicity, and powder particle
viability, this concentration was used for isolating lactose-negative de­
size distribution. In addition, AWLG powder did not have a greater de­
rivatives of RD1M5. After streaking a mutagenized RD1M5 culture on
gree of browning than AWL after two months’ storage (data not shown).
TTC agar plates, only 11 dark red colonies were found (dark red color =
These findings indicate that only lactate has an important influence on
unable to produce lactic acid), indicating that RD1M5 readily reverts to
the yield of the process and properties of the AW powder. Thorakkattu
producing lactic acid. Subsequently, the capacity of these 11 red col­
(2020) observed that the addition of 0.3% galactose to deproteinized
onies to metabolize lactose and galactose was investigated, and finally
whey (DPW) and whey permeate could increase the hygroscopicity of
one isolate, LAC1, was identified, that had lost its ability to metabolize
the spray-dried powders obtained from these. In this study, galactose
lactose, while retaining the ability to metabolize galactose (see supple­
mentary material). Therefore, this strain was used for removing galac­
Table 3
tose in AW in combination with G6006 to remove lactate from AW.
lactose yield, hygroscopicity, and particle size distribution of three kinds of
powders.
3.7. Co-cultivation of strains G6006 and LAC1 to remove lactate and
Analysis Unit RAW AWL AWLG
galactose from AW
Raw AW AW without lactate
AW without and with reduced
In the initial experiment, it was observed that LAC1 exhibited a slow lactate galactose content
metabolism rate of galactose in pH-adjusted AW (data not shown),
Lactose yield (%) % 31.64 49.56 ± 50.45 ± 3.12
possibly due to the insufficient amounts of proteins/peptides to support
± 1.25 0.78
the growth of LAC1. To accelerate the removal of galactose while Hygroscopicity at g/ 5.88 ± 3.32 ± 3.17 ± 0.23
removing lactate, LAC1 with varying initial OD600 was co-cultivated 40% relative 100 0.02 0.08
with G6006, which can produce amino acids, which may be utilized humidity g
by LAC1 and therefore contribute to the growth of LAC1. As seen from Hygroscopicity at g/ 28.15 18.45 ± 16.20 ± 0.42
80% relative 100 ± 0.18 1.22
Fig. 5a, when the initial OD600 of LAC1 is 1, 58.7% of the galactose can humidity g
be consumed within 18 h. Further increasing the inoculum size of LAC1 D [4,3] μm 243.0 112.0 95.3
slowed down the metabolism rate of galactose. However, when pro­ d (0.5) 154.1 55.8 47.8
longing the fermentation time by 18 h, only an additional 14.9% of the

Fig. 5. The effect of initial cell density (OD600) of LAC1 co-cultured with G6006 on (a) galactose removal and (b) lactate removal from AW.

8
G. Zhao et al.
was found to have a minor impact on the hygroscopic properties of the
spray-dried powder, possibly because of the source of the AW, which
could affect its composition and overall properties. As expected, mi­
crobial processing significantly increased the lactose yield after spray
drying by up to about 49.56 ± 0.78% (p < 0.01), as compared to 31.64
± 1.25 % for the RAW. In addition, the results for hygroscopicity
(Table 3) were also significantly different for the RAW and AWL pow­
ders, with values of 5.88 ± 0.02 % (p < 0.001) and 3.32 ± 0.08,
respectively, for relative humidity at 40%, and of 28.15 ± 0.18 % (p <
0.01) and 18.45 ± 1.22 % for relative humidity at 80%. These findings
are consistent with results previously reported by Bédas et al., (2017),
and can be explained by the fact that lactate is highly hygroscopic: the
more lactate removed, the greater the reduction in hygroscopicity will
be (Saffari & Langrish,2014). Murti et al., (2009) reported that as
moisture content increases, the rate of stickiness development also in­
creases. Therefore, hygroscopicity can be used to evaluate a powder’s
tendency towards stickiness and caking. The lower hygroscopicity of
AWL and AWLG powder thus indicates that it is less prone to lumping
and caking during long-term storage. Also, the absence of lactate
influenced the size distributions of powders, and resulted in finer par­
ticles with a size distribution with D[4,3] of 112.0 μm and (0.5) of 55.8
μm, compared to D[4,3] of 243.0 μm and d(0.5) of 154.0 μm for the
powder derived from the untreated AW.
4. Conclusions
For the first time, it shown that C. glutamicum can eliminate lactate
from AW. To reduce the time needed to remove lactate, G6006 derived
from C. glutamicum ATCC 13032 through ALE was isolated. Co-
cultivating G6006 with LAC1, a galactose-metabolizing L. lactis strain,
enabled the removal of all lactate and part of the galactose. The
approach was validated through spray drying of the microbially treated
AW, which produced a stable, low-hygroscopicity powder. These find­
ings highlight C. glutamicum as an excellent catalyst for converting low-
value AW into a valuable resource in a cost-effective and environmen­
tally friendly manner.
CRediT authorship contribution statement
Ge Zhao: Investigation, Data curation, Methodology. Shuangqing
Zhao: Investigation, Data curation, Methodology. Line Hagner Niel­
sen: Investigation, Data curation, Methodology. Fa Zhou: Investigation.
Liuyan Gu: Software, Data curation. Belay Tilahun Tadesse: Investi­
gation. Christian Solem: Conceptualization, Visualization, Supervision,
Project administration, Writing – review & editing.
Declaration of Competing Interest
The authors declare the following financial interests/personal re­
lationships which may be considered as potential competing interests:
Christian Solem reports financial support was provided by Technical
University of Denmark. Christian Solem reports a relationship with
Technical University of Denmark that includes: employment.
Data availability
The data that has been used is confidential.
Acknowledgement
This work was supported by the DTU discovery.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.biortech.2023.129594.
9
Bioresource Technology 387 (2023) 129594
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